bluuebunny/crossref_metadata_2025 huggingface dataset:
Three different origins for apparent triploid/diploid mosaics
Authors: Daniel Art , Wu Zhanhe , Darmanian Artur , Collins Felicity , Jackson Julianne
Published: July 2003

Four apparent triploid/diploid mosaic cases were studied. Three of the cases were detected at prenatal diagnosis and the other was of an intellectually handicapped, dysmorphic boy. Karyotypes were performed in multiple tissues if possible, and the inheritance of microsatellites was studied with DNA from fetal tissues and parental blood. Non‐mosaic triploids have a different origin from these mosaics with simple digyny or diandry documented in many cases. Three different mechanisms of origin for these apparent mosaics were detected: (1) chimaerism with karyotypes from two separate zygotes developing into a single individual, (2) delayed digyny, by incorporation of a pronucleus from a second polar body into one embryonic blastomere, and (3) delayed dispermy, similarly, by incorporation of a second sperm pronucleus into one embryonic blastomere. In three of the four cases, there was segregation within the embryos of triploid and diploid cell lines into different tissues from which DNA could be isolated. In case 2 originating by digyny, the same sperm allele at each locus could be detected in both triploid and diploid tissues, which is supportive evidence for the involvement of a single sperm and for true mosaicism rather than chimaerism. Similarly, in case 4 originating by dispermy, the same single ovum allele at each locus could be detected in diploid and triploid tissues, confirming mosaicism. In the chimaeric case (case 3), the diploid line had the karyotype 47,XY,+16 while the triploid line was 69,XXY. This suggests a chimaera, since, in a true mosaic, the triploid line should also contain the additional chromosome 16. Supporting the interpretation of a chimaeric origin for this case, the DNA data showed that the triploidy was consistent with MII non‐disjunction (i.e. involving a diploid ovum). In the mosaic cases (1, 2, 4), there was no evidence of the involvement of a diploid sperm or a diploid ova, and in triploid/diploid mosaicism, an origin from a diploid gamete is excluded, since all such conceptuses would be simple triploids. In one of these triploid/diploid mosaics detected at prenatal diagnosis by CVS, the triploid line seemed to be sequestered into the extra‐fetal tissues (confined placental mosaicism). This fetus developed normally and a normal infant was born with no evidence of triploidy in newborn blood or cord blood at three months of age. Copyright © 2003 John Wiley & Sons, Ltd.
Imaging technologies for the detection of multiple stains in proteomics
Authors: Miura Kenji
Published: July 2003

Laser‐based scanners and charge‐coupled device (CCD) camera systems are evolving to have greater functional capabilities for capturing images from a range of staining technologies used in gel electrophoresis and electroblotting. Digitizing Coomassie Brilliant Blue (CBB) stained gels and silver stained gels has now become possible using a laser‐based gel scanner, the FLA‐5000 fluorescent image analyzer system. Also, a simultaneous dual fluorescent imaging function has been incorporated into the FLA‐5000 system, utilizing dichroic mirrors with both the optical system and the emission filter. In the workflow of routine proteomics research, the relationship between SYPRO dye staining and fluorescent detection using the FLA‐5000 system have become symbiotic. Additionally in many cases, subsequent staining of the gel with CBB is useful for future research, and thus imaging instruments should be able to handle both staining formats. Digitizing the CBB stained gel can now be easily performed by the FLA‐5000 fluorescent image analyzer system using a fluorescent board as an epi‐illumination background. A cooled CCD camera system has the potential of imaging not only chemiluminescent membranes but also digitizing molecular weight markers and fluorescent detection of SYPRO dye‐stained gels. With Multi Gauge software version 2.0 it is now a simple task to combine two images into one, as commonly required in dual detection experiments. The LAS‐3000 system was designed to capture chemiluminescent images and to digitize the images automatically. Thus, new capabilities added to gel imaging systems make them capable of detecting and displaying multiple signals more conveniently.
High‐content proteomics: Fluorescence multiplexing using an integrated, high‐sensitivity, multiwavelength charge‐coupled device imaging system
Authors: Lopez Mary F. , Mikulskis Alvydas , Golenko Eva , Herick Klaus , Spibey Carole A. , Taylor Ian , Bobrow Mark , Jackson Peter
Published: July 2003

