Abstract:
The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the Serine/Threoninc-Protein Kinase B-raf (BRAF) that are particularly advantageous for quantifying the BRAF protein directly in bio-logical samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed where the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded.

Description:
[0001]    This application claims priority to provisional application Ser. No. 62/023,615, filed Jul. 11, 2014,the contents of which are hereby incorporated by reference in their entirety. 
     
    
     INTRODUCTION 
       [0002]    Specific peptides derived from subsequences of the Serine/Threonine-Protein Kinase B-raf, (also referred to as Proto-Oncogene B-Raf, p94, v-Raf murine sarcoma viral oncogene homolog B 1, and BRAF, and referred to herein as “BRAF”) are provided. The peptide sequence and fragmentation/transition ions for each peptide are useful in a mass spectrometry-based Selected Reaction Monitoring (SRM) assay, which can also be referred to as a Multiple Reaction Monitoring (MRM) assay, and referred to herein as SRM/MRM. The use of peptides for SRM/MRM quantitative analysis of the BRAF protein is described. 
         [0003]    This SRM/MRM assay can be used to measure relative or absolute quantitative levels of one or more of the specific peptides from the BRAF protein and therefore provides methods of measuring the amount of the BRAF protein in a given protein preparation obtained from a biological sample by mass spectrometry. 
         [0004]    More specifically, the SRM/MRM assay can measure these peptides directly in complex protein lysate samples prepared from cells procured from patient tissue samples, such as formalin fixed cancer patient tissue. Methods of preparing protein samples from formalin-fixed tissue are described in U.S. Pat. No. 7,473,532, the contents of which are hereby incorporated by references in their entirety. The methods described in U.S. Pat. No. 7,473,532 may conveniently be carried out using Liquid Tissue® reagents and protocol available from Expression Pathology Inc. (Rockville, Md.). The most widely and advantageously available (brut of tissues from cancer patients tissue is formalin fixed, paraffin embedded tissue. Formaldehyde/formalin fixation of surgically removed tissue is by far the most common method of preserving cancer tissue samples worldwide and is the accepted convention for standard pathology practice. Aqueous solutions of formaldehyde are referred to as formalin. “100%” formalin consists of a saturated solution of formaldehyde (about 40% by volume or 37% by mass) in water, with a small amount of stabilizer, usually methanol to limit oxidation and degree of polymerization. The most common way in which tissue is preserved is to soak whole tissue for extended periods of time (8 hours to 48 hours) in aqueous formaldehyde commonly termed 10% neutral buffered formalin, followed by embedding the fixed whole tissue in paraffin wax for long term storage at room temperature. Thus molecular analytical methods to analyze formalin fixed cancer tissue will be the most accepted and heavily utilized methods for analysis of cancer patient tissue. 
         [0005]    Results from the SRM/MRM assay can be used to correlate accurate and precise quantitative levels of the BRAF protein within the specific tissue samples (e.g., cancer tissue sample) of the patient or subject from whom the tissue (biological sample) was collected and preserved. This not only provides diagnostic and prognostic information about the cancer, but also allows a physician or other medical professional to more accurately determine appropriate therapy for the patient. Such an assay that provides diagnostically, prognostically, and therapeutically important information about levels of protein expression in a diseased tissue or other patient sample is termed a companion diagnostic assay. For example, such an assay can be designed to diagnose the stage or degree of a cancer and determine a therapeutic agent to which a patient is most likely to respond. 
       SUMMARY 
       [0006]    The assays described herein measure relative or absolute levels of specific unmodified peptides from the BRAF protein and also can measure absolute or relative levels of specific modified peptides from the BRAF protein. Examples of modifications include phosphorylated amino acid residues and glycosylated amino acid residues that may be present on the peptides. 
         [0007]    Relative quantitative levels of the BRAF protein are determined by the SRM/MRM methodology by, for example, comparing SRM/MRM signature peak areas (e.g., signature peak area or integrated fragment ion intensity) of an individual BRAF peptide in different samples. Alternatively, it is possible to compare multiple SRM/MRM signature peak areas for multiple BRAF signature peptides, where each peptide has its own specific SRM/MRM signature peak, to determine the relative BRAF protein content in one biological sample and compare it with the BRAF protein content in one or more additional or different biological samples. In this way, the amount of a particular peptide, or peptides, from the BRAF protein, and therefore the amount of the BRAF protein, is determined relative to the same BRAF peptide, or peptides, across 2 or more biological samples under the same experimental conditions. In addition, relative quantitation can be determined for a given peptide, or peptides, from the BRAF protein within a single sample by comparing the signature peak area for that peptide by SRM/MRM methodology to the signature peak area for another and different peptide, or peptides, from a different protein, or proteins, within the same protein preparation from the biological sample. In this way, the amount of a particular peptide from the