Patent Publication Number: US-2009221430-A1

Title: Proteomic Methods For The Identification And Use Of Putative Biomarkers Associated With The Dysplastic State In Cervical Cells Or Other Cell Types

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims the priority of U.S. Provisional Application No. 60/780,983, filed Mar. 10, 2006, entitled, PROTEOMIC METHODS FOR THE IDENTIFICATION AND USE OF PUTATIVE BIOMARKERS ASSOCIATED WITH THE DYSPLASTIC STATE IN CERVICAL CELLS OR OTHER CELL TYPES, the whole of which is hereby incorporated by reference herein. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     N/A 
     BACKGROUND OF THE INVENTION 
     Carcinoma of the uterine cervix is the second most common neoplasm among women worldwide, and fifth leading cause of all cancer related deaths (Baldwin et al., 2003). Recent estimates indicate that approximately 500,000 new cases of cervical cancer are diagnosed annually (Munoz et al., 1989; NIH Statement, 1996). Cervical carcinoma develops slowly over a time period of several years through well-defined non-invasive stages. Preneoplastic lesions, classified as cervical intraepithelial neoplasia (CIN), are defined according to the degree of cellular abnormality and have the potential to progress to carcinoma in situ or invasive carcinoma. While only a small fraction of all dysplastic lesions would progress to invasive cervical cancer if left untreated, the overall risk escalates with increased grade of lesion (Melnikow et al., 1998). 
     It has been well established that early detection of these morphological changes significantly increases the chances for successful treatment. Since its introduction in the 1940&#39;s, the conventional Pap test has dramatically decreased the incidence and mortality rates associated with cervical cancer by identifying and classifying cellular changes associated with the progression to cancer. The abnormal morphological changes that precede squamous cell carcinoma have been classified according to numerous systems, including the commonly used 2001 Bethesda System (Tabbara et al., 1992), among others. Under the Bethesda System, the abnormal morphological changes include Atypical Squamous Cells of Undetermined Significance (ASC-US), Atypical Squamous Cells—Cannot Exclude High-Grade (ASC-H), Low-Grade Squamous Intraepithelial Lesions (LSIL) and High-Grade Squamous Intraepithelial Lesions (HSIL). The concept of cervical intraepithelial neoplasia, or CIN, was introduced in 1967 to embrace all grades of dysplasia and carcinoma in situ under a single disease heading. The diagnosis of CIN is based on histological sections and graded as I, II and III. These and other generally accepted classification schemes are more fully described in Chan et al., The Papanicolaou Test—Its Current Status (1990) Hong Kong Practitioner, 12, 1198-1203, which is incorporated by reference herein. More recent liquid-based cytology (LBC) preparations, such as the ThinPrep® Pap Test (Cytyc Corporation), have proven useful in reducing the number of inadequate Pap tests and the incidence of false negative diagnoses by enabling the improved homogeneous transfer of cells from the cervix to the slide (Roberts et al., 1997). The use of computer imaging to locate potential abnormal cells has also improved the detection of preneoplastic lesions of the cervix. Since the introduction of LBC methodology, the American Cancer Society estimates that the rate of invasive cervical cancer in the US has declined by 28%. The success of the Pap test, however, ultimately relies upon the ability of the technician to accurately identify and evaluate those characteristic cellular changes. 
     Therefore, despite recent improvements in sample collection, processing and image-directed slide review, a number of studies have reported low substantial inter-observer variability and Pap test discordance with histological follow-up, particularly within the ASCUS and LSIL diagnostic categories (Howell et al., 2004; Joste et al., 2005). Importantly, low sensitivity and poor reproducibility within these cytological categories have complicated the management of this subset of patients. Because a diagnosis that is made based upon the cytology sample establishes the basis for further treatment, an inaccurate diagnosis may lead to over-treatment of a healthy woman (i.e., colposcopy and biopsy) or under-treatment of a woman having a cervical lesion. False negative and positive results are therefore costly in terms of time and expense, and can generate significant anxiety in affected women. Improvements in diagnostic accuracy would therefore benefit patients and reduce related health system costs. 
     The subjectivity of cervical cytology may be reduced by integrating the use of objective markers to help determine the presence and severity of dysplastic cells. For example, high-risk human papillomavirus (HPV) has been shown to be present in 99% of all cervical cancers (Syrjanen et al., 1987), and the concept that persistent viral infection is required for progression to cervical neoplasia is well accepted (Cuschieri et al., 2005). However, while HPV DNA testing can provide an objective measurement, high-risk HPV testing cannot accurately discriminate between patients whose squamous intraepithelial lesions will persist or progress to invasive carcinoma and those whose lesions will regress spontaneously. It was reported in the ASCUS-LSIL Triage Study that 83% of women having an LSIL Pap result tested positive for high-risk HPV (ASCUS-LSIL Triage Study (ALTS) Group, 2003), a level too high to provide clinical utility in a patient triage strategy. Although a triage strategy that incorporates HPV detection within the ASCUS population has proven to be more sensitive for detecting underlying high-grade disease, decreased specificity was a primary concern (Shiffman et al., 2003). HPV screening is currently most appropriate in the triage of borderline or ASCUS cytology cases and in conjunction with Pap testing for women 30 years or older. In other words, in this age group, benefits of HPV testing independent of cytology classification have been observed. 
     The human papillomavirus contributes to neoplastic progression predominantly through the action of two viral oncoproteins, E6 and E7, which interact with various host regulatory proteins to influence the function or expression levels of host gene products, eventually leading to the disruption of the cell cycle (Shai et al., 2007). It has been previously demonstrated that the E6 oncoprotein interacts with the p53 tumor suppressor protein (Crook et al., 1991), while E7 binds to the retinoblastoma protein, pRb. p16 INK4a  is a cyclin-dependent kinase inhibitor that negatively regulates cell proliferation by inhibiting hyperphosphorylation of pRb via the cdk4/6 complex. Overexpression of the p16 INK4a  protein has been well documented in cervical cancer and is a consequence of pRb targeted inactivation from E7. While it has been proposed that p16 INK4a  is a useful biomarker for the identification of dysplastic cervical epithelial cells, its specificity has been questioned and other surrogate markers may exist that also have clinical utility due to their ability to quantify cellular changes that are indicative of active HPV oncogene expression rather than viral presence only. The differential expression of specific cellular proteins might therefore prove useful in identifying those clinically important cases of HPV infection that have a more significant risk of progression towards cervical carcinoma. 
     BRIEF SUMMARY OF THE INVENTION 
     While molecular tests for the detection of HPV are very sensitive, the specificity of HPV testing is not currently high enough to perform well in a primary screening setting and is therefore most useful in the triage of ASCUS cytology cases. Incorporation of cellular biomarkers indicative of cervical cancer progression to and through the dysplastic state may help improve sensitivity, specificity, standardization and ultimately the quality of diagnosis. More recently, a variety of molecular approaches have been utilized to identify potential markers of cervical cancer. However, in all cases, cultured mammalian cell lines or cervical cancer tissue was utilized for discovery research purposes. Furthermore, the majority of these research efforts evaluated changes in gene expression, which may or may not directly translate to the protein level. Thus, significant opportunities exist for the identification of cervical markers specifically for the dysplastic state and their utilization in the development of convenient to use, robust and predictive tests having improved diagnostic value. 
     Laser Capture Microdissection (LCM) is a powerful tool that enables the isolation of specific cell types from a heterogeneous population. While this technology has routinely been used with tissue, few studies have applied this methodology to investigate cytological specimens in conjunction with protein analysis. LCM was utilized to select approximately 10,000 high-grade (HSIL) dysplastic cells per specimen from ThinPrep Pap Test prepared slides. Following cell capture, samples were processed and analyzed using a highly sensitive linear ion trap with Fourier transform mass spectrometer (LTQ-FTMS). Multiple individual specimens having a clinical diagnosis of either Within Normal Limits (WNL) or HSIL were evaluated and compared in order to identify proteins that exhibited differential changes in expression, either upregulated and down-regulated. 
     Described herein are the specimen processing and proteomic methods of the invention, which are used to detect and identify potential biomarkers for cervical dysplasia, and the potential biomarkers for cervical dysplasia identified thereby. These same specimen processing and proteomic analysis methods can also be used to enrich any type of clinical sample, preferably an easily accessible clinical sample, for putative dysplastic cells and to analyze the enriched population for novel biomarkers. Information obtained from this type of analysis would be most useful in identifying protein expression profiles or protein signatures that become apparent in dysplastic conditions, before the cells are committed to the cancerous state. A significant aspect of this invention therefore relates to the proteomic characterization of high-grade dysplastic cells. The differential expression of proteins in high-grade dysplastic cells versus morphologically normal cells (of cervical or other tissue) can lead to the potential identification of novel biomarkers most useful in the detection, diagnosis and stratification of the dysplastic condition. 
     Thus, in general, the invention provides a method for the identification of biomarkers for the classification of cells in a manner that can complement or replace any cytological or histological analysis. An exemplary method of identifying a potential cervical dysplasia biomarker for the classification of cells in conjunction with a cytological or histological analysis includes the steps of: a) providing a cervical sample from a patient; b) carrying out the cytological or histological analysis on a specimen from the cervical sample; c) marking high-grade dysplastic cells generically identified by the cytological or histological analysis (e.g., Pap Test stained cells); d) carrying out laser capture microdissection (LCM) of the marked cells; e) lysing the captured cells; f) separating the proteins in the lysed cell preparation (e.g., by SDS-PAGE) and digesting the separated proteins (e.g., with trypsin); g) analyzing the digested samples (e.g., by LTQ/FT LC/MS/MS); h) determining a profile of protein abundance in each of the digested samples of marked cells; i) comparing the protein abundance profiles of said high risk patients with similarly determined protein abundance profiles of healthy individuals; and j) identifying any proteins that are present in the abundance profiles of said high risk patients but not in, or at a reduced level in, the abundance profiles of said healthy individuals, wherein any protein so identified is said potential cervical dysplasia biomarker. In a preferred embodiment, the patient from whom the cervical sample is obtained is suspected of being at high risk of developing a cervical cancer. 
     Furthermore, provided herein, in Tables 1-4, are panels of proteins identified in samples from individual women at risk of developing cervical cancer, wherein the samples have previously been enriched for cells in a dysplastic state. The proteins in these panels, either individually or as relative ratios, are potential biomarkers for the identification of a dysplasia in cervical tissue. Preferentially, the relative ratios of a combination or combinations of biomarkers are utilized for improved diagnostic performance. The methods of the invention also would be useful to detect and to identify potential biomarkers for any dysplastic condition in similarly enriched cell samples. 
     Using the methods of the invention, potential biomarker proteins for a predisposition to high-grade cervical dysplasia have been characterized in individual subjects. Use of proteins identified according to the principles of the invention as biomarkers for the classification of cervical dysplasia is within the invention. In addition, the invention provides a sensitive method for early detection of dysplasia and for monitoring of the related potentially cancerous state. 
     Thus, in one aspect, the invention is directed to a method for assessing the presence of a cervical dysplastic lesion in a human subject, the method including comparing the level of abundance, in a sample from the subject, of at least one marker of the invention selected from the group consisting of the markers listed in Tables 1-4; and the normal level of abundance of the at least one marker in a control sample, wherein a significantly higher level of abundance of the at least one marker in the sample from the subject compared to the level of abundance of the at least one marker in the control sample is an indication of the presence of a cervical dysplastic lesion in the subject. 
     Preferably, the level of abundance of the at least one marker in the sample from the subject is three or more times the abundance level of the at least one marker in the control sample. The level of abundance of the at least one marker can be determined by detecting the amount of marker protein present in the sample, for example by using an assay selected from the group consisting of an antibody based assay, a protein array assay and a mass spectrometry based assay. Alternatively, the level of abundance of the at least one marker can be determined by detecting the amount of mRNA that encodes a marker protein present in the sample. The control sample level of abundance of the at least one marker can be determined from a standard table or curve. In particularly preferred embodiments, a plurality of markers (e.g., three or more or five or more) is detected. 
     The invention additionally provides a test method for assessing the cervical carcinogenic potential of a compound. This method comprises the steps of: obtaining a sample comprising dysplastic cervical cells; maintaining separate aliquots of the dysplastic cells in the presence and absence of a compound to be tested; and comparing the expressed abundance of a marker of the invention in each of the aliquots. A significantly higher level of expression or abundance of a marker according to the invention in the aliquot maintained in the presence of the compound, relative to that of the aliquot maintained in the absence of the compound, is an indication that the compound possesses cervical carcinogenic potential. 
     In addition, the invention further provides methods for assessing the potential of a test composition as an inhibitor of the dysplastic state, e.g., in cervical cells, in a patient. These methods comprise the steps of: obtaining a sample comprising dysplastic cervical cells; separately maintaining aliquots of the sample in the presence and absence of a test composition; comparing the abundance of a marker of the invention in each of the aliquots; and identifying a composition as an inhibitor of the dysplastic, e.g., cervical dysplastic, state where the composition significantly lowers the level of expression of a marker of the invention in the aliquot containing the composition relative to the levels of expression of the marker in the presence of the other compositions. Compositions so identified can be administered appropriately to a patient having dysplasia for treating or for inhibiting the further development of the dysplasia. 
     Markers according to the invention may likewise be used to assess the efficacy of a therapy for inhibiting cervical dysplasia in a patient. In this method, the level of expression of one or more markers of the invention in a pair of samples (one subjected to the therapy, the other not subjected to the therapy) is assessed. As with the method of assessing the potential of test compounds, if the therapy induces a significantly lower level of expression of a marker of the invention, then the therapy can be considered potentially efficacious for inhibiting cervical dysplasia. As above, if samples from a selected patient are used in this method, then alternative therapies can be assessed in vitro in order to select a therapy most likely to be efficacious for inhibiting cervical dysplasia in the patient. Furthermore, the methods of the invention may be used to evaluate a patient before, during and after therapy, for example, to evaluate the reduction in tumor burden. 
     In another aspect, the invention relates to various diagnostic and test kits for detecting the presence of a marker protein in a subject sample (e.g., a cervical sample). In one embodiment, the invention provides a kit for assessing whether a human subject is afflicted with a cervical dysplasia. The kit comprises one or more reagents for assessing expression of at least one marker of the invention. For antibody-based kits, for example, a kit comprises, e.g., (1) a first antibody (e.g., attached to a solid support) that binds to a marker protein; and, optionally, (2) a second, different antibody that binds to either the protein or the first antibody and is conjugated to a detectable label. In another embodiment, the invention provides a kit for assessing the suitability of a chemical or biologic agent for inhibiting the progression of cells in the dysplastic state to the cancerous state in a patient. Such a kit comprises reagents for assessing expression of at least one marker of the invention and may also comprise one or more of such agents. In a further embodiment, the invention provides kits for assessing the presence of dysplastic cells. Such kits may comprise an antibody, an antibody derivative, or an antibody fragment that binds specifically with a marker protein, or a fragment of the protein. Such kits may also comprise a plurality of antibodies, antibody derivatives, or antibody fragments wherein the plurality of such antibody agents binds specifically with a marker protein, or a fragment of the protein. 
     Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention relates to methods for detecting and identifying potential biomarkers of high-grade cervical dysplasia in an individual human subject. The invention also relates to newly discovered biomarkers, as set forth in Tables 1-4, which are associated with the dysplastic state of cervical cells. It has been discovered that a differential level of expression of any of these markers or combination of these markers correlates with a dysplastic condition in a human subject, e.g., a patient. 
     As used herein, each of the following terms has the meaning associated with it in this section. 
     A “marker” is a protein, or associated gene or other nucleic acid, whose altered level of expression (or abundance) in a tissue or cell from its expression level in normal or healthy tissue or cell is associated with a disease state, such as cancer. 
     “Proteins of the invention” encompass marker proteins and their fragments; variant marker proteins and their fragments (including those with side chain modifications); peptides and polypeptides comprising an at least 15 amino acid segment of a marker or variant marker protein; and fusion proteins comprising a marker or variant marker protein, or an at least 15 amino acid segment of a marker or variant marker protein. 
     The term “probe” refers to any molecule that is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or marker protein. Probes can be either synthesized by one skilled in the art or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. 
     A “cervical sample” or “patient cervical sample” comprises cervical cells and/or cervical-associated body fluid obtained from a human subject, e.g., a patient. 
     A “cervical-associated” body fluid is a fluid that, when in the body of a subject, contacts or passes through cervical cells or into which cervical cells or proteins shed from cervical cells are capable of passing. The cells may be found in a cervical smear collected, for example, by a cervical brush. Exemplary cervical-associated body fluids include blood fluids, lymph, ascitic fluids, gynecological fluids, cystic fluid, urine, and fluids collected by vaginal rinsing. 
     The “normal” level of expression (or WNL level) of a marker is the level of expression or abundance of the marker in a cervical sample of a subject not afflicted with a cervical dysplasia. 
     An “over-expression” or “significantly higher level of expression” of a marker refers to an abundance or expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least three, and more preferably four, five or ten times the expression level of the marker in a control sample (e.g., sample from a healthy subjects not having the marker-associated condition) and preferably, the average expression level of the marker in several control samples. 
     A “significantly lower level of expression” of a marker refers to an abundance or expression level in a test sample that is at least three, and more preferably four, five or ten times lower than the expression level of the marker in a control sample (e.g., sample from a healthy subjects not having the marker-associated condition) and preferably, the average expression level of the marker in several control samples. 
     A “kit” is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., a probe, for specifically detecting the abundance or expression of a marker of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. 
     Unless otherwise specified herein, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody. 
     The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. It will be appreciated that the methods and kits of the present invention may also include known cervical dysplasia markers or other materials known to bind to proteins such as small molecules, substrate mimetics, other non-antibody binding proteins, RNA or DNA aptamers, etc. 
     The present invention is based, in part, on newly identified biomarkers, which are differentially expressed in dysplastic cervical cells as compared to their expression in normal or healthy cervical cells. The enhanced or reduced expression of one or more of these markers in cervical cells is herein correlated with the dysplastic state of the tissue. The invention provides compositions, kits and methods for assessing the dysplastic state of cells (e.g., cells obtained from a human, cultured human cells, archived or preserved human cells and in vivo cells) as well as for treating patients afflicted with the dysplastic state. 
     The invention thus includes a method of assessing the dysplastic state of cervical cells in a human subject. This method comprises comparing the level of expression of one or more markers of the invention (listed in Tables 1-4) in a cervical sample from a subject (i.e., cervical cells and/or cervical-associated body fluid) and the normal level of expression of the one or more markers in a control, e.g., a human subject not afflicted with cervical dysplasia. For, example, a significantly higher level of expression, or abundance, of the marker in the patient sample as compared to the normal level of expression is an indication that the subject has a dysplastic condition. It is also within the invention to use a combination of the identified biomarkers and to assess the differential expression of these markers as a change in their relative ratios. 
     Furthermore, the invention encompasses in general an approach to targeted clinical proteomics wherein a potentially cancerous lesion from a patient is sampled and then the sample is enriched for a specific dysplastic cell type. In this way, one can correlate morphological changes in the tissue with biomarkers and establish the relationship of the biomarker to the stage of disease. For example, one can identify a biomarker in a cell type associated with a specific staging of the disease and then carry out imaging of the cell type with an antibody to that protein. In this manner, the antibody can be used as a backup to the cytology procedures and to reduce error rates. Also such an antibody can be used for imaging studies of the distribution of cancerous or precancerous cells as the disease progresses. 
     Although an immunocytochemistry based assay is described herein, the methods according to the invention also encompass using any other method known to or later developed by those of ordinary skill in the art within a cellular and/or morphological context. For example, the use of immunohistochemistry, flow cytometry, etc., as well as soluble formats (e.g., ELISA) are encompassed herein. 
     In addition to cervical disease, the methods of the invention have application for other diseases and carcinomas such as those of the breast, lung, colon, anus, stomach, nasal tissue, mouth, esophagus and skin. The expectation from theory and general practice is that all squamous/adeno (i.e., “skin”) derived cancers have a pre-invasive phase. The detection of this pre-invasive phase is dependent on the accessibility of the organ. 
     The colon has pre-cancerous polyps, the anus has pre-invasive skin changes, and similar esophageal changes are observed. Anal and colon lesions may be detected by direct vision via endoscope or colonoscopy, and esophageal lesions by endoscopy. Cells from these pre-cancerous lesions can be obtained via biopsy or washings. 
     The method of the invention can also be practiced employing a device in which a membrane based on the Pap smear is used to collect a layer of cells from the cancer tissue (in cervix, mouth, lung, nose, eye, kidney tubules, colon, etc., and the membrane is then transferred to an automated device, such as an LCM device, where the target cells are collected. The target cells can be identified, e.g., by a flourescently labelled antibody discovered in an earlier phase of the study. The sensitivity and specificity of such an assay can be increased by combining the Pap smear membrane aspect with LCM. In this manner, one can generate a total abnormal cell count as well as a histogram of the distribution of label. 