The detection of proteins in 2‐D gels and their subsequent identification by MS is still the “gold standard” in proteomics. Fluorescent detection has increasingly replaced colorimetric and radiometric detection on gels and blots. The reasons for this are multiple and varied and include higher sensitivity, better quantitation, increased dynamic range, speed, safety and ease of use. Unlike other methods, fluorescent protein detection is also typically very consistent in response from protein to protein and in many cases is compatible with MS methods for protein identification. The superior sensitivity and benefits achieved by fluorescent techniques have spurred the development of instrumentation capable of delivering precise, sensitive, high‐resolution image acquisition over a wide variety of excitation and emission wavelengths. This report focuses on applications using the highly sensitive, charge‐coupled device based ProXPRESS™ multilabel imager, readily configurable for image acquisition over a wide variety of wavelengths (380–700 nm and ultraviolet (UV)) using xenon lamp or UV excitation. The ability to simultaneously detect enzyme activities or protein modifications with different color fluorescent probes in addition to total protein amounts (multiplexing) allows the further mining of proteomic data content from a single set of protein samples. To this end, the development of instrumentation that enables a multiplexing strategy will become central to in‐depth proteomic studies. The ProXPRESS maximizes the efficiency of experimental strategies that require flexibility and multicolor fluorescence detection.
Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations
Authors: Champion Kathleen M. , Nishihara Julie C. , Aldor Ilana S. , Moreno G. Tony , Andersen Dana , Stults Kathy L. , Vanderlaan Martin
Published: July 2003

Two‐dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin™) in fermentations were conducted. The resulting two‐dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH‐producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.
Inhibitor affinity chromatography: Profiling the specific reactivity of the proteome with immobilized molecules
Authors: Lolli Graziano , Thaler Florian , Valsasina Barbara , Roletto Fulvia , Knapp Stefan , Uggeri Mauro , Bachi Angela , Matafora Vittoria , Storici Paola , Stewart Albert , Kalisz Henryk M. , Isacchi Antonella
Published: July 2003

An inhibitor affinity chromatography (IAC) method has been developed for the analysis of inhibitor‐protein interactions as a complementary approach to two‐dimensional electrophoresis for functional proteomics studies. The procedure was developed utilizing a cyclin‐dependent kinase 2 (Cdk2) inhibitor coupled to a polymeric resin and validated using a number of proteins interacting with the inhibitor with different specificities. Cdk2 and the other kinases bound and eluted from the resin in accordance with the relative in vitro potency of the inhibitor for each enzyme. Molecular interactions with the Cdk2 inhibitor were compared for HCT116 cancer cells versus rat pancreatic acinar cells. Proteins interacting with the ligand on the IAC matrix were identified by mass spectrometry. Isothermal calorimetry was used to confirm and quantitatively evaluate the binding affinity of some of the interacting proteins. Heat‐shock protein (Hsp) 70 and Hsp27 were the strongest interactors with the inhibitor, displaying binding affinities comparable to those of Cdk2. These results support the use of IAC as a general method for the rapid identification and qualitative evaluation of the in vivo targets and potential side effects of a given drug.
Proteomic analysis of rat heart in ischemia and ischemia‐reperfusion using fluorescence two‐dimensional difference gel electrophoresis
Authors: Sakai Jun , Ishikawa Hironori , Kojima Shinichi , Satoh Hideshi , Yamamoto Setsuko , Kanaoka Masaharu
Published: July 2003