BRAF protein, and therefore the amount of the BRAF protein, is determined relative one to another within the same sample. These approaches permit quantitation of an individual peptide, or peptides, from the BRAF protein to the amount of another peptide, or peptides, between samples and within samples wherein the amounts as determined by signature peak area are relative one to another, regardless of the absolute weight to volume or weight to weight amounts of the BRAF peptide in the protein preparation from the biological sample. Relative quantitative data about individual signature peak areas between different samples can be normalized to the amount of protein analyzed per sample. Relative quantitation can be performed across many peptides from multiple proteins and the BRAF protein simultaneously in a single sample and/or across many samples to gain insight into relative protein amounts of one peptide/protein with respect to other peptides/proteins. 
         [0008]    Absolute quantitative levels of the BRAF protein are determined by, for example, the SRM/MRM methodology whereby the SRM/MRM signature peak area of an individual peptide from the BRAF protein in one biological sample is compared to the SRM/MRM signature peak area of a spiked internal standard. In one embodiment, the internal standard is a synthetic version of the same exact BRAF peptide that contains one or more amino acid residues labeled with one or more heavy isotopes. Such an isotope labeled internal standard is synthesized so that, when analyzed by mass spectrometry, it generates a predictable and consistent SRM/MRM signature peak that is different and distinct front the native BRAF peptide signature peak and which therefore can be used as a comparator peak. Thus, when the internal standard is spiked into a protein preparation from a biological sample in known amounts and analyzed by mass spectrometry, the SRM/MRM signature peak area of the native peptide is compared to the SRM/MRM signature peak area of the internal standard peptide, and this numerical comparison indicates either the absolute molarity and/or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantitative data for fragment peptides are displayed according to the amount of protein analyzed per sample. Absolute quantitation can be performed across many peptides, and thus proteins, simultaneously in a single sample and/or across many samples to gain insight into absolute protein amounts in individual biological samples and in entire cohorts of individual samples. 
         [0009]    The SRM/MRM assay method can be used to aid diagnosis of the stage of a cancer and/or the patient prognosis, for example, directly in patient-derived tissue, such as formalin fixed tissue, and to aid in determining which therapeutic agent would be most advantageous for use in treating that patient. Cancer tissue that is removed from a patient either through surgery, such as for therapeutic removal of partial or entire tumors, or through biopsy procedures conducted to determine the presence or absence of suspected disease, is analyzed to determine whether or not a specific protein, or proteins, and which forms of proteins, are present in that patient tissue. Moreover, the expression level of a protein, or multiple proteins, can be determined and compared to a “normal” or reference level found in healthy tissue. Normal or reference levels of proteins found in healthy tissue may be derived from, for example, the relevant tissues of one or more individuals that do not have cancer. Alternatively, normal or reference levels may be obtained for individuals with cancer by analysis of relevant tissues not affected by the cancer. 
         [0010]    Assays of protein levels (e.g., BRAF levels) can also be used to diagnose the stage of cancer and provide prognostic information about a patient or subject diagnosed with cancer by employing the BRAF levels. The level of an individual BRAF peptide is defined as the molar amount of the peptide determined by the SRM/MRM assay per total amount of protein lysate analyzed. Information regarding BRAF can thus be used to aid in determining the stage or grade of a cancer and/or patient prognosis by correlating the level of the BRAF protein (or fragment peptides of the BRAF protein) with levels observed in normal tissues. Once the stage and/or grade, and/or BRAF protein expression characteristics of the cancer has been determined, that information can be matched to a list of therapeutic agents (chemical and biological) developed to specifically treat cancer tissue that is characterized by, for example, abnormal expression of the protein or protein(s) (e.g., BRAF) that were assayed. The therapeutic agents Vemurafenib and Sorafenib are particularly useful for targeting BRAF-expressing cancer cells. Other BRAF inhibitors include GDC-0879, PLX-4720, dabrafenib, and LGX818. Matching information from a BRAF protein assay to a list of therapeutic agents that specifically targets, for example, the BRAF protein or cells/tissue expressing the protein, defines what has been termed a personalized medicine approach to treating disease. The assay methods described herein form the foundation of a personalized medicine approach by using analysis of proteins from the patient&#39;s own tissue as a source for diagnostic and treatment decisions. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]      FIG. 1 , parts A to C, shows an example of an SRM/MRM assay of a single peptide from the BRAF protein performed on a Liquid Tissue lysate from a formalin fixed biological sample with quantitation of the BRAF peptide conducted on a triplequadrupole mass spectrometer. The specific characteristics about how to measure this peptide in biological samples that have been fixed in formalin is shown. 
       