     The invention also includes an array comprising a marker of the present invention. The array can be used to assay abundance of, e.g., one or more proteins in the array. In one embodiment, the array can be used to assay protein abundance in an individual sample from a patient to ascertain the specificity of proteins in the array. In this manner, a large number of proteins can be simultaneously assayed for expression or abundance level. This allows a profile to be developed showing a battery of proteins specifically expressed in one or more sample sites. 
     In addition to such qualitative determination, the invention allows the quantitation of protein expression. Thus, not only sample site specificity, but also the level of abundance of a battery of proteins in individual samples is ascertainable. Thus, proteins can be grouped on the basis of their expression site per se and level of expression at that site. 
     In another embodiment, the array can be used to monitor the time course of expression of one or more proteins in the array. This can occur in various biological contexts, as disclosed herein, related to the development of cervical cancer. 
     Markers of the invention are selective for as an indication of the presence of a cervical dysplastic lesion. By “an indication of the presence of a cervical dysplastic lesion” it is intended that the marker of interest is overexpressed in high-grade cervical disease but is not overexpressed in conditions classified as WNL, ASCUS, LSIL, CINI, immature metaplastic cells, and other conditions that are not considered to be clinical disease. Thus, detection of the markers of the invention permits the differentiation of samples indicative of underlying high-grade cervical disease from samples that are indicative of benign proliferation, or mild dysplasia. As used herein, “mild dysplasia” refers to LSIL and CINI where no high-grade lesion is present. The methods of the invention also distinguish cells indicative of high-grade disease from normal cells, immature metaplastic cells, and other cells that are not indicative of clinical disease. In this manner, the methods of the invention permit the accurate identification of high-grade cervical disease, even in cases mistakenly classified as normal, CINI, LSIL, or ASCUS by traditional Pap testing (i.e., “false negatives”). In some embodiments, the methods for diagnosing high-grade cervical disease are performed as a reflexive response to an abnormal or atypical Pap smear. That is, the methods of the invention may be performed in response to a patient having an abnormal or atypical Pap smear result. In other aspects of the invention, the methods are performed as a primary screening test for high-grade cervical disease in the general population of women, just as the conventional Pap test is performed currently. 
     The markers of the invention include any gene or protein that is selectively over expressed in cervical disease, as defined herein above. Such markers are capable of identifying cells within a cytology cell suspension that are an indication of the presence of a cervical dysplastic lesion. The biomarkers of the invention detect cells of CINII conditions and above, but do not detect CINI where there is no underlying high-grade disease. 
     As discussed above, a significant percentage of patients presenting with Pap smears classified as WNL, CINI, or ASCUS actually have lesions characteristic of high-grade cervical disease. Thus, the methods of the present invention permit the identification of high-grade cervical disease in all patient populations, including these “false negative” patients, and facilitate the detection of rare abnormal cells in a patient sample. The diagnosis can be made independent of cell morphology and HPV infection status, although the methods of the invention can also be used in conjunction with conventional diagnostic techniques, e.g., Pap test, molecular testing for high-risk types of HPV, etc. 
     Assessing the presence of a cervical dysplastic lesion is intended to include, for example, diagnosing or detecting the presence of cervical disease, monitoring the progression of the disease, and identifying or detecting cells or samples that are indicative of high-grade cervical disease. The terms diagnosing, detecting, and identifying high-grade cervical disease are used interchangeably herein. By “high-grade cervical disease” is intended those conditions classified by colposcopy as premalignant pathology or moderate to severe dysplasia. Underlying high-grade cervical disease includes histological identification of CINII, CINIII and HSIL. 
     In particular embodiments, the diagnostic methods of the invention comprise collecting a cervical sample from a patient, contacting the sample with at least one antibody specific for a marker of interest, and detecting antibody binding. Samples that exhibit over expression of a marker of the invention, as determined by detection of antibody binding, are deemed positive for high-grade cervical disease. In particular embodiments, the body sample is a monolayer of cervical cells. In some aspects of the invention, the monolayer of cervical cells is provided on a glass slide. 
     By “body sample” is intended any sampling of cells, tissues, or bodily fluids in which expression of a biomarker can be detected. Examples of such body samples include but are not limited to blood, lymph, urine, gynecological fluids, biopsies, and smears. Body samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to aspirate bodily fluids. Methods for collecting various body samples are well known in the art. In particular embodiments, the body sample comprises cervical fluid or cervical cells, as cervical tissue samples or as cervical cells in suspension, particularly in a liquid-based preparation. In one embodiment, cervical samples are collected according to liquid-based cytology specimen preparation guidelines such as, for example, the ThinPrep® System (Cytyc Corporation, Marlborough, Mass.). Body samples may be transferred to a glass slide for viewing under magnification. Fixative and staining solutions may be applied to the cells on the glass slide for preserving the specimen and for facilitating examination. In one embodiment the cervical sample will be collected and processed to provide a monolayer sample, as set forth in U.S. Pat. No. 5,143,627, herein incorporated by reference. 
     Any methods available in the art for identification or detection of the markers are encompassed herein. The over expression of a biomarker of the invention can be detected on a nucleic acid level or a protein level. In order to determine over expression, the body sample to be examined may be compared with a corresponding body sample that originates from a healthy person. That is, the “normal” level of expression is the level of expression of the biomarker in cervical cells of a human subject or patient not afflicted with high-grade cervical disease. Such a sample can be present in standardized form. In some embodiments, particularly when the body sample comprises a monolayer of cervical cells, determination of biomarker over expression requires no comparison between the body sample and a corresponding body sample that originates from a healthy person. In this situation, the monolayer of cervical cells from a single patient may contain as few as 1-2 abnormal cells per 50,000 normal cells present. Detection of these abnormal cells, identified by their over expression of a biomarker of the invention, precludes the need for comparison to a corresponding body sample that originates from a healthy person. 
     Methods for detecting markers of the invention comprise any methods that determine the quantity or the presence of the biomarkers either at the nucleic acid or protein level. Such methods are well known in the art and include but are not limited to western blots, northern blots, southern blots, ELISA, immunoprecipitation, immunofluorescence, flow cytometry, immunocytochemistry, nucleic acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In particular embodiments, over expression of a biomarker is detected on a protein level using, for example, antibodies that are directed against specific biomarker proteins. These antibodies can be used in various methods such as Western blot, ELISA, immunoprecipitation, or immunocytochemistry techniques. Likewise, immunostaining of cervical smears can be combined with conventional Pap stain methods so that morphological information and immunocytochemical information can be obtained. In this manner, the detection of the biomarkers can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening. 
     In another aspect, the invention relates to various diagnostic and test kits. In one embodiment, the invention provides a kit for assessing whether a patient is afflicted with high grade cervical dysplasia. The kit comprises a reagent for assessing expression of a marker of the invention. In another embodiment, the invention provides a kit for assessing the suitability of a chemical or biologic agent for inhibiting cervical dysplasia in a patient. Such kits comprise a reagent for assessing expression of a marker of the invention, and may also comprise one or more of such agents. In a further embodiment, the invention provides kits for assessing the presence of cervical dysplastic cells or treating cervical dysplasia. Such kits comprise an antibody, an antibody derivative, or an antibody fragment that binds specifically with a marker protein, or a fragment of the protein. Such kits may also comprise a plurality of antibodies, antibody derivatives, or antibody fragments wherein the plurality of such antibody agents binds specifically with a marker protein, or a fragment of the protein. 
     In an alternative embodiment, the invention provides a kit for assessing the presence of high-grade cervical dysplastic cells wherein the kit comprises a nucleic acid probe that binds specifically with a marker nucleic acid or a fragment of the nucleic acid. The kit may also comprise a plurality of probes, wherein each of the probes binds specifically with a marker nucleic acid, or a fragment of the nucleic acid. Suitable reagents for binding with a marker nucleic acid (e.g., a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like) include complementary nucleic acids. For example, the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like. 
     For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a marker protein; and, optionally, (2) a second, different antibody that binds to either the protein or the first antibody and is conjugated to a detectable label. 
     For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a marker protein or (2) a pair of primers useful for amplifying a marker nucleic acid molecule. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. 
     It is recognized that certain marker proteins are secreted from cervical cells (i.e., one or both of normal and cancerous cells) to the extracellular space surrounding the cells. These markers are preferably used in certain embodiments of the compositions, kits, and methods of the invention, owing to the fact that such marker proteins can be detected in a cervical-associated body fluid sample, which may be more easily collected from a human patient than a tissue biopsy sample. In addition, preferred in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. An exemplary technique is disclosed in U.S. Pat. No. 6,665,050, hereby incorporated by reference herein. 
     A preferred agent for detecting marker protein of the invention is an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment or derivative thereof (e.g., Fab or F(ab′).sub.2) can be used. In a preferred embodiment, expression of a marker is assessed using a labeled antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugated with a substrate or with the protein or ligand of a protein-ligand pair such as biotin-streptavidin), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) that binds specifically with a marker protein or fragment thereof, including a marker protein which has undergone all or a portion of its normal post-translational modification. 
     An exemplary method for detecting the presence or absence of a marker protein or nucleic acid in a biological sample involves obtaining a biological sample (e.g., a cervical-associated body fluid) from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA, or CDNA). The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo. Exemplary in vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. 
     Exemplary in vitro techniques for detection of a marker protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. Exemplary in vitro techniques for detection of genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against the protein or fragment thereof. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. 
     A general principle of such diagnostic and prognostic assays involves preparing a sample or reaction mixture that may contain a marker, and a probe, under appropriate conditions and for a time sufficient to allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. These assays can be conducted in a variety of ways. 
     For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid phase support, also referred to as a substrate, and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample from a subject, which is to be assayed for presence and/or concentration of marker, can be anchored onto a carrier or solid phase support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid phase and a sample from a subject can be allowed to react as an unanchored component of the assay. 
     There are many established methods for anchoring assay components to a solid phase. These include, without limitation, marker or probe molecules which are immobilized through conjugation of biotin and streptavidin. Such biotinylated assay components can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemicals). In certain embodiments, the surfaces with immobilized assay components can be prepared in advance and stored. 
     Other suitable carriers or solid phase supports for such assays include any material capable of binding the class of molecule to which the marker or probe belongs. Well-known supports or carriers include, but are not limited to, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. 
     In order to conduct assays with the above mentioned approaches, the non-immobilized component is added to the solid phase upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid phase. The detection of marker/probe complexes anchored to the solid phase can be accomplished in a number of methods outlined herein. In a preferred embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with detectable labels discussed herein and which are well-known to one skilled in the art. 
     It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see, for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, “donor” molecule is selected such that, upon excitation with incident light of appropriate wavelength, its emitted fluorescent energy will be absorbed by a fluorescent label on a second, “acceptor” molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the “donor” protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the “acceptor” molecule label may be differentiated from that of the “donor.” Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the “acceptor” molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). 
     In another embodiment, determination of the ability of a probe to recognize a marker can be accomplished without labeling either assay component (probe or marker) by utilizing a technology such as real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander, S. and Urbaniczky, C., 1991, Anal Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA” or “surface plasmon resonance” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore®). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules. 
     Alternatively, in another embodiment, analogous diagnostic and prognostic assays can be conducted with marker and probe as solutes in a liquid phase. In such an assay, the complexed marker and probe are separated from uncomplexed components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, marker/probe complexes may be separated from uncomplexed assay components through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A. P., 1993, Trends Biochem Sci. 118(8):284-7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the marker/probe complex as compared to the uncomplexed components may be exploited to differentiate the complex from uncomplexed components, for example through the utilization of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, N. H., 1998, J. Mol. Recognit. 11(1-6):141-8; Hage, D. S., and Tweed, S. A. J Chromatogr B Biomed Sci Appl 1997 699(1-2):499-525). Gel electrophoresis may also be employed to separate complexed assay components from unbound components (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, 1987-1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, non-denaturing gel matrix materials and conditions in the absence of reducing agent are typically preferred. Appropriate conditions to the particular assay and components thereof will be well known to one skilled in the art. 
     In a particular embodiment, the level of marker mRNA can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art. The term “biological sample” is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject. Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cervical cells (see, e.g., Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley &amp; Sons, New York, 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155). 
     The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding a marker of the present invention. Other suitable probes for use in the diagnostic assays of the invention are described herein. Hybridization of an mRNA with the probe indicates that the marker in question is being expressed. 
     In one format, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in, detecting the level of mRNA encoded by the markers of the present invention. 
     An alternative method for determining the level of mRNA marker in a sample involves the process of nucleic acid amplification, e.g., by rtPCR (the experimental embodiment set forth in Mullis, 1987., U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5′ or 3′ regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers. 
     For in situ methods, mRNA does not need to be isolated from the cervical cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the marker. 
     As an alternative to making determinations based on the absolute expression level of the marker, determinations may be based on the normalized expression level of the marker. Expression levels are normalized by correcting the absolute expression level of a marker by comparing its expression to the expression of a gene that is not a marker, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cervical cancer sample, or between samples from different sources. 
     Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a marker, the level of expression of the marker is determined for 10 or more samples of normal versus high-grade dysplastic cell isolates, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the genes assayed in the larger number of samples is determined and this is used as a baseline expression level for the marker. The expression level of the marker determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that marker. This provides a relative expression level. 
     Preferably, the samples used in the baseline determination will be from high-grade dysplastic or from non-cervical cancer cells of cervical tissue. The choice of the cell source is dependent on the use of the relative expression level. Using expression found in normal tissues as a mean expression score aids in validating whether the marker assayed is cervical specific (versus normal cells). In addition, as more data is accumulated, the mean expression value can be revised, providing improved relative expression values based on accumulated data. Expression data from cervical dysplastic cells provides a means for grading the severity of the dysplasia. 
     Methods 
     Preliminary Method Evaluation: Prior to the protein discovery work utilizing clinical specimens, several pilot studies were completed to assess the compatibility of PreservCyt® Solution (Cytyc Corporation) and ThinPrep® Pap Stain (Cytyc Corporation) with subsequent sample processing methods and mass spectroscopy analysis. PreservCyt fixative is a proprietary methanol-based buffered preservative solution designed to support cells during transport and slide preparation on the ThinPrep 2000 or 3000 Processor. PreservCyt Solution has routinely been utilized for the collection, storage, and processing of gynecological samples as well as Fine Need Aspirates (FNA), mucoid specimens, body fluids, and superficial brushings and scrapings. The ThinPrep Pap Stain is a specialized cocktail of individual stains (including hematoxylin, Orange G, Eosin) which has been specifically optimized for the visualization and diagnosis of cervical cytology specimens. 
     An in-solution cell staining protocol was developed to evaluate HeLa cells that had been cultured, harvested at confluency, and placed into PreservCyt solution. Cells in solution were split and subsequently processed through a series of incubation and wash steps utilizing protocols adapted from existing slide-based staining procedures to stain cells with either hematoxylin or the ThinPrep Pap Stain. Because hematoxylin stained tissue has been successfully utilized in mass spectroscopy, protein recovery for cells processed using hematoxylin was directly compared to cells processed with the ThinPrep stain. No significant differences in protein recovery were observed between the two staining methods. Because the ThinPrep Pap stain provides improved morphological discrimination of dysplastic cells proved to be compatible with subsequent mass spectroscopy methods, this stain was utilized in the processing of all cervical specimens. 
     Sample Procurement Residual ThinPrep cervical specimens having a diagnosis of Within Normal Limits (WNL) or HSIL were evaluated for overall cellularity as well as the percentage of high-grade cells (HSIL specimens). An initial ThinPrep slide (control slide) was prepared on the ThinPrep 2000 instrument from residual clinical samples and subsequently ThinPrep Pap stained and coverslipped. This control slide was utilized to confirm clinical diagnosis and select specimens suitable for inclusion in the study. In addition to requirements of adequate cellularity, all specimens met additional inclusion criteria such as a minimal prevalence of polymorphonuclear neutrophils (PMN&#39;s) and bacteria. Finally, selected specimens were processed approximately 6 weeks or less from the date of collection in an effort to minimize potential protein degradation. 
     For specimens having adequate cellularity, the ThinPrep processor and filter routinely applies approximately 70,000 cells to the slide in a homogeneous thin layer. Because the actual number of high-grade cells can vary substantially between specimens, multiple slides were prepared from selected cases having the highest percentage of dysplastic cells and Pap stained. Abnormal cells from HSIL specimens were identified and marked on the back side of slides using a xylene resistant pen (0.20 mm Pigma pen, Sakura Color Products Corp.) in conjunction with Cytyc&#39;s ThinPrep Imaging System Review Scope®. Reference marks served as locator guides for the LCM operator to identify the high-grade cells or cell clusters of interest. A cell count (number of high-grade cells) was performed at this time to ensure that a minimum of 12,000 cells were available per specimen. A total of 116 residual ThinPrep samples having an initial clinical diagnosis of HSIL were acquired and evaluated; of these 10 samples (˜9%) satisfied all inclusion requirements and were prepared for LCM. A total of nine HSIL (abnormal), one AGUS (abnormal) and 13 WNL (normal) samples were analyzed for this study. (AGUS=atypical glandular cells of undetermined significance) 
     Sample Assessment: To assist in the interpretation of proteomic results, a macroscopic cellular assessment was completed for each WNL and HSIL specimen control slide. Evaluations included estimates for the percentage of superficial, intermediate, and parabasal cells as well as endocervical and metaplastic cellular components. This information was documented for WNL specimens to better understand how potential differences in protein profiles might be attributed to differences in cellular content. For HSIL specimens, patient follow-up information was also requested to permit the segregation of potential patterns. Finally, a 0.25 ml aliquot was removed from each HSIL specimen for HPV genotype analysis using the Roche Linear Array HPV Genotyping Test. A 4 ml aliquot was removed from WNL specimens and subject to analysis using Digene&#39;s HCII test for the detection of low-risk or high-risk HPV. 
     Laser Capture Microdissection (LCM): Coverslips were removed from Pap stained slides with xylene and air-dried. Prior to LCM, Prep Strips were applied to remove poorly adhered material and help reduce overall background. Immediately before cell capture, a drop of xylene was applied to the slide to allow visualization of the cells for coordinate selection. Cycles of xylene application, coordinate selection, and drying were utilized to identify and capture high-grade cells present on the slide. Cells were collected using CapSure polyethylene membrane caps and the LCM caps subsequently placed in eppendorf tubes. Approximately 12,000 cells per specimen were selected using the Autopix LCM System® from Arcturus (Mountain View, Calif.). 
     Quality control was performed to assess both the background and accuracy of cell removal during the LCM process. This was accomplished by imaging representative areas of the slide before and after LCM. In summary, two slides from each case were selected for quality control and a total of 8 before and after images were taken from each slide (2 images per slide quadrant). Finally, a full image of the LCM cap was taken for all caps. Images were reviewed by a cytotechnologist to quantify the accuracy of selective abnormal cell removal as well as the approximate number of normal cells unintentionally removed (background). Background for the majority of slides was determined to be less than five percent for all samples. 
     SDS-PAGE: Lysis buffer (2% SDS) was added to the Eppendorf tube to solubilize LCM captured cells. Protein extract was subjected to SDS-PAGE to separate proteins by molecular weight. The gels were divided into three sections and in-gel tryptic digestion performed.
 