Ischemia‐reperfusion injury is a major complication occurring in acute myocardial infarction, cardiopulmonary bypass surgery, and heart transplantation. The aim of this study was to identify proteins that were involved in ischemia‐reperfusion injury using fluorescence two‐dimensional difference gel electrophoresis. We compared the 100 000×g precipitate fractions of normal, ischemic and ischemia‐reperfused rat hearts and detected six spots which changed more than two‐fold in expression level and two additional spots related to these spots. Using peptide mass fingerprinting by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry, we identified five of these spots as protein disulfide isomerase A3 (PDA3), one as 60 kDa heat shock protein (HSP60) and two as elongation factor Tu (EF‐Tu). HSP60 was increased during ischemia and decreased to normal expression level after reperfusion. EF‐Tu was increased in ischemia but not decreased by reperfusion. We also found that several protein spots of PDA3 shifted towards a higher isoelectric point in ischemia and ischemia‐reperfusion. Our data strongly suggested that PDA3 underwent dephosphorylation during ischemia and reperfusion and serine 343 of PDA3 was one of the phosphorylation sites.
Using standard positions and image fusion to create proteome maps from collections of two‐dimensional gel electrophoresis images
Authors: Luhn Sven , Berth Matthias , Hecker Michael , Bernhardt Jörg
Published: July 2003

Databases for two‐dimensional protein gels pose new challenges in extracting meaningful information from large numbers of experiments. In order to create expression profiles, positions of corresponding protein spots across all gel images have to be established. In larger gel sets errors may accumulate rapidly during this spot matching process, effectively limiting the number of samples available for data mining. Here we present a novel approach for organizing spot data based on the concept of a standard position for a protein species. Standard positions are meaningful average positions that are determined using all occurrences of a protein species. They can be extended to spots that are not annotated via interpolation. The standard position of a spot can serve as a unifying index across all gels in a database, thus allowing creation and analysis of expression profiles that span the whole collection. The standard position gives a much more accurate estimation of a spot's position on a gel than can be obtained using theoretical isoelectric point and molecular weight. Positional indexing is a complement to a priori identifications (e.g. by mass spectrometry or Edman degradation). Moreover it can be used in advance to select spots that are worth identifying because they show relevant expression profiles. Furthermore, we show how to combine all spots that occur on any of the gels into one synthetic but nevertheless realistic‐looking image. This composite image is produced such that all spots have their standard positions. It can serve as a proteome reference map for an organism. As an application, we have computed a reference map from 23 gel images of Bacillus subtilis, using an enhanced prerelease version of the gel analysis software Delta2D (DECODON, Greifswald, Germany).
Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology
Authors: Steinberg Thomas H. , Agnew Brian J. , Gee Kyle R. , Leung Wai‐Yee , Goodman Terrie , Schulenberg Birte , Hendrickson Jill , Beechem Joseph M. , Haugland Richard P. , Patton Wayne F.
Published: July 2003

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post‐genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel‐separated phosphoproteins from complex samples. The newly developed Pro‐Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine‐, phosphothreonine‐, and phosphotyrosine‐containing proteins directly in sodium dodecyl sulfate (SDS)‐polyacrylamide gels and two‐dimensional (2‐D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro‐Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry. Pro‐Q Diamond dye technology can detect as little as 1–2 ng of β‐casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro‐Q Diamond phosphoprotein stain with SYPRO® Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2‐D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post‐translational modification patterns within a single 2‐D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500–1000‐fold concentration range.
Redox proteomics: Identification of oxidatively modified proteins
Authors: Ghezzi Pietro , Bonetto Valentina
Published: July 2003