    
    
     DETAILED DESCRIPTION 
       [0012]    In principle, any predicted peptide derived from BRAF protein, prepared for example by digesting with a protease of known specificity (e.g. trypsin), can be used as a surrogate reporter to determine the abundance of BRAF protein in a sample using a mass spectrometry-based SRM/MRM assay. Similarly, any predicted peptide sequence containing an amino acid residue at a site that is known to be potentially modified in the BRAF protein also might potentially be used to assay the extent of modification of the BRAF protein in a sample. 
         [0013]    BRAF fragment peptides may be generated by a variety of methods including by the use of the Liquid Tissue protocol provided in U.S. Pat. No. 7,473,532. The Liquid Tissue protocol and reagents are capable of producing peptide samples suitable for mass spectroscopic analysis from formalin fixed paraffin embedded tissue by proteolytic digestion of the proteins in the tissue/biological sample. In the Liquid Tissue protocol the tissue/biological is heated in a buffer for an extended period of time (e.g., from about 80° C. to about 100° C. for a period of time from about 10 minutes to about 4 hours) to reverse or release protein cross-linking. The buffer employed is a neutral buffer, (e.g., a Tris-based buffer, or a buffer containing a detergent). Following heat treatment the tissue/biological sample is treated with one or more proteases, including but not limited to trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C, for a time sufficient to disrupt the tissue and cellular structure of said biological sample and to liquefy the sample (e.g., a period of time from 30 minutes to 24 hours at a temperature from 37° C. to 65° C.). The result of the heating and proteolysis is a liquid, soluble, dilutable biomolecule lysate. 
         [0014]    Surprisingly, it was found that many potential peptide sequences from the BRAF protein are unsuitable or ineffective for use in mass spectrometry-based SRM/MRM assays for reasons that are not immediately evident. This is particularly true for peptides derived from formalin fixed tissue. As it was not possible to predict the most suitable peptides for MRM/SRM assay, it was necessary to experimentally identify modified and unmodified peptides in actual Liquid Tissue lysates to develop a reliable and accurate SRM/MRM assay for the BRAF protein. While not wishing to be bound by any theory, it is believed that some peptides might, for example, be difficult to detect by mass spectrometry as they do not ionize well or produce fragments that are not distinct from other proteins. Peptides may also fail to resolve well in separation (e.g., chromatography), or may adhere to glass or plastic ware. 
         [0015]    BRAF peptides found in various embodiments of this disclosure (e.g., Tables 1 and 2) were derived from the BRAF protein by protease digestion of all the proteins within a complex Liquid Tissue lysate prepared from cells procured from formalin fixed cancer tissue, Unless noted otherwise, in each instance the protease was trypsin, The Liquid Tissue lysate was then analyzed by mass spectrometry to determine those peptides derived from the BRAF protein that are detected and analyzed by mass spectrometry. Identification of a specific preferred subset of peptides for mass-spectrometric analysis is based on 1) experimental determination of which peptide or peptides from a protein ionize in mass spectrometry analyses of Liquid Tissue lysates, and 2) the ability of the peptide to survive the protocol and experimental conditions used in preparing a Liquid Tissue lysate. This latter property extends not only to the amino acid sequence of the peptide but also to the ability of a modified amino acid residue within a peptide to survive in modified form during the sample preparation. 
         [0016]    Protein lysates from cells procured directly from formalin (formaldehyde) fixed tissue were prepared using the Liquid Tissue reagents and protocol that entails collecting cells into a sample tube via tissue microdissection followed by heating the cells in the Liquid Tissue buffer for an extended period of time. Once the formalin-induced cross linking has been negatively affected, the tissue/cells are then digested to completion in a predictable manner using a protease, such as, for example, trypsin (although other proteases can be used). Each protein lysate is turned into a collection of peptides by digestion of intact polypeptides with the protease. Each Liquid Tissue lysate was analyzed (e.g., by ion trap mass spectrometry) to perform multiple global proteomic surveys of the peptides, where the data was presented as identification of as many peptides as could be identified by mass spectrometry from all cellular proteins present in each protein lysate. An ion trap mass spectrometer or another form of a mass spectrometer that is capable of performing global profiling for identification of as many peptides as possible from a single complex protein/peptide lysate is employed. Ion trap mass spectrometers however may advantageously be used conducting global profiling of peptides. Although an SRM/MRM assay can be developed and performed on any type of mass spectrometer, including a MALDI, ion trap, or triple quadrupole, advantageously a triple quadrupole instrument platform is used for an SRM/MRM assay. That type of a mass spectrometer is suitable instrument for analyzing a single isolated target peptide within a very complex protein lysate that may consist of hundreds of thousands to millions of individual peptides from all the proteins contained within a cell. 
         [0017]    Once as many peptides as possible were identified in a single MS analysis of a single lysate under the conditions employed, then that list of peptides was collated and used to determine the proteins that were detected in that lysate. That process was repeated for multiple Liquid Tissue lysates, and the very large list of peptides was collated into a single dataset. That type of dataset can be considered to represent the peptides that can be detected in the type of biological sample that was analyzed (after protease digestion), and specifically in a Liquid Tissue lysate of the biological sample, and thus includes the peptides for specific proteins, such as for example the BRAF protein. 
         [0018]    In one embodiment, the BRAF tryptic peptides identified as useful in the determination of absolute or relative amounts of the BRAF protein include one or more, two or more, three or more, or four or more of the peptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, all of which are listed in Table 1. Each of those peptides was detected by mass spectrometry in Liquid Tissue lysates prepared from formalin fixed, paraffin embedded tissue. Thus, each peptide is a candidate for use in developing a quantitative SRM/MRM assay for the BRAF protein in human biological samples, including directly in formalin fixed patient tissue. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 1  
               