LTQ FT Mass Spectroscopy (MS): Proteolytic samples were analyzed by on-line liquid chromatography using a Thermo Electron linear ion trap with Fourier transfer mass spectrometer (LTQ-FT) with a Dionex nanoLC instrument and a 75 μm ID×15 cm C-18 capillary column (flow rate of 300 mL per minute). Mass spectrometry was performed as 1 full FT-MS scan followed by 8 sequential LTQ-MS/MS scans throughout the 90-minute separation.
 
Protein Identification and Quantitation: ProteinProphet probability software was utilized first to identify proteins based upon corresponding peptide sequences with &gt;95% confidence, followed by confirmation from accurate mass assignment (within 5 ppm). The peak area from the extract ions (i.e. disease and normal) were used for comparison (differential quantitation).
 
     Experimental 
     Method: Cervical specimens were evaluated for overall cellularity as well as the percentage of having a diagnosis of high-grade squamous intraepithelial lesion (HSIL) cells. Multiple slides were prepared from selected cases, and subsequently imaged utilizing Pap stained and ThinPrep Imaging System. Cells selected for LCM were marked using the Review Scope. Approximately 12,000 high-grade cells per specimen were captured via LCM using the Autopix System®. Cells were then lysed with SDS and proteins separated via SDS-PAGE in preparation for in-gel digestion. The resulting peptides were analyzed by on-line liquid chromatography with a LTQ-FTMS. Proteins with different quantitation levels between normal and HSIL samples were identified by comparing the intensities of the representative peptide ions after normalization with intrinsic house keeping proteins and/or cell numbers.
 
Results: Diagnostic cells of interest from ThinPrep cervical cytology specimens were identified, selected via LCM, and successfully processed for proteomic analysis using mass spectroscopy. To validate this approach, reproducibility and dynamic range were first studied. Less than 30% variation for a given sample was observed for the entire process, and good linearity (r2=0.95) from 3,000 to 24,000 cells was obtained. Following this, 10 disease (HSIL) and 10 normal LCM samples were globally investigated. 2,184 proteins with at least 2 peptide identifications, and including one peptide with accurate mass, a total of 4300 unique proteins were identified. Many proteins were found to be up- or down-regulated with at least a 3-fold difference, particularly in nuclear and mitochondrial regions, based on Gene Ontology software. Due to the sensitivity and dynamic range of this approach, very few cells were required for analysis, and quantitation without labeling was successfully employed. Protein profiles unique to high-grade dysplastic cells can yield potential biomarkers for molecular diagnostic applications.
 
     These results are illustrated in the following Tables. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Relative Abundant Proteins With Significant Biological Interest Upregulated in 
               
               
                 High-Grade Cervical Specimens 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Number Up- 
                   
               
               
                 Gene/Protein Name 
                 GI Accession 
                 Regulated Pairs 
                 UniGene ID 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Heterogeneous nuclear ribonucleoproteins A2/B1 
                 133257 
                 9 
                 HS.487774 
               
               
                 Heterogeneous nuclear ribonucleoproteins C1/C2 
                 20455507 
                 9 
                 HS.508848 
               
               
                 Heterogeneous nuclear ribonucleoprotein K 
                 48429103 
                 8 
                 HS.522257 
               
               
                 Heterogenous nuclear ribonucleoprotein U 
                 6226894 
                 7 
                 HS.166463 
               
               
                 Polypyrimidine tract-binding protein 1 
                 131528 
                 5 
                 HS.172550 
               
               
                 Heterogeneous nuclear ribonucleoprotein A1 
                 133254 
                 4 
                 HS.534921, HS.546261 
               
               
                 Heterogeneous nuclear ribonucleoprotein F 
                 1710628 
                 4 
                 HS.808 
               
               
                 Heterogeneous nuclear ribonucleoprotein H 
                 1710632 
                 4 
                 HS.202166 
               
               
                 Heterogeneous nuclear ribonucleoprotein R 
                 12230547 
                 4 
                 HS.373763 
               
               
                 Heterogeneous nuclear ribonucleoprotein D0 
                 13124489 
                 3 
                 HS.480073 
               
               
                 Heterogeneous nuclear ribonucleoprotein M 
                 55977747 
                 3 
                 HS.465808 
               
               
                 Heterogeneous nuclear ribonucleoprotein H 
                 2500576 
                 2 
                 HS.278857 
               
               
                 Heterogeneous nuclear ribonucleoprotein L 
                 133274 
                 1 
                 HS.446623 
               
               
                 Heterogeneous nuclear ribonucleoprotein A3 
                 51338779 
                 1 
                 HS.516539 
               
               
                 Transgelin-2 
                 586000 
                 10 
                 HS.517168 
               
               
                 Nucleolin 
                 128841 
                 6 
                 HS.79110 
               
               
                 ADP, ATP carrier protein, fibroblast isoform 
                 113459 
                 9 
                 HS.522767 
               
               
                 Voltage-dependent anion-selective channel protein 2 
                 1172554 
                 8 
                 HS.355927 
               
               
                 Voltage-dependent anion-selective channel protein 1 
                 130683 
                 4 
                 HS.519320 
               
               
                 Superoxide dismutase [Mn], mitochondrial precursor 
                 134665 
                 4 
                 HS.487046 
               
               
                 60S ribosomal protein L7 
                 133021 
                 6 
                 HS.421257 
               
               
                 60S acidic ribosomal protein P0 
                 133041 
                 4 
                 HS.546285, HS.448 
               
               
                 40S ribosomal protein S5 
                 22002064 
                 3 
                 HS.378103 
               
               
                 Keratin, type I cytoskeletal 18 
                 125083 
                 4 
                 HS.406013 
               
               
                 Peptidyl-prolyl cis-trans isomerase A 
                 51702775 
                 4 
                 HS.356331, HS.517 
               
               
                   
                   
                   
                 HS.356331 
               
               
                 Peptidyl-prolyl cis-trans isomerase B precursor 
                 118090 
                 5 
                 HS.434937 
               
               
                 GTP-binding nuclear protein Ran 
                 51338598 
                 4 
                 HS.10842, HS.519656 
               
               
                 Ras-related protein Rab-7 
                 1709999 
                 3 
                 HS.15738 
               
               
                 Heat shock protein HSP 90-alpha 
                 123678 
                 4 
                 HS.523560, HS.525600 
               
               
                 Heat shock protein HSP 90-beta 
                 17865718 
                 5 
                 HS.509736 
               
               
                 Heat shock 70 kDa protein 1 
                 462325 
                 4 
                 HS.405994, HS.520028 
               
               
                   
                   
                   
                 HS.274402, HS.490287 
               
               
                   
                   
                   
                 HS.546245, HS.533257 
               
               
                   
                   
                   
                 HS.522463, HS.439552 
               
               
                   
                   
                   
                 HS.52 
               
               
                 Pyruvate kinase, isozymes M1/M2 
                 20178296 
                 6 
                 HS.198281 
               
               
                 Ubiquitin-activating enzyme E1 
                 24418865 
                 3 
                 HS.533273 
               
               
                 Myosin-9 
                 6166599 
                 3 
                 HS.474751 
               
               
                 SET protein 
                 46397790 
                 3 
                 HS.436687 
               
               
                 Histone H2A.z 
                 121994 
                 4 
                 HS.119192 
               
               
                 T-complex protein 1, beta subunit 
                 6094436 
                 3 
                 HS.189772 
               
               
                 Erythrocyte band 7 integral membrane protein 
                 114823 
                 4 
                 HS.253903 
               
               
                 Moesin 
                 127234 
                 4 
                 HS.87752 
               
               
                 Peroxiredoxin 1 
                 548453 
                 7 
                 HS.180909 
               
               
                 Sodium/potassium-transporting ATPase alpha-1 chain 
                 114374 
                 3 
                 HS.371889 
               
               
                 precursor 
               
               
                 Elongation factor Tu, mitochondrial precursor 
                 1706611 
                 3 
                 HS.12084 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Relative Abundant Proteins Upregulated in High-Grade Cervical Specimens 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Number Up- 
                   
               
               