Reactive oxygen and nitrogen species may cause various types of chemical modifications on specific proteins, Such modifications if irreversible are often associated with permanent loss of function and may lead to the elimination or to the accumulation of the damaged proteins. Reversible modifications, particularly at the cysteine residues, may have a dual role of protection from cysteine irreversible oxidation and modulation of protein function (redox regulation). Here we will review the techniques available for identifying proteins based on their redox state. In particular, we will focus on protein carbonylation, tyrosine nitration and thiol‐disulfide chemistry of cysteines, with special emphasis on glutathionylation, because these are the fields where the tools of proteome analysis have been applied.
Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells
Authors: Fratelli Maddalena , Demol Hans , Puype Magda , Casagrande Simona , Villa Pia , Eberini Ivano , Vandekerckhove Joel , Gianazza Elisabetta , Ghezzi Pietro
Published: July 2003

Protein glutathionylation is a post‐translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L‐[35S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two‐dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.
Multiplex proteomic analysis by two‐dimensional differential in‐gel electrophoresis
Authors: Knowles Michael R. , Cervino Sandra , Skynner Heather A. , Hunt Stephen P. , de Felipe Carmen , Salim Kamran , Meneses‐Lorente Georgina , McAllister George , Guest Paul C.
Published: July 2003

This paper describes the use of fluorescence two‐dimensional differential in‐gel electrophoresis in a multiplex analysis of two distinct proteomes. As a model system, cerebral cortex tissues were analyzed from neurokinin1 receptor knockout (NK1R−/−) and wild type (NK1R+/+) mice in an attempt to identify molecular pathways involved in the function of this protein. Paired NK1R−/− and NK1R+/+ samples were labeled with fluorescent Cy3 and Cy5 dyes and electrophoresed on the same two‐dimensional gels. Scanning the gels at wavelengths specific for each dye revealed the two different proteomes which were overlaid and the differences in abundance of specific protein spots were determined by the Amersham Biosciences DeCyder Differential In‐gel Analysis software. A Cy2‐labeled sample pool was co‐electrophoresed with all Cy3‐ and Cy5‐labeled sample pairs as an internal standard providing a link for inter‐gel comparisons and for more robust statistical analysis of the data. Eight spots were found to be upregulated and two downregulated in the NK1R−/− mice compared to NK1R+/+ controls. Matrix assisted laser desorption/ionisation‐time of flight (MALDI‐TOF) mass fingerprinting was used to identify the proteins. The results illustrate the power of this multiplex proteomics technology and illustrate how proteomics can be used to understand gene function.
A sensitive two‐color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels
Authors: Jing Debra , Agnew Jakyoung , Patton Wayne F. , Hendrickson Jill , Beechem Joseph M.
Published: July 2003

DNA‐binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein‐nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous 32P‐labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility‐shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR® Green EMSA dye, while proteins are subsequently detected with SYPRO® Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel‐shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein‐nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.
Evaluation of saturation labelling two‐dimensional difference gel electrophoresis fluorescent dyes
Authors: Shaw Joanne , Rowlinson Rachel , Nickson Janice , Stone Tim , Sweet Alison , Williams Karen , Tonge Robert
Published: July 2003

Two‐dimensional difference gel electrophoresis (2‐D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye™ DIGE Cy™3 and Cy5 dyes, which aim to overcome these limitations through saturation‐labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation‐labelled gels for MS analysis. The saturation‐labelling dyes were superior in sensitivity to their minimal‐labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2‐D spot pattern was significantly altered from that of unlabelled or minimal‐labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation‐labelled gels eliminating the need for post‐stained preparative gels.
An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence‐based protein detection technologies
Authors: Schulenberg Birte , Arnold Brad , Patton Wayne F.
Published: July 2003

Unfortunately, conventional large‐format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large‐format two‐dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide‐based matrix has been developed that, upon polymerization, facilitates the handling of one‐dimensional and two‐dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer‐reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high‐molecular‐weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro‐Q Diamond phosphoprotein stain, Pro‐Q Emerald glycoprotein stain and SYPRO® Ruby protein gel stain, respectively.
Two‐dimensional electrophoresis replica blotting: A valuable technique for the immunological and biochemical characterization of single components of complex extracts
Authors: Petersen Arnd
Published: July 2003