               
                   
                   
               
               
                   
                 SEQ ID 
                 Peptide sequence 
               
               
                   
                   
               
             
             
               
                   
                 SEQ ID NO: 1 
                 LTQEHIEALLDK 
               
               
                   
                   
               
               
                   
                 SEQ TD NO: 2 
                 TVVPAR 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 3 
                 IQDGEK 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 4 
                 LLFQGFR 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 5 
                 NQFGQR 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 6 
                 NEVGVLR 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 7 
                 SNNIFLHEDLTVK 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 8 
                 SASEPSLNR 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 9 
                 FGGEHNPPSIYLEAYEEYTSK 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 10 
                 SSSSSEDR 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 11 
                 TPIQAGGYGAFPVH 
               
               
                   
                   
               
             
          
         
       
     
         [0019]    The BRAF tryptic peptides listed in Table 1 were detected from multiple Liquid Tissue lysates of multiple different formalin fixed tissues of different human organs including prostate, colon, and breast. Each of those peptides is considered useful for quantitative SRM/MRM assay of the BRAF protein in formalin fixed tissue. Further data analysis of these experiments indicated no preference for any specific peptides from any specific organ site. Thus, these peptides may be used for conducting SRM/MRM assays of the BRAF protein on a Liquid Tissue lysate from any formalin fixed tissue originating from any biological sample or from any organ site in the body. 
         [0020]    In order to most efficiently implement an SRM/MRM assay for each peptide derived from the BRAF protein it is desirable to utilize information in addition to the peptide sequence in the analysis. That additional information may be used in directing and instructing the mass spectrometer (e.g. a triple quadrupole mass spectrometer) to perform the correct and focused analysis of specific targeted peptide(s), such that the assay may be effectively performed. 
         [0021]    The additional information about target peptides in general, and about specific BRAF peptides, may include one or more of the mono isotopic mass of the peptide, its precursor charge state, the precursor m/z value, the m/z transition ions, and the ion type of each transition ion. Table 2 shows additional peptide information that may be used to develop an SRM/MRM assay for the BRAF protein for two (2) of the BRAF peptides from the list in Table 1. Similar additional information described for the two (2) BRAF peptides shown by example in Table 2 may be prepared, obtained, and applied to the analysis of the other peptides contained in Table 1. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2  
               