                 Gene/Protein Name 
                 GI Accession 
                 Regulated Pairs 
                 UniGene ID 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Malate dehydrogenase, mitochondrial precursor 
                 6648067 
                 8 
                 HS.520967 
               
               
                 Keratin, type II cytoskeletal 7 
                 20178293 
                 8 
                 HS.411501 
               
               
                 Vimentin 
                 55977767 
                 8 
                 HS.533317 
               
               
                 Endoplasmin precursor 
                 119360 
                 7 
                 HS.459507, HS.192 
               
               
                 60 kDa heat shock protein, mitochondrial precursor 
                 129379 
                 7 
                 HS.471014, HS.113 
               
               
                 Transketolase 
                 1729976 
                 7 
                 HS.89643 
               
               
                 Protein disulfide-isomerase A3 precursor 
                 2507461 
                 7 
                 HS.308709 
               
               
                 Stress-70 protein, mitochondrial precursor 
                 21264428 
                 7 
                 HS.547532, HS.184 
               
               
                 Dolichyl-diphosphooligosaccharide-protein 
                 132559 
                 6 
                 HS.518244 
               
               
                 glycosyltransferase 67 kDa subunit precursor 
               
               
                 Histone H1.2 
                 417101 
                 6 
                 HS.7644 
               
               
                 Core histone macro-H2A.1 
                 12643340 
                 6 
                 HS.420272 
               
               
                 Neutrophil defensin 3 precursor 
                 30316323 
                 6 
                 HS.294176, HS.380 
               
               
                 Tubulin beta-1 chain 
                 56757569 
                 6 
                 HS.294176 
               
               
                 14-3-3 protein tau 
                 112690 
                 5 
                 HS.74405 
               
               
                 Annexin A5 
                 113960 
                 5 
                 HS.480653 
               
               
                 Apolipoprotein A-I precursor 
                 113992 
                 5 
                 HS.534983, HS.93194 
               
               
                 Calreticulin precursor 
                 117501 
                 5 
                 HS.515162 
               
               
                 Adenylyl cyclase-associated protein 1 
                 399184 
                 5 
                 HS.370581 
               
               
                 Calnexin precursor 
                 543920 
                 5 
                 HS.529890 
               
               
                 L-plastin 
                 1346733 
                 5 
                 HS.381099 
               
               
                 Annexin A4 
                 1703319 
                 5 
                 HS.422986 
               
               
                 Annexin A11 
                 1703322 
                 5 
                 HS.530291 
               
               
                 Keratin, type II cytoskeletal 8 
                 2506774 
                 5 
                 HS.533782 
               
               
                 Myeloblastin precursor 
                 6174926 
                 5 
                 HS.928 
               
               
                 Nebulin 
                 19856971 
                 5 
                 HS.134602, HS.529265 
               
               
                 Trifunctional enzyme alpha subunit, mitochondrial 
                 20141376 
                 5 
                 HS.516032 
               
               
                 precursor 
               
               
                 Hemoglobin alpha chain 
                 57013850 
                 5 
               
               
                 Alpha-2-macroglobulin precursor 
                 112911 
                 4 
                 HS.212838 
               
               
                 ATP synthase alpha chain, mitochondrial precursor 
                 114517 
                 4 
                 HS.298280 
               
               
                 Leukocyte elastase precursor 
                 119292 
                 4 
                 HS.99863 
               
               
                 Protein disulfide-isomerase A4 precursor 
                 119530 
                 4 
                 HS.93659 
               
               
                 Haptoglobin precursor 
                 123508 
                 4 
                 HS.513711 
               
               
                 Lamin A/C 
                 125962 
                 4 
                 HS.491359 
               
               
                 Ig mu chain C region 
                 127514 
                 4 
                 HS.525648 
               
               
                 Myeloperoxidase precursor 
                 129825 
                 4 
                 HS.458272 
               
               
                 Fibrinogen beta chain precursor 
                 399492 
                 4 
                 HS.300774 
               
               
                 40S ribosomal protein S3 
                 417719 
                 4 
                 HS.546286 
               
               
                 Alpha-1-antitrypsin precursor 
                 1703025 
                 4 
                 HS.525557 
               
               
                 Clathrin heavy chain 1 
                 1705916 
                 4 
                 HS.491351 
               
               
                 Peroxisomal multifunctional enzyme type 2 
                 1706396 
                 4 
                 HS.406861 
               
               
                 Probable ATP-dependent RNA helicase p47 
                 2500529 
                 4 
                 HS.254042 
               
               
                 Fibronectin precursor 
                 2506872 
                 4 
                 HS.203717 
               
               
                 Thioredoxin-dependent peroxide reductase, 
                 2507171 
                 4 
                 HS.523302 
               
               
                 mitochondrial precursor 
               
               
                 WD-repeat protein 1 
                 12643636 
                 4 
                 HS.128548 
               
               
                 Epiplakin 
                 14194713 
                 4 
                 HS.200412 
               
               
                 Histone H1.5 
                 19856407 
                 4 
                 HS.131956 
               
               
                 Isocitrate dehydrogenase [NADP], mitochondrial 
                 20141568 
                 4 
                 HS.513141 
               
               
                 precursor 
               
               
                 Fibrinogen gamma chain precursor 
                 20178280 
                 4 
                 HS.546255 
               
               
                 Filamin B 
                 38257363 
                 4 
                 HS.476448 
               
               
                 DNA-dependent protein kinase catalytic subunit 
                 38258929 
                 4 
                 HS.491682 
               
               
                 Hook homolog 1 
                 41688595 
                 4 
                 HS.378836 
               
               
                 Alpha-actinin 1 
                 46397817 
                 4 
                 HS.509765 
               
               
                 40S ribosomal protein S8 
                 50403622 
                 4 
                 HS.512675 
               
               
                 Histone H3.3 
                 55977062 
                 4 
               
               
                 Tubulin alpha-1 chain 
                 55977476 
                 4 
                 HS.75318 
               
               
                 Hemoglobin beta chain 
                 56749856 
                 4 
                 HS.523443 
               
               
                 Alpha-1-acid glycoprotein 1 precursor 
                 112877 
                 3 
                 HS.494894 
               
               
                 Filamin A 
                 113001 
                 3 
                 HS.195464 
               
               
                 Fructose-bisphosphate aldolase A 
                 113606 
                 3 
                 HS.513490 
               
               
                 Nucleophosmin 
                 114762 
                 3 
                 HS.519452, HS.535499 
               
               
                   
                   
                   
                 HS.196534 
               
               
                 Cathepsin G precursor 
                 115725 
                 3 
                 HS.421724 
               
               
                 Complement C3 precursor 
                 116594 
                 3 
                 HS.529053 
               
               
                 Elongation factor 2 
                 119172 
                 3 
                 HS.515070 
               
               
                 Ig gamma-4 chain C region, Ig gamma-2 chain C region 
                 121043 
                 3 
                 null 
               
               
                 Ig gamma-4 chain C region, Ig gamma-2 chain C region 
                 121047 
                 3 
                 HS.534324 
               
               
                 Hemoglobin delta chain 
                 122713 
                 3 
                 HS.36977 
               
               
                 ATP-dependent DNA helicase II, 70 kDa subunit 
                 125729 
                 3 
                 HS.292493 
               
               
                 Ig kappa chain V-III region HAH precursor 
                 125817 
                 3 
               
               
                 Lamin B1 
                 125953 
                 3 
                 HS.89497 
               
               
                 40S ribosomal protein SA 
                 125969 
                 3 
                 HS.374553 
               
               
                 Myosin regulatory light chain 2, nonsarcomeric 
                 127169 
                 3 
                 HS.190086 
               
               
                 T-complex protein 1, alpha subunit 
                 135538 
                 3 
                 HS.487054 
               
               
                 Serotransferrin precursor 
                 136191 
                 3 
                 HS.518267 
               
               
                 Thymidine phosphorylase precursor 
                 136588 
                 3 
                 HS.531314, HS.546 
               
               
                 Vitronectin precursor 
                 139653 
                 3 
                 HS.2257 
               
               
                 Zinc-alpha-2-glycoprotein precursor 
                 141596 
                 3 
                 HS.546239 
               
               
                 Prohibitin 
                 464371 
                 3 
                 HS.514303 
               
               
                 Macrophage capping protein 
                 729022 
                 3 
                 HS.516155 
               
               
                 Glutamine synthetase 
                 1169929 
                 3 
                 HS.518525 
               
               
                 Ras GTPase-activating-like protein IQGAP1 
                 1170586 
                 3 
                 HS.430551 
               
               
                 Nicotinamide phosphoribosyltransferase 
                 1172027 
                 3 
                 HS.489615 
               
               
                 ATP synthase oligomycin sensitivity conferral protein, 
                 1352049 
                 3 
                 HS.409140 
               
               
                 mitochondrial precursor 
               
               
                 Fructose-1,6-bisphosphatase 
                 1352403 
                 3 
                 HS.494496 
               
               
                 Fibrinogen alpha/alpha-E chain precursor 
                 1706799 
                 3 
                 HS.351593 
               
               
                 Splicing factor, proline-and glutamine-rich 
                 1709851 
                 3 
                 HS.355934 
               
               
                 Calgizzarin 
                 1710818 
                 3 
                 HS.417004 
               
               
                 Protein disulfide-isomerase A6 precursor 
                 2501205 
                 3 
                 HS.212102, HS.372429 
               
               
                 Probable RNA-dependent helicase p72 
                 3122595 
                 3 
                 HS.528305 
               
               
                 Keratin, type I cuticular HA1 
                 6016413 
                 3 
                 HS.41696 
               
               
                 Histone H2B.e 
                 7387742 
                 3 
                 HS.182432 
               
               
                 Chloride intracellular channel protein 1 
                 12643390 
                 3 
                 HS.414565 
               
               
                 Serine protease inhibitor Kazal-type 5 precursor 
                 13959398 
                 3 
                 HS.331555 
               
               
                 Plectin 1 
                 14195007 
                 3 
                 HS.434248 
               
               
                 Talin 1 
                 14916725 
                 3 
                 HS.375001 
               
               
                 78 kDa glucose-regulated protein precursor 
                 14916999 
                 3 
                 HS.522394 
               
               
                 Crumbs protein homolog 1 precursor 
                 17374421 
                 3 
                 HS.126135 
               
               
                 Ryanodine receptor 3 
                 18202506 
                 3 
                 HS.445841 
               
               
                 Probable ATP-dependent helicase DDX48 
                 20532400 
                 3 
                 HS.389649 
               
               
                 Isocitrate dehydrogenase [NADP] cytoplasmic 
                 21903432 
                 3 
                 HS.11223 
               
               
                 Vinculin 
                 21903479 
                 3 
                 HS.500101 
               
               
                 60S ribosomal protein L4 
                 22002063 
                 3 
                 HS.186350, HS.4328 
               
               
                 Fibulin-1 precursor 
                 30581038 
                 3 
                 HS.24601 
               
               
                 Ras-related protein Rab-11B 
                 38258938 
                 3 
                 HS.433888 
               
               
                 Dolichyl-diphosphooligosaccharide-Protein 
                 46397832 
                 3 
                 HS.523145 
               
               
                 glycosyltransferase 48 kDa subunit precursor 
               
               
                 Lysozyme C precursor 
                 48428995 
                 3 
                 HS.524579 
               
               
                 Keratin, type II cuticular HB5 
                 48474780 
                 3 
                 HS.182507 
               
               
                 Nuclear mitotic apparatus protein 1 
                 50400858 
                 3 
                 HS.523873 
               
               
                 40S ribosomal protein S4, X isoform 
                 50403628 
                 3 
                 HS.446628 
               
               
                 T-complex protein 1, delta subunit 
                 52001478 
                 3 
                 HS.421509 
               
               
                 Tubulin alpha-ubiquitous chain 
                 55977474 
                 3 
                 HS.524390 
               
               
                 Citrate synthase, mitochondrial precursor 
                 57015285 
                 3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Lower Probability Proteins Upregulated in High-Grade Cervical Specimens 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Number Up- 
                   
               
               
                 Gene/Protein Name 
                 GI Accession 
                 Regulated Pairs 
                 UniGene ID 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Alpha-1-antichymotrypsin precursor 
                 112874 
                 2 
                 HS.534293 
               
               
                 Aspartate aminotransferase, mitochondrial precursor 
                 112983 
                 2 
                 HS.460929 
               
               
                 Ig alpha-1 chain C region 
                 113584 
                 2 
                 null 
               
               
                 Alcohol dehydrogenase [NADP+] 
                 113600 
                 2 
                 HS.474584 
               
               
                 Antithrombin-III precursor 
                 113936 
                 2 
                 HS.75599 
               
               
                 Annexin A3 
                 113954 
                 2 
                 HS.480042 
               
               
                 Annexin A6 
                 113962 
                 2 
                 HS.412117 
               
               
                 Annexin A8 
                 113967 
                 2 
                 HS.463110, HS.524 
               
               
                 Apolipoprotein B-100 precursor 
                 114014 
                 2 
                 HS.120759 
               
               
                 ATP synthase beta chain, mitochondrial precursor 
                 114549 
                 2 
                 HS.406510 
               
               
                 Carbonic anhydrase II 
                 115456 
                 2 
                 HS.155097 
               
               
                 Calpain small subunit 1 
                 115612 
                 2 
                 HS.515371 
               
               
                 Ceruloplasmin precursor 
                 116117 
                 2 
                 HS.282557 
               
               
                 Clusterin precursor 
                 116533 
                 2 
                 HS.436657 
               
               
                 Alpha enolase 
                 119339 
                 2 
                 HS.517145 
               
               
                 Ferritin light chain 
                 120523 
                 2 
                 HS.433670 
               
               
                 Guanine nucleotide-binding protein G(i), alpha-2 subunit 
                 121023 
                 2 
                 HS.77269 
               
               
                 Ig gamma-1 chain C region 
                 121039 
                 2 
                 HS.375600 
               
               
                 Solute carrier family 2, facilitated glucose transporter, 
                 121751 
                 2 
                 HS.473721 
               
               
                 member 1 
               
               
                 Histone H1.3 
                 121925 
                 2 
                 HS.136857 
               
               
                 Histone H2A.o 
                 121970 
                 2 
                 HS.530461 
               
               
                 Histone H2A.x 
                 121992 
                 2 
                 HS.477879 
               
               
                 Ig heavy chain V-I region HG3 precursor 
                 123799 
                 2 
               
               
                 Ig heavy chain V-III region TEI, Ig heavy chain V-III 
                 123845 
                 2 
               
               
                 region BRO 
               
               
                 Keratin, type I cytoskeletal 15 
                 125081 
                 2 
                 HS.80342 
               
               
                 ATP-dependent DNA helicase II, 80 kDa subunit 
                 125731 
                 2 
                 HS.388739 
               
               
                 Leukotriene A-4 hydrolase 
                 126353 
                 2 
                 HS.524648 
               
               
                 Galectin-3 
                 126678 
                 2 
                 HS.531081 
               
               
                 Tyrosine-protein phosphatase, non-receptor type 6 
                 131469 
                 2 
                 HS.63489 
               
               
                 Prolactin-inducible protein precursor 
                 134170 
                 2 
                 HS.99949 
               
               
                 Transferrin receptor protein 1 
                 136378 
                 2 
                 HS.529618 
               
               
                 Transthyretin precursor 
                 136464 
                 2 
                 HS.427202 
               
               
                 Vitamin D-binding protein precursor 
                 139641 
                 2 
                 HS.418497 
               
               
                 Fatty acid-binding protein, epidermal 
                 232081 
                 2 
                 HS.408061 
               
               
                 Tumor necrosis factor, alpha-induced protein 2 
                 416700 
                 2 
                 HS.525607 
               
               
                 60S ribasomal protein L9 
                 417677 
                 2 
                 HS.412370, HS.513083 
               
               
                 Fibrillin 1 precursor 
                 544279 
                 2 
                 HS.146447 
               
               
                 Keratin, type I cytoskeletal 17 
                 547751 
                 2 
                 HS.2785 
               
               
                 Serine/threonine protein phosphatase PP1-gamma 
                 548573 
                 2 
                 HS.79081 
               
               
                 catalytic subunit 
               
               
                 Myeloid cell nuclear differentiation antigen 
                 730038 
                 2 
                 HS.153837 
               
               
                 UTP-glucose-1-phosphate uridylyltransferase 1 
                 731050 
                 2 
                 null 
               
               
                 Neutrophil gelatinase-associated lipocalin precursor 
                 1171700 
                 2 
                 HS.204238 
               
               
                 14-3-3 protein beta/alpha 
                 1345590 
                 2 
                 HS.279920 
               
               
                 60S ribosomal protein L6 
                 1350762 
                 2 
                 HS.546283, HS.5286 
               
               
                 Phosphatidylethanolamine-binding protein 
                 1352726 
                 2 
                 HS.433863 
               
               
                 F-actin capping protein alpha-1 subunit 
                 1705650 
                 2 
                 HS.514934 
               
               
                 Coatomer alpha subunit 
                 1705996 
                 2 
                 HS.162121 
               
               
                 Hemopexin precursor 
                 1708182 
                 2 
                 HS.426485 
               
               
                 Hexokinase type III 
                 1708363 
                 2 
                 HS.411695 
               
               
                 Malate dehydrogenase, cytoplasmic 
                 1708967 
                 2 
                 HS.526521 
               
               
                 130 kDa leucine-rich protein 
                 1730078 
                 2 
                 HS.368084 
               
               
                 3-hydroxyacyl-CoA dehydrogenase type II 
                 2492759 
                 2 
                 HS.171280 
               
               
                 Laminin alpha-2 chain precursor 
                 2506805 
                 2 
                 HS.200841 
               
               
                 Protein disulfide-isomerase precursor 
                 2507460 
                 2 
                 HS.464336 
               
               
                 Pyridoxal kinase 
                 2811007 
                 2 
                 HS.284491 
               
               
                 Enoyl-CoA hydratase, mitochondrial precursor 
                 2851395 
                 2 
                 HS.76394 
               
               
                 DEAD-box protein 3, X-chromosomal 
                 3023628 
                 2 
                 HS.380774 
               
               
                 Actin-related protein ⅔ complex subunit 2 
                 3121764 
                 2 
                 HS.529303 
               
               
                 2,4-dienoyl-CoA reductase, mitochondrial precursor 
                 3913456 
                 2 
                 HS.492212 
               
               
                 ATP-dependent RNA helicase A 
                 3915658 
                 2 
                 HS.191518 
               
               
                 Carcinoembryonic antigen-related cell adhesion 
                 5921734 
                 2 
                 HS.74466 
               
               
                 molecule 7 precursor 
               
               
                 Lactotransferrin precursor 
                 6175096 
                 2 
                 HS.529517 
               
               
                 Transaldolase 
                 6648092 
                 2 
                 HS.438678 
               
               
                 Eukaryotic translation initiation factor 3 subunit 10 
                 6685537 
                 2 
                 HS.523299 
               
               
                 Keratin, type I cuticular HA6 
                 6685565 
                 2 
                 HS.248189 
               
               
                 Aconitate hydratase, mitochondrial precursor 
                 6686275 
                 2 
                 HS.474982 
               
               
                 Hsc70-interacting protein 
                 6686278 
                 2 
                 HS.546303 
               
               
                 Keratin, type I cuticular HA5 
                 6686303 
                 2 
                 HS.73082 
               
               
                 Poly(rC)-binding protein 2 
                 6707736 
                 2 
                 HS.546271 
               
               
                 Dolichyl-diphosphooligosaccharide-protein 
                 9297108 
                 2 
                 HS.370895 
               
               
                 glycosyltransferase 63 kDa subunit precursor 
               
               
                 6-phosphofructokinase, liver type 
                 9988057 
                 2 
                 HS.255093 
               
               
                 T-complex protein 1, theta subunit 
                 9988062 
                 2 
                 HS.125113 
               
               
                 Coatomer gamma subunit 
                 12229771 
                 2 
                 HS.518250 
               
               
                 Zinc finger protein 208 
                 12585543 
                 2 
                 HS.419763 
               
               
                 Proteasome subunit alpha type 7 
                 12643540 
                 2 
                 HS.233952 
               
               
                 Myeloid/lymphoid or mixed-lineage leukemia protein 4 
                 12643900 
                 2 
                 HS.92236 
               
               
                 54 kDa nuclear RNA- and DNA-binding protein 
                 13124797 
                 2 
                 HS.533282 
               
               
                 Catenin delta-1 
                 14916543 
                 2 
                 HS.166011 
               
               
                 Growth hormone inducible transmembrane protein 
                 15213977 
                 2 
                 HS.352656 
               
               
                 Glucose-6-phosphate isomerase 
                 17380385 
                 2 
                 HS.466471 
               
               
                 Proteasome activator complex subunit 2 
                 18203506 
                 2 
                 HS.512410, HS.434081 
               
               
                 EF-hand domain-containing protein 2 
                 20140139 
                 2 
                 HS.465374 
               
               
                 Complement C4 precursor 
                 20141171 
                 2 
                 HS.534847, HS.546241 
               
               
                   
                   
                   
                 HS.534847, HS.546241 
               
               
                 Argininosuccinate synthase 
                 20141195 
                 2 
                 HS.160786 
               
               
                 Collagen-binding protein 2 precursor 
                 20141241 
                 2 
                 HS.241579 
               
               
                 Dermcidin precursor 
                 20141302 
                 2 
                 HS.350570 
               
               
                 Tubulin alpha-6 chain 
                 20455322 
                 2 
                 HS.436035 
               
               
                 ATP synthase B chain, mitochondrial precursor 
                 20455474 
                 2 
                 HS.514870 
               
               
                 Carcinoembryonic antigen-related cell adhesion 
                 20455477 
                 2 
                 HS.466814 
               
               
                 molecule 6 precursor 
               
               
                 26S proteasome non-ATPase regulatory subunit 3 
                 20532405 
                 2 
                 HS.12970 
               
               
                 Importin beta-1 subunit 
                 20981701 
                 2 
                 HS.532793 
               
               
                 D-3-phosphoglycerate dehydrogenase 
                 21264510 
                 2 
                 HS.487296 
               
               
                 Major vault protein 
                 21542417 
                 2 
                 HS.513488 
               
               
                 Niban-like protein 
                 22256935 
                 2 
                 HS.522401 
               
               
                 Cytosolic nonspecific dipeptidase 
                 23396498 
                 2 
                 HS.149185 
               
               
                 Normal mucosa of esophagus specific gene 1 protein 
                 23396774 
                 2 
                 HS.112242 
               
               
                 Myosin If (Myosin-IE) 
                 23831195 
                 2 
                 HS.408451 
               
               
                 N-acetylglucosamine kinase 
                 24638065 
                 2 
                 HS.7036 
               
               
                 Aldehyde dehydrogenase family 7 member A1 
                 25108887 
                 2 
                 HS.483239 
               
               
                 Myosin XVIIIB 
                 32699565 
                 2 
                 HS.417959 
               
               
                 Myosin VI 
                 33860183 
                 2 
                 HS.149387 
               
               
                 Ras-related protein Rab-5C 
                 38258923 
                 2 
                 HS.514182 
               
               
                 ARP ⅔ complex 20 kDa subunit 
                 38372625 
                 2 
                 HS.323342 
               
               
                 Neuroblast differentiation associated protein AHNAK 
                 39932547 
                 2 
                 HS.502756 
               
               
                 Cytochrome c 
                 42560196 
                 2 
                 HS.437060 
               
               
                 Poly(rC)-binding protein 1 
                 42560548 
                 2 
                 HS.2853 
               
               
                 Eukaryotic initiation factor 4A-I 
                 46397463 
                 2 
                 HS.129673 
               
               
                 Keratin, type II cuticular HB1 
                 46397468 
                 2 
                 HS.185568 
               
               
                 40S ribosomal protein S20 
                 46397703 
                 2 
                 HS.8102 
               
               
                 Actin-like protein 3 
                 47117647 
                 2 
                 HS.433512 
               
               
                 10 kDa heat shock protein, mitochondrial 
                 47606335 
                 2 
                 HS.1197 
               
               
                 Myosin light polypeptide 6 
                 47606436 
                 2 
                 HS.505705 
               
               
                 14-3-3 protein gamma 
                 48428721 
                 2 
                 HS.520974 
               
               
                 Keratin, type II cuticular HB6 
                 48474260 
                 2 
                 HS.278658 
               
               
                 Keratin, type II cuticular HB2 
                 48474984 
                 2 
                 HS.134640 
               
               
                 40S ribosomal protein S16 
                 50403607 
                 2 
                 HS.397609 
               
               
                 40S ribosomal protein S13 
                 50403608 
                 2 
                 HS.446588 
               
               
                 40S ribosomal protein S14 
                 50403752 
                 2 
                 H3.381126 
               
               
                 Ciliary dynein heavy chain 5 
                 51316044 
                 2 
                 HS.520106, HS.212 
               
               
                 Histone H4 
                 51317339 
                 2 
               
               
                 Ras-related protein Rab-1A 
                 51338603 
                 2 
                 HS.310645 
               
               
                 Small nuclear ribonucleoprotein Sm D1 
                 51338665 
                 2 
                 HS.464734 
               
               
                 14-3-3 protein epsilon 
                 51702210 
                 2 
                 HS.513851 
               
               
                 60S ribosomal protein L30 
                 51702805 
                 2 
                 HS.400295 
               
               
                 Phosphoglycerate kinase 1 
                 52788229 
                 2 
                 HS.78771 
               
               
                 Guanine nucleotide-binding protein beta subunit 2-like 1 
                 54037168 
                 2 
                 HS.5662 
               
               
                 Tropomyosin alpha 4 chain 
                 54039751 
                 2 
                 HS.466088 
               
               
                 Tubulin beta-2 chain 
                 56757569 
                 2 
               
               
                 Spectrin alpha chain, brain 
                 56757656 
                 2 
               
               
                 Protein-glutamine gamma-glutamyltransferase K 
                 57015359 
                 2 
               
               
                 Staphylococcal nuclease domain containing protein 1 
                 60415926 
                 2 
               
               
                 Interleukin enhancer-binding factor 2 
                 62510764 
                 2 
               
               
                 Probable ubiquitin ligase protein MYCBP2 
                 68052838 
                 2 
               
               
                 Mannose-6-phosphate receptor binding protein 1 
                 68846601 
                 2 
               
               
                 Erythrocyte membrane protein band 4.2 
                 112798 
                 1 
                 HS.368642 
               
               
                 4F2 cell-surface antigen heavy chain 
                 112803 
                 1 
                 HS.502769 
               
               
                 5′-nucleotidase precursor 
                 112825 
                 1 
                 HS.153952 
               
               
                 Alpha-2-antiplasmin precursor 
                 112907 
                 1 
                 HS.159509 
               
               
                 ADP/ATP translocase 3 
                 113463 
                 1 
                 HS.246506, HS.350 
               
               
                   
                   
                   
                 HS.246506 
               
               
                 Serum albumin precursor 
                 113576 
                 1 
                 HS.418167 
               
               
                 Fructose-bisphosphate aldolase C 
                 113613 
                 1 
                 HS.155247 
               
               
                 Angiotensinogen precursor 
                 113880 
                 1 
                 HS.19383 
               
               
                 Annexin A1 
                 113944 
                 1 
                 HS.494173 
               
               
                 Amine oxidase 
                 113978 
                 1 
                 HS.183109 
               
               
                 Apolipoprotein A-II precursor 
                 114000 
                 1 
                 HS.237658 
               
               
                 Apolipoprotein A-IV precursor 
                 114006 
                 1 
                 HS.1247 
               
               
                 Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 
                 114312 
                 1 
                 HS.506759 
               
               
                 Band 3 anion transport protein 
                 114787 
                 1 
                 HS443948 
               
               
                 Carbonic anhydrase I 
                 115449 
                 1 
                 HS.23118 
               
               
                 Calpain 1, large [catalytic] subunit 
                 115574 
                 1 
                 HS.502842, HS.492718 
               
               
                 Cathepsin D precursor 
                 115717 
                 1 
                 HS.546248, HS.121575 
               
               
                 Carcinoembryonic antigen-related cell adhesion 
                 115940 
                 1 
                 HS.220529 
               
               
                 molecule 5 Precursor 
               
               
                 Complement factor H precursor 
                 116131 
                 1 
                 HS.2637, HS.363396 
               
               
                 Calcyclin 
                 116509 
                 1 
                 HS.275243 
               
               
                 Complement C5 precursor 
                 116607 
                 1 
                 HS.494997 
               
               
                 Neutrophil collagenase precursor 
                 116862 
                 1 
                 HS.161839 
               
               
                 Matrix metalloproteinase-9 precursor 
                 116863 
                 1 
                 HS.297413 
               
               
                 Cytochrome c oxidase subunit IV isoform 1, 
                 117086 
                 1 
                 HS.433419 
               
               
                 Mitochondrial precursor 
               
               
                 Cytochrome P450 1A1 
                 117139 
                 1 
                 HS.72912 
               
               
                 Cytochrome P450 3A7 
                 117159 
                 1 
                 HS.111944 
               
               
                 Cystatin A 
                 118177 
                 1 
                 HS.518198 
               
               
                 Aldehyde dehydrogenase, mitochondrial precursor 
                 118504 
                 1 
                 HS.436437 
               
               
                 Glutamate dehydrogenase 1, mitochondrial precursor 
                 118541 
                 1 
                 HS.500409 
               
               
                 NAD(P)H dehydrogenase [quinone] 1 
                 118607 
                 1 
                 HS.406515 
               
               
                 Elongation factor 1-gamma 
                 119165 
                 1 
                 HS.144835, HS.444467 
               
               
                 Eosinophil granule major basic protein precursor 
                 119239 
                 1 
                 HS.512633 
               
               
                 Electron transfer flavoprotein alpha-subunit, 
                 119636 
                 1 
                 HS.39925 
               
               
                 Mitochondrial precursor 
               
               
                 Ferritin heavy chain 
                 120516 
                 1 
                 HS.446345, HS.533597 
               
               
                   
                   
                   
                 HS.448738, HS.500761 
               
               
                   
                   
                   
                 HS.523854 
               
               
                 Glucosidase II beta subunit precursor 
                 120629 
                 1 
                 HS.512640 
               
               
                 Gelsolin precursor 
                 121116 
                 1 
                 HS.522373 
               
               
                 Histone H2A.a 
                 121968 
                 1 
                 HS.121017, HS.2481 
               
               
                 HLA class I histocompatibility antigen, A-2 alpha chain 
                 122138 
                 1 
               
               
                 precursor 
               
               
                 Heparin cofactor II precursor 
                 123055 
                 1 
                 HS.474270 
               
               
                 Ig heavy chain V-II region NEVVM, Ig heavy chain V-II 
                 123828 
                 1 
               
               
                 Region ARH-77 precursor 
               
               
                 Ig heavy chain V-III region VH26 precursor 
                 123843 
                 1 
                 null 
               
               
                 Ig heavy chain V-III region TIL 
                 123844 
                 1 
               
               
                 Ig heavy chain V-III region HIL 
                 123850 
                 1 
               
               
                 Targeting protein for Xklp2 
                 124096 
                 1 
                 HS.384598 
               
               
                 Insulin-like growth factor I receptor precursor 
                 124240 
                 1 
                 HS.20573 
               
               
                 Integrin beta-2 precursor 
                 124966 
                 1 
                 HS.375957 
               
               
                 Inter-alpha-trypsin inhibitor heavy chain H2 precursor 
                 125000 
                 1 
                 HS.75285 
               
               
                 Ig kappa chain C region 
                 125145 
                 1 
                 HS.449621 
               
               
                 Creatine kinase, ubiquitous mitochondrial precursor 
                 125315 
                 1 
                 HS.425633 
               
               
                 Ig kappa chain V-III region SIE 
                 125819 
                 1 
               
               
                 Ig kappa chain V-IV region JI precursor, Ig kappa 
                 125833 
                 1 
               