               
                   
               
               
                   
                   
                 Mono 
                 Precursor 
                   
                   
                   
               
               
                   
                 Peptide 
                 Isotopic 
                 Charge 
                 Precursor 
                 Transition 
                 Ion 
               
               
                 SEQ ID 
                 sequence 
                 Mass 
                 State 
                 m/z 
                 m/z 
                 Type 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SEQ ID 
                 LTQEHIEA 
                 1408.7562 
                 2 
                 705.385 
                 688.387 
                 y6  
               
               
                 NO: 1 
                 LLDK 
                   
                 2 
                 705.385 
                 801.471 
                 Y7  
               
               
                   
                   
                   
                 2 
                 705.385 
                 938.53 
                 Y8  
               
               
                   
                   
                   
                 2 
                 705.385 
                 1067.573 
                 Y9  
               
               
                   
                   
                   
                 2 
                 705.385 
                 1195.631 
                 y10 
               
               
                   
               
               
                 SEQ ID 
                 LLFQGFR 
                 879.4966 
                 2 
                 440.756 
                 379.208 
                 Y3 
               
               
                 NO: 4 
                   
                   
                 2 
                 440.756 
                 507.267 
                 y4  
               
               
                   
                   
                   
                 2 
                 440.756 
                 654.335 
                 y5  
               
               
                   
                   
                   
                 2 
                 440.756 
                 767.419 
                 y6  
               
               
                   
                   
                   
                 2 
                 440.756 
                 880.503 
                 y7  
               
               
                   
               
             
          
         
       
     
         [0022]    The method described below was used to: 1) identify candidate peptides from the BRAF protein that can be used for a mass spectrometry-based SRM/MRM assay for the BRAF protein, 2) develop an individual SRM/MRM assay, or assays, for target peptides from the BRAF protein in order to correlate and 3) apply quantitative assays to cancer diagnosis and/or choice of optimal therapy. 
       Assay Method 
       [0000]    
       
         1. Identification of SRM/MRM candidate fragment peptides for the BRAF protein
       a. Prepare a Liquid Tissue protein lysate from a formalin fixed biological sample using a protease or proteases, (that may or may not include trypsin), to digest proteins   b. Analyze all protein fragments in the Liquid Tissue lysate on an ion trap tandem mass spectrometer and identify all fragment peptides from the BRAF protein, where individual fragment peptides do not contain any peptide modifications such as phosphorylations car glycosylations   c. Analyze all protein fragments in the Liquid Tissue lysate on an ion trap tandem mass spectrometer and identify all fragment peptides from the BRAF protein that carry peptide modifications such as for example phosphorylated or glycosylated residues   d. All peptides generated by a specific digestion method from the entire, full length BRAF protein potentially can be measured, but preferred peptides used for development of the SRM/MRM assay are those that are identified by mass spectrometry directly in a complex Liquid Tissue protein lysate prepared from a formalin fixed biological sample   e. Peptides that are specifically modified (phosphorylated, glycosylated, etc.) in patient tissue and which ionize, and thus detected, in a mass spectrometer when analyzing a Liquid Tissue lysate from a formalin fixed biological sample are identified as candidate peptides for assaying peptide modifications of the BRAF protein   
     
         2. Mass Spectrometry Assay for Fragment Peptides from the BRAF Protein
       a. SRM/MRM assay on a triple quadrupole mass spectrometer for individual fragment peptides identified in a Liquid Tissue lysate is applied to peptides from the BRAF protein
           i. Determine optimal retention time for a fragment peptide for optimal chromatography conditions including but not limited to gel electrophoresis, liquid chromatography, capillary electrophoresis, nano-reversed phase liquid chromatography, high performance liquid chromatography, or reverse phase high performance liquid chromatography   ii. Determine the mono isotopic mass of the peptide, the precursor charge state for each peptide, the precursor m/z value for each peptide, the m/z transition ions for each peptide, and the ion type of each transition ion for each fragment peptide in order to develop an SRM/MRM assay for each peptide.   iii. SRM/MRM assay can then be conducted using the information from (i) and (ii) on a triple quadrupole mass spectrometer where each peptide has a characteristic and unique SRM/MRM signature peak that precisely defines the unique SRM/MRM assay as performed on a triple quadrupole mass spectrometer   
           b. Perform SRM/MRM analysis so that the amount of the fragment peptide of the BRAF protein that is detected, as a function of the unique SRM/MRM signature peak area from an SRM/MRM mass spectrometry analysis, can indicate both the relative and absolute amount of the protein in a particular protein lysate.
           i. Relative quantitation may be achieved by:
               1. Determining increased or decreased presence of the BRAF protein by comparing the SRM/MRM signature peak area from a given BRAF peptide detected in a Liquid Tissue lysate from one formalin fixed biological sample to the same SRM/MRM signature peak area of the same BRAF fragment peptide in at least a second, third, fourth or more Liquid Tissue lysates from least a second, third, fourth or more formalin fixed biological samples   2. Determining increased or decreased presence of the BRAF protein by comparing the SRM/MRM signature peak area from a given BRAF peptide detected in a Liquid Tissue lysate from one formalin fixed biological sample to SRM/MRM signature peak areas developed from fragment peptides from other proteins, in other samples derived from different and separate biological sources, where the SRM/MRM signature peak area comparison between the 2 samples for a peptide fragment are normalized to amount of protein analyzed in each sample.   3. Determining increased or decreased presence of the BRAF protein by comparing the SRM/MRM signature peak area for a given BRAF peptide to the SRM/MRM signature peak areas from other fragment peptides derived from different proteins within the same Liquid Tissue lysate from the formalin fixed biological sample in order to normalize changing levels of BRAF protein to levels of other proteins that do not change their levels of expression under various cellular conditions.   4. These assays can be applied to both unmodified fragment peptides and for modified fragment peptides of the BRAF protein, where the modifications include but are not limited to phosphorylation and/or glycosylation, and where the relative levels of modified peptides are determined in the same manner as determining relative amounts of unmodified peptides.   
               ii. Absolute quantitation of a given peptide may be achieved by comparing the SRM/MRM signature peak area for a given fragment peptide from the BRAF protein in an individual biological sample to the SRM/MRM signature peak area of an internal fragment peptide standard spiked into the protein lysate from the biological sample
               1. The internal standard is a labeled synthetic version of the fragment peptide from the BRAF protein that is being interrogated. This standard is spiked into a sample in known amounts, and the SRM/MRM signature peak area can be determined for both the internal fragment peptide standard and the native fragment peptide in the biological sample separately, followed by comparison of both pea areas   2. This can be applied to unmodified fragment peptides and modified fragment, peptides, where the modifications include but are not limited to phosphorylation and/or glycosylation, and where the absolute levels of modified peptides can be determined in the same manner as determining absolute levels of unmodified peptides.   
               
           
     
         3. Apply Fragment Peptide Quantitation to Cancer Diagnosis and Treatment
       a. Perform relative and/or absolute quantitation of fragment peptide levels of the BRAF protein and demonstrate that the previously-determined association, as well understood in the field of cancer, of BRAF protein expression to the stage/grade/status of cancer in patient tumor tissue is confirmed   b. Perform relative and/or absolute quantitation of fragment peptide levels of the BRAF protein and demonstrate correlation with clinical outcomes from different treatment strategies, wherein this correlation has already been demonstrated in the field or can be demonstrated in the future through correlation studies across cohorts of patients and tissue from those patients. Once either previously established correlations or correlations derived in the future are confirmed by this assay then the assay method can be used to determine optimal treatment strategy   
     
       
     