               
                 chain V-IV region precursor 
               
               
                 L-lactate dehydrogenase B chain 
                 126041 
                 1 
                 HS.446149 
               
               
                 Laminin alpha-1 chain precursor 
                 126363 
                 1 
                 HS.270364 
               
               
                 Neutrophil cytosol factor 1 
                 127946 
                 1 
                 HS.458275, HS.1583 
               
               
                   
                   
                   
                 HS.448231 
               
               
                 Probable RNA-dependent helicase p68 
                 129383 
                 1 
                 HS.279806 
               
               
                 Junction plakoglobin 
                 130257 
                 1 
                 HS.514174 
               
               
                 Plasminogen precursor 
                 130316 
                 1 
                 HS.143436 
               
               
                 Phosphoglycerate mutase 1 
                 130348 
                 1 
                 HS.447492, HS.502841 
               
               
                   
                   
                   
                 HS.511830 
               
               
                 Poly [ADP-ribose] polymerase-1 
                 130781 
                 1 
                 HS.177766 
               
               
                 Proteasome subunit alpha type 2 
                 130850 
                 1 
                 HS.333786 
               
               
                 Placental ribonuclease inhibitor 
                 132573 
                 1 
                 HS.530687 
               
               
                 60S acidic ribosomal protein P1 
                 133051 
                 1 
                 HS.356502 
               
               
                 Proactivator polypeptide precursor 
                 134218 
                 1 
                 HS.523004 
               
               
                 Tryptophanyl-tRNA synthetase 
                 135191 
                 1 
                 HS.497599 
               
               
                 Thrombospondin-1 precursor 
                 135717 
                 1 
                 HS.164226 
               
               
                 Acetyl-CoA acetyltransferase, mitochondrial precursor 
                 135755 
                 1 
                 HS.232375 
               
               
                 Thioredoxin 
                 135773 
                 1 
                 HS.435136 
               
               
                 Tumor necrosis factor receptor superfamily member 1A 
                 135959 
                 1 
                 HS.279594 
               
               
                 precursor 
               
               
                 Tropomyosin alpha 3 chain 
                 136085 
                 1 
                 HS.406293, HS.146070 
               
               
                   
                   
                   
                 HS.546881, HS.449194 
               
               
                   
                   
                   
                 HS.518123 
               
               
                 60S ribosomal protein L12 
                 266921 
                 1 
                 HS.408054 
               
               
                 14-3-3 protein sigma 
                 398953 
                 1 
                 HS.523718 
               
               
                 Sodium channel protein type VII alpha subunit 
                 399254 
                 1 
                 HS.182889 
               
               
                 FK506-binding protein 4 
                 399866 
                 1 
                 HS.524183 
               
               
                 S100 calcium-binding protein A7 
                 400892 
                 1 
                 HS.112408 
               
               
                 Von Willebrand factor precursor 
                 401413 
                 1 
                 HS.440848 
               
               
                 C4b-binding protein alpha chain precursor 
                 416733 
                 1 
                 HS.1012 
               
               
                 Azurocidin precursor 
                 416746 
                 1 
                 HS.72885 
               
               
                 Long-chain-fatty-acid-CoA ligase 1 
                 417241 
                 1 
                 HS.406678 
               
               
                 Beta-catenin 
                 461854 
                 1 
                 HS.476018 
               
               
                 Metallothionein-IK 
                 462636 
                 1 
                 HS.433391, HS.188518 
               
               
                 ATP synthase gamma chain, mitochondrial precursor 
                 543875 
                 1 
                 HS.271135 
               
               
                 60S ribosomal protein L18 
                 548749 
                 1 
                 HS.515517 
               
               
                 Antigen peptide transporter 1 
                 549042 
                 1 
                 HS.352018 
               
               
                 Complement factor B precursor 
                 584908 
                 1 
                 HS.69771 
               
               
                 Trichohyalin 
                 586120 
                 1 
                 null 
               
               
                 Alu subfamily SX sequence contamination warning entry 
                 728838 
                 1 
               
               
                 Peroxisomal farnesylated protein 
                 729723 
                 1 
                 HS.517232 
               
               
                 Proteasome subunit beta type 10 precursor 
                 730376 
                 1 
                 HS.9661 
               
               
                 40S ribosomal protein S19 
                 730640 
                 1 
                 HS.438429 
               
               
                 T-complex protein 1, zeta subunit 
                 730922 
                 1 
                 HS.82916 
               
               
                 Ubiquitin carboxyl-terminal hydrolase 8 
                 731046 
                 1 
                 HS.443731 
               
               
                 Succinate dehydrogenase [ubiquinone] flavoprotein 
                 1169337 
                 1 
                 HS.440475 
               
               
                 subunit, mitochondrial precursor 
               
               
                 FKBP12-rapamycin complex-associated protein 
                 1169735 
                 1 
                 HS.338207 
               
               
                 Grancalcin 
                 1170014 
                 1 
                 HS.377894 
               
               
                 Glutathione S-transferase Mu 4 
                 1170096 
                 1 
                 HS.348387 
               
               
                 Proteasome activator complex subunit 1 
                 1170519 
                 1 
                 HS.75348 
               
               
                 Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic 
                 1171953 
                 1 
                 HS.518316 
               
               
                 subunit, alpha isoform 
               
               
                 40S ribosomal protein S10 
                 1173177 
                 1 
                 HS.406620, HS.5391 
               
               
                 CENP-F kinetochore protein 
                 1345731 
                 1 
                 HS.497741 
               
               
                 Fibrillin 2 precursor 
                 1345961 
                 1 
                 HS.519294 
               
               
                 Keratin, type II cytoskeletal 1 
                 1346343 
                 1 
                 HS.80828 
               
               
                 Keratin, type II cytoskeletal 6A 
                 1346344 
                 1 
                 HS.367762 
               
               
                 Keratin, type II cytoskeletal 6B 
                 1346345 
                 1 
                 HS.524438 
               
               
                 Keratin, type II cytoskeletal 6C 
                 1346346 
                 1 
                 HS.367762 
               
               
                 Diacylglycerol kinase, gamma 
                 1346372 
                 1 
                 HS.171499 
               
               
                 Galectin-7 
                 1346431 
                 1 
                 HS.99923 
               
               
                 Retinoic acid receptor RXR-gamma 
                 1350913 
                 1 
                 HS.26550 
               
               
                 Tissue factor pathway inhibitor 2 precursor 
                 1351226 
                 1 
                 HS.438231 
               
               
                 Complement component C9 precursor 
                 1352108 
                 1 
                 HS.1290 
               
               
                 Eosinophil peroxidase precursor 
                 1352738 
                 1 
                 HS.279259 
               
               
                 Acyl-CoA dehydrogenase, very-long-chain specific, 
                 1703068 
                 1 
                 HS.437178 
               
               
                 Mitochondrial precursor 
               
               
                 Adapter-related protein complex 1 beta 1 subunit 
                 1703167 
                 1 
                 HS.368794 
               
               
                 Flavin reductase 
                 1706870 
                 1 
                 HS.515785 
               
               
                 Rho GDP-dissociation inhibitor 1 
                 1707892 
                 1 
                 HS.159161 
               
               
                 Rho GDP-dissociation inhibitor 2 
                 1707893 
                 1 
                 HS.504877 
               
               
                 Krueppel-related zinc finger protein 3 
                 1708212 
                 1 
                 HS.502330 
               
               
                 Hydroxymethylglutaryl-CoA synthase, mitochondrial 
                 1708234 
                 1 
                 HS.59889 
               
               
                 precursor 
               
               
                 Heat shock-related 70 kDa protein 2 
                 1708307 
                 1 
                 HS.432648 
               
               
                 Serpin B8 
                 1709895 
                 1 
                 HS.368077 
               
               
                 Regulator of G-protein signaling 3 
                 1710136 
                 1 
                 HS.494875 
               
               
                 Squamous cell carcinoma antigen 2 
                 1710877 
                 1 
                 HS.227948, HS.123035 
               
               
                 Translocon-associated protein, delta subunit precursor 
                 1711550 
                 1 
                 HS.409223 
               
               
                 Vascular endothelial growth factor receptor 3 precursor 
                 1718189 
                 1 
                 HS.415048 
               
               
                 Tetratricopeptide repeat protein 3 
                 1730008 
                 1 
                 HS.368214 
               
               
                 Nucleoprotein TPR 
                 1730009 
                 1 
                 HS.279640 
               
               
                 Ketohexokinase 
                 1730044 
                 1 
                 HS.159525 
               
               
                 Glycine amidinotransferase, mitochondrial precursor 
                 1730201 
                 1 
                 HS.75335 
               
               
                 Polymeric-immunoglobulin receptor precursor 
                 1730570 
                 1 
                 HS.497589 
               
               
                 Desmoglein-2 precursor 
                 2493421 
                 1 
                 HS.412597 
               
               
                 Spectrin beta chain, brain 1 
                 2493434 
                 1 
                 HS.503178 
               
               
                 Calcyphosine 
                 2493439 
                 1 
                 HS.26685 
               
               
                 I-plastin 
                 2493466 
                 1 
                 HS.203637 
               
               
                 Delta3,5-delta2,4-dienoyl-CoA isomerase, mitochondrial 
                 2494238 
                 1 
                 HS.196176 
               
               
                 precursor 
               
               
                 Fascin 
                 2498357 
                 1 
                 HS.118400 
               
               
                 Spliceosome RNA helicase BAT1 
                 2500529 
                 1 
               
               
                 Septin-2 
                 2500769 
                 1 
                 HS.335057 
               
               
                 Vesicle-associated membrane protein 3 
                 2501082 
                 1 
                 HS.66708 
               
               
                 Zinc finger protein 239 
                 2501707 
                 1 
                 HS.25040 
               
               
                 Peroxiredoxin 2 
                 2507169 
                 1 
                 HS.432121 
               
               
                 CD44 antigen precursor 
                 2507241 
                 1 
                 HS.502328 
               
               
                 NADPH--cytochrome P450 reductase 
                 2851393 
                 1 
                 HS.354056 
               
               
                 Myosin heavy chain, skeletal muscle, perinatal 
                 3041707 
                 1 
                 HS.534028 
               
               
                 Myosin heavy chain, fast skeletal muscle, embryonic 
                 3043372 
                 1 
                 HS.440895 
               
               
                 Actin-related protein ⅔ complex subunit 1B 
                 3121763 
                 1 
                 HS.489284 
               
               
                 Actin-related protein ⅔ complex subunit 3 
                 3121765 
                 1 
                 HS.524741 
               
               
                 ARP2/3 complex 16 kDa subunit 
                 3121767 
                 1 
                 HS.518609 
               
               
                 Dihydropyrimidinase-related protein 2 
                 3122051 
                 1 
                 HS.173381 
               
               
                 Type I inositol-1,4,5-trisphosphate 5-phosphatase 
                 3122245 
                 1 
                 HS.523360 
               
               
                 Thiosulfate sulfurtransferase 
                 3122965 
                 1 
                 HS.474783 
               
               
                 Collagen alpha 1(XII) chain precursor 
                 3182940 
                 1 
                 HS.101302 
               
               
                 Protein tyrosine kinase 2 beta 
                 3183003 
                 1 
                 HS.491322 
               
               
                 Polyadenylate-binding protein 1 
                 3183544 
                 1 
                 HS.387804 
               
               
                 Dehydrogenase/reductase SDR family member 2 
                 3915733 
                 1 
                 HS.272499 
               
               
                 Transformer-2 protein homolog 
                 4033480 
                 1 
                 HS.445652 
               
               
                 Sorbitol dehydrogenase 
                 4033691 
                 1 
                 HS.878 
               
               
                 Serine/threonine protein phosphatase 2A, 65 kDa 
                 5915686 
                 1 
                 HS.546276 
               
               
                 regulatory subunit A, alpha isoform 
               
               
                 Collagen alpha 3(VI) chain precursor 
                 5921193 
                 1 
                 HS.233240 
               
               
                 Endoplasmic reticulum protein ERp29 precursor 
                 6015110 
                 1 
                 HS.75841 
               
               
                 Glutathione S-transferase A4-4 
                 6016167 
                 1 
                 HS.485557 
               
               
                 Ig lambda chain V-III region LOI 
                 6016518 
                 1 
               
               
                 Phytanoyl-CoA dioxygenase, peroxisomal precursor 
                 6093646 
                 1 
                 HS.498732 
               
               
                 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 
                 6093730 
                 1 
                 HS.477866 
               
               
                 precursor 
               
               
                 Selenium-binding protein 1 
                 6094240 
                 1 
                 HS.334841 
               
               
                 Transitional endoplasmic reticulum ATPase 
                 6094447 
                 1 
                 HS.529782 
               
               
                 Voltage-dependent P/Q-type calcium channel alpha-1A 
                 6166047 
                 1 
                 HS.408449 
               
               
                 subunit 
               
               
                 Dimethylaniline monooxygenase 
                 6166183 
                 1 
                 HS.445350 
               
               
                 NDRG1 protein 
                 6166568 
                 1 
                 HS.372914 
               
               
                 Gamma-synuclein 
                 6175048 
                 1 
                 HS.349470 
               
               
                 Tripeptidyl-peptidase I precursor 
                 6175068 
                 1 
                 HS.523454 
               
               
                 UDP-glucose 6-dehydrogenase 
                 6175086 
                 1 
                 HS.28309 
               
               
                 Heat shock 70 kDa protein 4 
                 6226869 
                 1 
                 HS.90093 
               
               
                 Membrane associated progesterone receptor 
                 6647589 
                 1 
                 HS.90061 
               
               
                 component 1 
               
               
                 Calcineurin-binding protein Cabin 1 
                 6685261 
                 1 
                 HS.517478 
               
               
                 Claudin-4 
                 6685272 
                 1 
                 HS.520942 
               
               
                 Keratin, type I cuticular HA4 
                 6685564 
                 1 
                 HS.296942 
               
               
                 TNF receptor associated factor 3 
                 6686035 
                 1 
                 HS.510528 
               
               
                 Microsomal glutathione S-transferase 3 
                 7387731 
                 1 
                 HS.191734 
               
               
                 Histone H2B.q 
                 7387736 
                 1 
                 HS.2178 
               
               
                 Histone H2B.q 
                 7404367 
                 1 
               
               
                 NK-tumor recognition protein 
                 8039798 
                 1 
                 HS.529509 
               
               
                 Forkhead box protein O3A 
                 8134467 
                 1 
                 HS.220950 
               
               
                 Mannose-6-phosphate receptor binding protein 1 
                 8134735 
                 1 
                 HS.140452 
               
               
                 Acid ceramidase precursor 
                 8247915 
                 1 
                 HS.527412 
               
               
                 Regulator of G-protein signaling 9 
                 8475983 
                 1 
                 HS.132327 
               
               
                 Tenascin X precursor 
                 9087217 
                 1 
                 HS.42853, HS.485104 
               
               
                 Histone H2B.e 
                 9973351 
                 1 
               
               
                 U6 snRNA-associated Sm-like protein LSm2 
                 10720079 
                 1 
                 HS.103106 
               
               
                 150 kDa oxygen-regulated protein precursor 
                 10720185 
                 1 
                 HS.277704 
               
               
                 Zinc finger protein 221 
                 11136126 
                 1 
                 HS.274445 
               
               
                 Programmed cell death protein 6 
                 12230420 
                 1 
                 HS.50823, HS.379186 
               
               
                 Zinc finger protein 43 
                 12585553 
                 1 
                 HS.534365 
               
               
                 Vacuolar ATP synthase subunit B, brain isoform 
                 12643271 
                 1 
                 HS.295917 
               
               
                 Absent in melanoma 1 protein 
                 12643308 
                 1 
                 HS.486074 
               
               
                 Cathepsin Z precursor 
                 12643324 
                 1 
                 HS.252549 
               
               
                 Glutathione S-transferase kappa 1 
                 12643338 
                 1 
                 HS.390667 
               
               
                 Sorting nexin-3 
                 12643620 
                 1 
                 HS.12102 
               
               
                 Vacuolar proton translocating ATPase 116 kDa subunit 
                 12643719 
                 1 
                 HS.495985 
               
               
                 A isoform 3 
               
               
                 Gamma-aminobutyric acid type B receptor, subunit 1 
                 12643873 
                 1 
                 HS.167017 
               
               
                 precursor 
               
               
                 Coronin-1C 
                 12643898 
                 1 
                 HS.330384 
               
               
                 Voltage-dependent anion-selective channel protein 3 
                 12643945 
                 1 
                 HS.491597 
               
               
                 Desmoplakin 
                 12644130 
                 1 
                 HS.519873 
               
               
                 Probable DNA dC-)dU editing enzyme APOBEC-3A 
                 12644206 
                 1 
                 HS.348983 
               
               
                 Diacylglycerol kinase, zeta 
                 12644407 
                 1 
                 HS.502461 
               
               
                 Myosin light chain kinase, smooth muscle and non- 
                 12644418 
                 1 
                 HS.477375 
               
               
                 Muscle isozymes 
               
               
                 Coatomer gamma subunit 
                 13124090 
                 1 
                 HS.532231 
               
               
                 Targeting protein for Xklp2 
                 13124096 
                 1 
                 HS.244580 
               
               
                 F-box only protein 3 
                 13124239 
                 1 
                 HS.406787 
               
               
                 Long-chain-fatty-acid-CoA ligase 5 
                 13431659 
                 1 
                 HS.11638 
               
               
                 Myosin Ic 
                 13431674 
                 1 
                 HS.286226 
               
               
                 Keratin, type I cytoskeletal 14 
                 13432173 
                 1 
                 HS.355214 
               
               
                 Myosin-11 
                 13432177 
                 1 
                 HS.460109 
               
               
                 Inter-alpha-trypsin inhibitor heavy chain H4 precursor 
                 13432192 
                 1 
                 HS.518000 
               
               
                 Serine-protein kinase ATM 
                 13878337 
                 1 
                 HS.435561 
               
               
                 Lysyl oxidase homolog 2 precursor 
                 13878585 
                 1 
                 HS.116479 
               
               
                 Collagen alpha 1(VI) chain precursor 
                 13878903 
                 1 
                 HS.474053 
               
               
                 Envoplakin 
                 14194715 
                 1 
                 HS.500635 
               
               
                 Vinexin 
                 14423996 
                 1 
                 HS.528572 
               
               
                 Myosin Ixb 
                 14548118 
                 1 
                 HS.123198 
               
               
                 Ras-GTPase-activating protein binding protein 2 
                 14916573 
                 1 
                 HS.303676 
               
               
                 Glutathione reductase, mitochondrial precursor 
                 14916998 
                 1 
                 HS.271510 
               
               
                 Splicing factor 3B subunit 1 
                 15214275 
                 1 
                 HS.471011 
               
               
                 ADAMTS-12 precursor 
                 17366354 
                 1 
                 HS.481865 
               
               
                 Inositol 1,4,5-trisphosphate receptor type 3 
                 17366458 
                 1 
                 HS.65758 
               
               
                 Spectrin beta chain, brain 2 
                 17367904 
                 1 
                 HS.26915 
               
               
                 Reticulon 4 
                 17369290 
                 1 
                 HS.429581 
               
               
                 Ryanodine receptor 2 
                 17380312 
                 1 
                 HS.109514 
               
               
                 Transcriptional regulator ATRX 
                 17380440 
                 1 
                 HS.533526 
               
               
                 Acyl-coenzyme A oxidase 1, peroxisomal 
                 17380467 
                 1 
                 HS.464137 
               
               
                 Heat shock protein 75 kDa, mitochondrial precursor 
                 17865679 
                 1 
                 HS.30345 
               
               
                 Histone H3/b, Histone H3.1 
                 18202621 
                 1 
                 HS.546315, HS.532 
               
               
                   
                   
                   
                 HS.248176, HS.44 
               
               
                   
                   
                   
                 HS.132854, HS.24 
               
               
                   
                   
                   
                 HS.484990, HS.70 
               
               
                   
                   
                   
                 HS.533292, HS.24 
               
               
                 Tripartite motif protein 8 
                 18202744 
                 1 
                 HS.336810 
               
               
                 Echinoderm microtubule-associated protein-like 4 
                 18202954 
                 1 
                 HS.432438 
               
               
                 PINCH protein 
                 18266876 
                 1 
                 HS.469593 
               
               
                 Heat-shock protein beta-1 
                 19855073 
                 1 
                 HS.520973 
               
               
                 Ryanodine receptor 1 
                 19857096 
                 1 
                 HS.466664 
               
               
                 DNA replication licensing factor MCM5 
                 19858646 
                 1 
                 HS.517582 
               
               
                 Zinc finger protein 268 
                 19863363 
                 1 
                 HS.183291, HS.186387 
               
               
                 Cytochrome P450 3A43, Cytochrome P450 3A3 
                 20137481 
                 1 
                 HS.306220 
               
               
                 Interleukin-4 induced protein 1 precursor 
                 20138284 
                 1 
                 HS.467133 
               
               
                 Methylcrotonoyl-CoA carboxylase beta chain, 
                 20138731 
                 1 
                 HS.167531 
               
               
                 Mitochondrial precursor 
               
               
                 Neurogenic locus notch homolog protein 2 precursor 
                 20138948 
                 1 
                 HS.487360, HS.502564 
               
               
                 Myoferlin 
                 20139241 
                 1 
                 HS.500572 
               
               
                 N-acetylated-alpha-linked acidic dipeptidase II 
                 20139300 
                 1 
                 HS.503560 
               
               
                 Sciellin 
                 20139986 
                 1 
                 HS.115166 
               
               
                 WD-repeat protein 10 
                 20140806 
                 1 
                 HS.477537 
               
               
                 Apoptotic protease activating factor 1 
                 20141188 
                 1 
                 HS.546236 
               
               
                 Monocyte differentiation antigen CD14 precursor 
                 20141203 
                 1 
                 HS.163867 
               
               
                 Elongation factor 1-delta 
                 20141357 
                 1 
                 HS.333388 
               
               
                 Short chain 3-hydroxyacyl-CoA dehydrogenase, 
                 20141424 
                 1 
                 HS.438289 
               
               
                 Mitochondrial precursor 
               
               
                 Lumican precursor 
                 20141464 
                 1 
                 HS.406475 
               
               
                 Protein-glutamine gamma-glutamyltransferase 
                 20141877 
                 1 
                 HS.517033 
               
               
                 Zinc finger protein 41 
                 20141930 
                 1 
                 HS.496074 
               
               
                 Complement C1q subcomponent, C chain precursor 
                 20178281 
                 1 
                 HS.467753 
               
               
                 S100 calcium-binding protein A2 
                 20178319 
                 1 
                 HS.516484 
               
               
                 Lysyl-tRNA synthetase 
                 20178333 
                 1 
                 HS.3100 
               
               
                 Aldo-keto reductase family 1 member C3 
                 20532372 
                 1 
                 HS.78183 
               
               
                 ATP-dependent RNA helicase DDX18 
                 20532388 
                 1 
                 HS.363492 
               
               
                 Laminin alpha-5 chain precursor 
                 20532393 
                 1 
                 HS.473256 
               
               
                 Proteasome subunit beta type 3 
                 20532411 
                 1 
                 HS.82793 
               
               
                 Wnt-3a protein precursor 
                 20532424 
                 1 
                 HS.336930 
               
               
                 6-phosphogluconate dehydrogenase, decarboxylating 
                 20981679 
                 1 
                 HS.464071 
               
               
                 ELAV-like protein 1 
                 20981691 
                 1 
                 HS.184492 
               
               
                 Glutathione S-transferase Mu 3 
                 21264423 
                 1 
                 HS.2006 
               
               
                 Hsp90 co-chaperone Cdc37 
                 21542000 
                 1 
                 HS.160958 
               
               
                 Ankyrin 3 
                 21759000 
                 1 
                 HS.499725 
               
               
                 Coactosin-like protein 
                 21759076 
                 1 
                 HS.289092 
               
               
                 GPI transamidase component PIG-S 
                 21759353 
                 1 
                 HS.462550 
               
               
                 Cadherin EGF LAG seven-pass G-type receptor 3 
                 22095552 
                 1 
                 HS.533070 
               
               
                 precursor 
               
               
                 ATP synthase g chain, mitochondrial 
                 22096328 
                 1 
                 HS.486360 
               
               
                 Vacuolar ATP synthase catalytic subunit A, ubiquitous 
                 22096378 
                 1 
                 HS.477155 
               
               
                 isoform 
               
               
                 SAM domain and HD domain-containing protein 1 
                 22257047 
                 1 
                 HS.472630 
               
               
                 Translocon-associated protein, alpha subunit precursor 
                 22261821 
                 1 
                 HS.114033 
               
               
                 Chromodomain-helicase-DNA-binding protein 6 
                 23396493 
                 1 
                 HS.522898, HS.371979 
               
               
                 Ciliary dynein heavy chain 11 
                 23396581 
                 1 
                 HS.520245, HS.432390 
               
               
                 Kinesin-like protein KIF13B 
                 23396625 
                 1 
                 HS.444767 
               
               
                 Keratin, type I cytoskeletal 16 
                 23503075 
                 1 
                 HS.432448 
               
               
                 NADP-dependent leukotriene B4 12- 
                 23503081 
                 1 
                 HS.546348 
               
               
                 hydroxydehydrogenase 
               
               
                 Protein KIAA1404 
                 23821814 
                 1 
                 HS.371794 
               
               
                 Mucin 5B precursor 
                 23821885 
                 1 
                 HS.534332, HS.534862 
               
               
                   
                   
                   
                 HS.523395, HS.534332 
               
               
                 Biliverdin reductase A precursor 
                 23830892 
                 1 
                 HS.488143 
               
               
                 WD-repeat protein 9 
                 23831562 
                 1 
                 HS.314338 
               
               
                 Loss of heterozygosity 11 chromosomal region 2 gene A 
                 24211888 
                 1 
                 HS.152944 
               
               
                 protein 
               
               
                 Midasin 
                 24212017 
                 1 
                 HS.529948 
               
               
                 Basement membrane-specific heparan sulfate 
                 24212664 
                 1 
                 HS.467545 
               
               
                 proteoglycan core protein precursor 
               
               
                 Exocyst complex component Sec15B 
                 24418685 
                 1 
                 HS.303454 
               
               
                 PR-domain protein 11 
                 25008957 
                 1 
                 HS.147331 
               
               
                 Netrin-1 precursor 
                 25090820 
                 1 
                 HS.128002 
               
               
                 Phosphatidylinositol-binding clathrin assembly protein 
                 25090897 
                 1 
                 HS.163893 
               
               
                 XPA-binding protein 2 
                 25091548 
                 1 
                 HS.9822 
               
               
                 Putative GTP-binding protein PTD004 
                 25453240 
                 1 
                 HS.157351 
               
               
                   
                   
                   
                 HS.447547, HS.454 
               
               
                 Vacuolar protein sorting 35 
                 25453321 
                 1 
                 HS.467824 
               
               
                 Flotillin-1 
                 26006960 
                 1 
                 HS.179986 
               
               
                 Talin-2 
                 26400725 
                 1 
                 HS.511686 
               
               
                 Sulfide:quinone oxidoreductase, mitochondrial precursor 
                 27151704 
                 1 
                 HS.511251 
               
               
                 Ras-related protein Rab-6C 
                 27734458 
                 1 
                 HS.535586, HS.440 
               
               
                 Junctophilin 1 
                 27805492 
                 1 
                 HS.160574 
               
               
                 Bullous pemphigoid antigen 1, isoforms 6/9/10 
                 27923958 
                 1 
                 HS.485616 
               
               
                 Bullous pemphigoid antigen 1 isoforms 1/2/3/4/5/8 
                 27923959 
                 1 
                 HS.485616 
               
               
                 Zonadhesin precursor 
                 27924006 
                 1 
                 HS.307004 
               
               
                 HECT domain containing protein 1 
                 28380056 
                 1 
                 HS.210850 
               
               
                 Wnt inhibitory factor 1 precursor 
                 29337245 
                 1 
                 HS.284122 
               
               
                 DNA-directed RNA polymerases III 80 kDa polypeptide 
                 29428028 
                 1 
                 HS.460298 
               
               
                 Nesprin 1 
                 29839561 
                 1 
                 HS.12967 
               
               
                 Nesprin-2 
                 29839588 
                 1 
                 HS.525392 
               
               
                 Map kinase phosphatase-like protein MK-STYX 
                 29840801 
                 1 
                 HS.11615 
               
               
                 Putative Polycomb group protein ASXL1 
                 30172872 
                 1 
                 HS.374043 
               
               
                 GDNF family receptor alpha 4 precursor 
                 30173123 
                 1 
                 HS.302025 
               
               
                 Glucosamine--fructose-6-phosphate aminotransferase 
                 30923274 
                 1 
                 HS.468864 
               
               
                 [isomerizing] 1 
               
               
                 Programmed cell death 6-interacting protein 
                 31076831 
                 1 
                 HS.475896 
               
               
                 Egl nine homolog 3 
                 32129515 
                 1 
                 HS.135507 
               
               
                 ADAMTS-9 precursor 
                 32130427 
                 1 
                 HS.476604 
               
               
                 Myosin Id 
                 32172416 
                 1 
                 HS.462777 
               
               
                 GRIP and coiled-coil domain-containing protein 2 
                 32469733 
                 1 
                 HS.436505 
               
               
                 Dedicator of cytokinesis protein 3 
                 32469734 
                 1 
                 HS.476284 
               
               
                 Transcription elongation factor B polypeptide 1 
                 32699511 
                 1 
                 HS.546305 
               
               
                 Early endosome antigen 1 
                 34222508 
                 1 
                 HS.506309 
               
               
                 X-linked interleukin-1 receptor accessory protein-like 1 
                 34222654 
                 1 
                 HS.495893 
               
               
                 precursor 
               
               
                 HLA class I histocompatibility antigen, A-3 alpha chain 
                 34223717 
                 1 
                 HS.181244 
               
               
                 precursor 
               
               
                 Zinc finger protein 430 
                 34925658 
                 1 
                 HS.466289 
               
               
                 Heat shock 70 kDa protein 6 
                 34978357 
                 1 
                 HS.3268 
               
               
                 Sphingosine-1-phosphate lyase 1 
                 37999486 
                 1 
                 HS.499984 
               
               
                 Proteasome subunit alpha type 5 
                 38258905 
                 1 
                 HS.485246 
               
               
                 Zinc finger protein 397 
                 38258943 
                 1 
                 HS.464896 
               
               
                 Unc-112 related protein 2 
                 41018464 
                 1 
                 HS.180535 
               
               
                 Piccolo protein 
                 41019528 
                 1 
                 HS.12376, HS.455230 
               
               
                 Cohen syndrome protein 1 
                 42558898 
                 1 
                 HS.191540 
               
               
                 Liprin-alpha 1 
                 42558969 
                 1 
                 HS.530749 
               
               
                 Hornerin 
                 45476906 
                 1 
               
               
                 Zinc finger protein ZFPM2 
                 45476962 
                 1 
                 HS.431009 
               
               
                 Eukaryotic initiation factor 4A-II 
                 45645183 
                 1 
                 HS.478553 
               
               
                 Protein MICAL-2 
                 46396148 
                 1 
                 HS.501928 
               
               
                 Low-density lipoprotein receptor-related protein 10 
                 46396347 
                 1 
                 HS.525232 
               
               
                 precursor 
               
               
                 Periphilin 1 
                 46396942 
                 1 
                 HS.444157 
               
               
                 Vesicle trafficking protein SEC22b 
                 46397702 
                 1 
                 HS.534212 
               
               
                 Ras-related protein Rab-10 
                 46577638 
                 1 
               
               
                 Ubiquitin-conjugating enzyme E2 D3 
                 46577654 
                 1 
                 HS.518773, HS.472031 
               
               
                 Ras-related protein Rab-25 
                 46577696 
                 1 
                 HS.491308 
               
               
                 Galectin-3 binding protein precursor 
                 47115668 
                 1 
                 HS.514535 
               