         [0046]      FIG. 1  shows an example of a single SRM/MRM assay performed on a Liquid Tissue lysate from a formalin fixed biological sample. An SRM/MRM assay was developed for a single peptide for quantitation of the BRAF protein on a triplequadrupole mass spectrometer. Specific and unique characteristics about this BRAF peptide (sequence LLFQGFR) were developed by analysis of all BRAF peptides on both an ion trap and triple quadrupole mass spectrometers and are shown in  FIG. 1A . That information includes the monoisotopic mass of the peptide, its precursor charge state, the precursor m/z value, the transition m/z values of the precursor, and the ion types of each of the identified transitions. That information must be determined experimentally for each and every candidate SRM/MRM peptide directly in Liquid Tissue lysates from formalin fixed samples/tissue; because, interestingly, not all peptides from the BRAF protein can be detected in such lysates using SRM/MRM as described herein, indicating that BRAF peptides not detected cannot be considered candidate peptides for developing an SRM/MRM assay for use in quantitating peptides/proteins directly in Liquid Tissue lysates from formalin fixed samples/tissue. 
         [0047]    As shown in  FIG. 1B , this particular SRM/MRM assay was performed on a triple quadrupole mass spectrometer. The experimental sample in this experiment was a Liquid Tissue protein lysate prepared from a cell line that had been formalin fixed, paraffin embedded to act as a tissue surrogate. Data from the assay indicates the presence of the unique SRM/MRM signature peak for this BRAF peptide in the formalin fixed sample. 
         [0048]      FIG. 1C  shows the specific transition ion characteristics for this peptide used to quantitatively measure the above-mentioned peptide in formalin fixed biological samples. These data indicate absolute amounts of this BRAF peptide as a function of the molar amount of the peptide per microgram of protein lysate analyzed. Assessment of BRAF protein levels in tissues based on analysis of formalin fixed patient-derived tissue can provide diagnostic, prognostic, and therapeutically-relevant information about each particular patient. In one embodiment, this disclosure describes a method for measuring the level of the Serine/Threonine-Protein Kinase B-raf (BRAF) protein in a biological sample, comprising detecting and/or quantifying the amount of one or more modified or unmodified BRAF fragment peptides in a protein digest prepared from said biological sample using mass spectrometry; and calculating the level of modified or unmodified BRAF protein in said sample; and where the level is a relative level or an absolute level. In a related embodiment, quantifying one or more BRAF fragment peptides comprises determining the amount of the each of the BRAF fragment peptides in a biological sample by comparison to an added internal standard peptide of known amount, wherein each of the BRAF fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. In some embodiments the internal standard is an isotopically labeled internal standard peptide comprises one or more heavy stable isotopes selected from  18 O,  17 O,  34 S,  15 N,  13 C,  2 H or combinations thereof. 
         [0049]    The method for measuring the level of the BRAF protein in a biological sample described herein (or fragment peptides as surrogates thereof) may be used as a diagnostic and/or prognostic indicator of cancer in a patient or subject. In one embodiment, the results from measurements of the level of the BRAF protein may be employed to determine the diagnostic stage/grade/status and/or the prognostic status of a cancer by correlating (e.g., comparing) the level of BRAF protein found in a tissue with the level of that protein found in normal and/or cancerous or precancerous tissues. 
         [0050]    Because both nucleic acids and protein can be analyzed from the same Liquid Tissue™ biomolecular preparation it is possible to generate additional information about disease diagnosis and drug treatment decisions from the nucleic acids in same sample upon which proteins were analyzed. For example, if the BRAF protein is expressed by certain cells at increased levels, when assayed by SRM the data can provide information about the state of the cells and their potential for uncontrolled growth, potential drug resistance and the development of cancers can be obtained. At the same time, information about the status of the BRAF genes and/or the nucleic acids and proteins they encode (e.g., mRNA molecules and their expression levels or splice variations) can be obtained from nucleic acids present in the same Liquid Tissue™ bimolecular preparation can be assessed simultaneously to the SRM analysis of the BRAF protein. An gene and/or nucleic acid not from the BRAF and which is present in the same biomolecular preparation can be assessed simultaneously to the SRM analysis of the BRAF protein. In one embodiment, information about the BRAF protein and/or one, two, three, four or more additional proteins may be assessed by examining the nucleic acids encoding those proteins. Those nucleic acids can be examined, for example, by one or more, two or more, or three or more of: sequencing methods, polymerase chain reaction methods, restriction fragment polymorphism analysis, identification of deletions, insertions, and/or determinations of the presence of mutations, including but not limited to, single base pair polymorphisms, transitions, transversions, combinations thereof.