               
                 Tubulin tyrosine ligase-like protein 2 
                 47117620 
                 1 
               
               
                 Actin-like protein 2 
                 47117648 
                 1 
                 HS.393201 
               
               
                 40S ribosomal protein S3a 
                 47117764 
                 1 
                 HS.356572 
               
               
                 Nuclear receptor corepressor 1 
                 47117817 
                 1 
                 HS.462323, HS.307 
               
               
                 Dynamin-2 
                 47117856 
                 1 
                 HS.211463 
               
               
                 Histone H2A.q 
                 47117890 
                 1 
               
               
                 Transforming protein RhoA 
                 47606458 
                 1 
                 HS.247077 
               
               
                 Scavenger receptor class F member 2 precursor 
                 47606791 
                 1 
                 HS.474251 
               
               
                 Defender against cell death 1 
                 48428858 
                 1 
                 HS.82890 
               
               
                 Signal transducer and activator of transcription 3 
                 48429227 
                 1 
                 HS.463059 
               
               
                 Pantophysin 
                 48474786 
                 1 
                 HS.80919 
               
               
                 Serine/threonine protein phosphatase PP1-beta 
                 49065814 
                 1 
                 HS.468018 
               
               
                 catalytic subunit 
               
               
                 40S ribosomal protein S7 
                 49065831 
                 1 
                 HS.546287, HS.534 
               
               
                   
                   
                   
                 HS.33348 
               
               
                 Inositol hexaphosphate kinase 1 
                 50400597 
                 1 
                 HS.438691 
               
               
                 Ninein 
                 50400772 
                 1 
                 HS.310429 
               
               
                 Potassium channel tetramerisation domain containing 
                 50401124 
                 1 
                 HS.109438 
               
               
                 protein 12 
               
               
                 Signal-induced proliferation-associated 1 like protein 1 
                 50401319 
                 1 
                 HS.208846 
               
               
                 Signal-induced proliferation-associated 1 like protein 2 
                 50401690 
                 1 
                 HS.268774 
               
               
                 Ras-related protein Rab-11A 
                 50402542 
                 1 
                 HS.321541 
               
               
                 40S ribosomal protein S18 
                 50403625 
                 1 
                 HS.546290 
               
               
                 Receptor-type tyrosine-protein phosphatase S precursor 
                 50403770 
                 1 
                 HS.408456 
               
               
                 Bassoon protein 
                 51315800 
                 1 
                 HS.194684 
               
               
                 Drebrin-like protein 
                 51316115 
                 1 
               
               
                 Unc-13 homolog D 
                 51316668 
                 1 
                 HS.41045 
               
               
                 ADP-ribosylation factor 6 
                 51316984 
                 1 
                 HS.525330 
               
               
                 ADP-ribosylation factor 1, ADP-ribosylation factor 
                 51316985 
                 1 
                 HS.286221 
               
               
                 3, ADP-ribosylation factor 5 
               
               
                 Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta 
                 51317302 
                 1 
                 HS.430425 
               
               
                 subunit 1 
               
               
                 Ras-related protein Rap-1A 
                 51338607 
                 1 
                 HS.190334 
               
               
                 60S ribosomal protein L23 
                 51338639 
                 1 
                 HS.406300, HS.512542 
               
               
                 Small nuclear ribonucleoprotein Sm D2 
                 51338666 
                 1 
                 HS.515472 
               
               
                 F-box only protein 44 
                 51338823 
                 1 
                 HS.519716 
               
               
                 Histone H2B K 
                 51701495 
                 1 
                 HS.437275 
               
               
                 Zinc finger protein 237 
                 51702202 
                 1 
                 HS.530988 
               
               
                 Zinc finger protein 330 
                 51702204 
                 1 
                 HS.120766 
               
               
                 60S ribosomal protein L11 
                 51702795 
                 1 
                 HS.388664 
               
               
                 Vesicle-associated membrane protein 2 
                 51704192 
                 1 
                 HS.25348 
               
               
                 Puromycin-sensitive aminopeptidase 
                 51704228 
                 1 
                 HS.443837 
               
               
                 Interferon-induced 17 kDa protein precursor 
                 52001470 
                 1 
                 HS.458485 
               
               
                 Protein FAM49B 
                 52782794 
                 1 
                 HS.492869 
               
               
                 Kelch-like protein 17 
                 52783052 
                 1 
                 HS.109212 
               
               
                 60S ribosomal protein L38 
                 52783779 
                 1 
                 HS.380953 
               
               
                 Actin, cytoplasmic 2 
                 54036678 
                 1 
                 HS.514581 
               
               
                 Actin, alpha cardiac 
                 54036697 
                 1 
                 HS.118127 
               
               
                 Neutral alpha-glucosidase AB precursor 
                 54037162 
                 1 
                 HS.76847 
               
               
                 Eukaryotic translation initiation factor 5A 
                 54037409 
                 1 
                 HS.534314 
               
               
                 Ubiquitin-conjugating enzyme E2 L3 
                 54039805 
                 1 
                 HS.108104 
               
               
                 Annexin A7 
                 55584155 
                 1 
                 HS.386434 
               
               
                 Kin of IRRE-like protein 3 precursor 
                 55736065 
                 1 
                 HS.302350 
               
               
                 Tripartite motif protein 29 
                 55976299 
                 1 
                 HS.504115 
               
               
                 Vesicle-associated membrane protein 8 
                 55976764 
                 1 
                 HS.534373 
               
               
                 Abnormal spindle-like microcephaly-associated protein 
                 55976785 
                 1 
                 HS.121028 
               
               
                 Tubulin beta-2 chain 
                 55977480 
                 1 
                 HS.433615 
               
               
                 Tubulin alpha-3 chain 
                 55977864 
                 1 
                 HS.524395 
               
               
                 Zinc finger protein 219 
                 55977885 
                 1 
                 HS.250493 
               
               
                 Netrin-2 like protein precursor 
                 56404431 
                 1 
                 HS.158336 
               
               
                 Zinc finger protein 644 
                 56404958 
                 1 
                 HS.173001 
               
               
                 Serine/threonine-protein kinase 38-like 
                 56749668 
                 1 
               
               
                 Hemoglobin gamma-2 chain 
                 56749861 
                 1 
               
               
                 Keratin, type II cytoskeletal 5 
                 56757580 
                 1 
               
               
                 Dynein heavy chain, cytosolic 
                 57015308 
                 1 
               
               
                 Low-density lipoprotein receptor-related protein 1B 
                 57015418 
                 1 
               
               
                 precursor 
               
               
                 DNA polymerase eta 
                 59798441 
                 1 
               
               
                 TBC1 domain family member 21 
                 59798963 
                 1 
               
               
                 Keratin, type II cytoskeletal 6E 
                 59803089 
                 1 
               
               
                 Protein C19orf10 precursor 
                 61221730 
                 1 
               
               
                 Autophagy protein 7-like 
                 62286592 
                 1 
               
               
                 Delta-1-pyrroline-5-carboxylate dehydrogenase, 
                 62511241 
                 1 
               
               
                 Mitochondrial precursor 
               
               
                 Interleukin enhancer-binding factor 3 
                 62512150 
                 1 
               
               
                 Enoyl-CoA hydratase, mitochondrial precursor 
                 62906863 
                 1 
               
               
                 ATP synthase g chain, mitochondrial 
                 62906882 
                 1 
               
               
                 Keratin, type II cytoskeletal 1b 
                 66774007 
                 1 
               
               
                 Thymidine phosphorylase precursor 
                 67477361 
                 1 
               
               
                 Exportin-1 
                 68052989 
                 1 
               
               
                 Cathepsin B precursor 
                 68067549 
                 1 
               
               
                 Myosin-14 
                 71151982 
                 1 
               
               
                 ATP-dependent RNA helicase A 
                 71153504 
                 1 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Relative Abundant Proteins Downregulated in High-Grade Cervical Specimens 
               
            
           
           
               
               
               
            
               
                   
                   
                 Number Down- 
               
               
                 Gene/Protein Name 
                 GI Accession 
                 Regulated Pairs 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Calmodulin-related protein NB-1 
                 115502 
                 7 
               
               
                 Interleukin-1 receptor antagonist protein precursor 
                 124312 
                 7 
               
               
                 Arachidonate 12-lipoxygenase, 12S-type 
                 126400 
                 7 
               
               
                 Desmoplakin 
                 12644130 
                 7 
               
               
                 Periplakin 
                 14195005 
                 7 
               
               
                 Small proline-rich protein 3 
                 20138065 
                 7 
               
               
                 Plakophilin 3 
                 20139301 
                 7 
               
               
                 Keratin, type II cytoskeletal 4 
                 20141510 
                 7 
               
               
                 Squamous cell carcinoma antigen 1 
                 20141712 
                 7 
               
               
                 Desmoglein-3 precursor 
                 416918 
                 7 
               
               
                 Involucrin 
                 124731 
                 6 
               
               
                 Keratin, type II cytoskeletal 3 
                 125098 
                 6 
               
               
                 Hurpin 
                 12643252 
                 6 
               
               
                 Cystatin B 
                 1706278 
                 6 
               
               
                 Plakophilin 1 
                 20138951 
                 6 
               
               
                 Aldo-keto reductase family 1 member B10 
                 20531983 
                 6 
               
               
                 Mucin 5B precursor 
                 23821885 
                 6 
               
               
                 Chloride intracellular channel protein 3 
                 46397812 
                 6 
               
               
                 Puromycin-sensitive aminopeptidase 
                 51704228 
                 6 
               
               
                 Maspin precursor 
                 547892 
                 6 
               
               
                 Junction plakoglobin 
                 130257 
                 5 
               
               
                 Retinoic acid-binding protein II, cellular 
                 132401 
                 5 
               
               
                 Keratin, type II cytoskeletal 6A 
                 1346344 
                 5 
               
               
                 Galectin-7 
                 1346431 
                 5 
               
               
                 Envoplakin 
                 14194715 
                 5 
               
               
                 Fatty acid-binding protein, epidermal 
                 232081 
                 5 
               
               
                 Desmoglein-1 precursor 
                 416917 
                 5 
               
               
                 Desmocollin 2A/2B precursor 
                 461968 
                 5 
               
               
                 Protein-glutamine gamma-glutamyltransferase K 
                 57015359 
                 5 
               
               
                 Keratin, type I cytoskeletal 13 
                 6016411 
                 5 
               
               
                 Ig alpha-1 chain C region 
                 113584 
                 4 
               
               
                 Cystatin A 
                 118177 
                 4 
               
               
                 Placental ribonuclease inhibitor 
                 132573 
                 4 
               
               
                 Keratin, type II cytoskeletal 6B 
                 1346345 
                 4 
               
               
                 Protein-glutamine glutamyltransferase E precursor 
                 13638501 
                 4 
               
               
                 Low-density lipoprotein receptor-related protein 1 precursor 
                 1708865 
                 4 
               
               
                 Squamous cell carcinoma antigen 2 
                 1710877 
                 4 
               
               
                 Airway trypsin-like protease precursor 
                 17376886 
                 4 
               
               
                 Sciellin 
                 20139986 
                 4 
               
               
                 S100 calcium-binding protein A14 
                 20178118 
                 4 
               
               
                 Tubulin alpha-6 chain 
                 20455322 
                 4 
               
               
                 Antileukoproteinase 1 precursor 
                 113636 
                 3 
               
               
                 Annexin A1 
                 113944 
                 3 
               
               
                 Annexin A3 
                 113954 
                 3 
               
               
                 Annexin A8 
                 113967 
                 3 
               
               
                 Carbonyl reductase 
                 118519 
                 3 
               
               
                 Ezrin 
                 119717 
                 3 
               
               
                 Gelsolin precursor 
                 121116 
                 3 
               
               
                 Histone H2A.g 
                 121978, 121959, 
                 3 
               
               
                   
                 12585257 
               
               
                 Keratin, type I cytoskeletal 15 
                 125081 
                 3 
               
               
                 Ig kappa chain V-III region SIE, Ig kappa chain V-III region WOL 
                 125797, 125803 
                 3 
               
               
                 Phosphoglycerate mutase 1 
                 130348 
                 3 
               
               
                 Fatty acid synthase 
                 1345959 
                 3 
               
               
                 Breast cancer type 2 susceptibility protein 
                 14424438 
                 3 
               
               
                 Ketohexokinase 
                 1730044 
                 3 
               
               
                 Polymeric-immunoglobulin receptor precursor 
                 1730570 
                 3 
               
               
                 Serpin B12 
                 20140145 
                 3 
               
               
                 6-phosphogluconate dehydrogenase, decarboxylating 
                 20981679 
                 3 
               
               
                 Leukocyte elastase inhibitor 
                 266344 
                 3 
               
               
                 Long palate, lung and nasal epithelium carcinoma associated 
                 34395685 
                 3 
               
               
                 protein 1 precursor 
               
               
                 Eukaryotic initiation factor 4A-I 
                 46397463 
                 3 
               
               
                 Ras-related protein Rab-2A 
                 46577636 
                 3 
               
               
                 Keratin, type II cuticular HB2 
                 48474984 
                 3 
               
               
                 ERO1-like protein alpha precursor 
                 50400608 
                 3 
               
               
                 ADP-ribosylation factor 1, ADP-ribosylation factor 3 
                 51316985, 47117657 
                 3 
               
               
                 Tubulin alpha-ubiquitous chain 
                 55977474 
                 3 
               
               
                 Tubulin beta-2 chain 
                 55977480 
                 3 
               
               
                   
               
               
                 Criteria for inclusion in upregulated sample pairs HSIL/WNL (Total N = 11): 
               
               
                 1) Cutoff values of &gt;4 fold or &gt;4s in total peptide ratios 
               
               
                 2) &gt;2.5 fold for software integration analysis (specimen pairs 48/65, 51/66, 50/80) 
               
               
                 3) Quantitative data &gt;1.7 
               
               
                 Criteria for inclusion in downregulated sample pairs WNL/HSIL (Total N = 11): 
               
               
                 1) Cutoff values of &gt;4 fold or &gt;4s in total peptide ratios 
               
               
                 Table 1: Selected proteins with significant biological interest upregulated in 3+ specimen pairs 
               
               
                 Table 2: Relative abundant proteins upregulated in 3+ specimen pairs 
               
               
                 Table 3: Lower probability proteins upregulated in 1 or 2 specimen pairs 
               
               
                 Table 4: Relative abundant proteins downregulated in 3+ specimen pairs 
               
            
           
         
       
     
     REFERENCES 
     
         
         ASCUS-LSIL Triage Study (ALTS) Group (2003), Am J Obstet Gynecol, 188:1383-92 and 1393-400. 
         Baldwin et al. (2003) Nature Reviews Cancer 3:1-10. 
         Crook et al. (1991) Cell 67(3):547-56. 
         Cuschieri et-al. (2005) Journal of Clinical Pathology 58:946-950. 
         Howell et al. (2004) Diagn Cytopathol, 30(5):362-6. 
         Joste et al. (2005) Diagnostic Cytopathology 32(5):310-314. 
         Melnikow et al. (1998) Natural history of cervical squamous intraepithelial lesions: a meta-analysis. Obstet Gynecol 92:727-35. 
         Munoz et al. (1989) Epidemiology of Cervical Cancer In: “Human Papillomavirus,” New York, Oxford Press, pp 9-39. 
         National Institutes of Health, Consensus Development Conference Statement on Cervical Cancer, Apr. 1-3, 1996. 
         Roberts et al. (1997) The Medical Journal of Australia 167:466-469. 
         Shai et al. (2007) Cancer Research 67:1626-1635. 
         Shiffman et al. (2003) Arch Pathol Lab Med 127(8):946-9. 
         Syrjanen et al. (1987) Applied Pathol 5:121-135. 
         Tabbara et al. (1992) The Bethesda classification for squamous intraepithelial lesions: histologic, cytologic and viral correlates, Obstet Gynecol 79:338-346. 
       
    
     While the present invention has been described in conjunction with a preferred embodiment, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. It is therefore intended that the protection granted by Letters Patent hereon be limited only by the definitions contained in the appended claims and equivalents thereof.