The combination of the high resolution electrophoresis (2‐DE) with subsequent transfer onto a protein‐binding membrane (blotting), immunological detection, and/or N‐terminal sequencing is a powerful tool to identify and characterize single components of complex protein mixtures. Direct comparison of protein staining, immunological detection, and biochemical characterization of single protein spots was achieved by the replica blotting technique. The proteins were transferred from one two‐dimensional gel onto several blotting membranes one after another. A canon of methods has been employed to identify and characterize allergens from different allergen sources. We have studied single major allergens as well as related major allergens from different grass species (“allergen groups”) using patients’ sera and allergen‐specific monoclonal antibodies. The biochemical structure of the allergenic components has been analyzed by N‐terminal and internal protein sequencing, precise mass determinations by matrix‐assisted laser desorption/ionization mass spectrometry and investigations on post‐translational modifications such as glycosylation. Here, we give a general survey of methods, and we describe an array of techniques suitable for characterization and identification of components of complex extracts, even if there is little or no previous information available.
Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine‐reactive dye in combination with alkaline phosphatase‐ and horseradish peroxidase‐antibody conjugates
Authors: Martin Karen , Hart Courtenay , Liu Jixiang , Leung Wai‐Yee , Patton Wayne F.
Published: July 2003

Three‐color fluorescence detection methods are described based upon covalently coupling the dye 2‐methoxy‐2,4‐diphenyl‐2(2H)‐furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline‐phosphatase‐conjugated reporter molecules in combination with the fluorogenic substrate 9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one‐7‐yl) phosphate (DDAO‐phosphate) as well as horseradish peroxidase‐conjugated reporter molecules in combination with the new fluorogenic substrate Amplex® Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi‐illuminators combined with photographic or charge‐coupled device (CCD) cameras, and xenon‐arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser‐based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.
Protein microarrays using liquid phase fractionation of cell lysates
Authors: Yan Fang , Sreekumar Arun , Laxman Bharathi , Chinnaiyan Arul M. , Lubman David M. , Barder Timothy J.
Published: July 2003

We describe an approach in which protein microarrays are produced using a two‐dimensional (2‐D) liquid phase fractionation of cell lysates. The method involves a pI‐based fractionation using chromatofocusing in the first dimension followed by nonporous reversed‐phase high‐performance liquid chromatography (HPLC) of each pI fraction in the second dimension. This allows fractionation of cellular proteins in the liquid phase that could then be arrayed on nitrocellulose slides and used to study humoral response in cancer. Protein microarrays have been used to identify potential serum biomarkers for prostate cancer. It is shown that specific fractions are immunoreactive against prostate cancer serum but not against serum from healthy individuals. These proteins could serve as sero‐diagnostic markers for prostate cancer. Importantly, this method allows for use of post‐translationally modified proteins as baits for detection of humoral response. Proteins eliciting an immune response are identified using the molecular mass and peptide sequence data obtained using mass spectrometric analysis of the liquid fractions. The fractionation of proteins in the liquid phase make this method amenable to automation.
In vitro protein microarrays for detecting protein‐protein interactions: Application of a new method for fluorescence labeling of proteins
Authors: Kawahashi Yuko , Doi Nobuhide , Takashima Hideaki , Tsuda Chizuru , Oishi Yuko , Oyama Rieko , Yonezawa Masato , Miyamoto‐Sato Etsuko , Yanagawa Hiroshi
Published: July 2003

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein‐protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell‐free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore‐puromycin conjugate was incorporated into a protein at the C‐terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein‐protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high‐throughput format and also combined with in vitro display technologies such as the streptavidin‐biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227–230), our method should be useful for large‐scale analysis of protein‐protein interactions.
Quantitative analysis of protein phosphorylation status and protein kinase activity on microarrays using a novel fluorescent phosphorylation sensor dye
Authors: Martin Karen , Steinberg Thomas H. , Cooley Laurie A. , Gee Kyle R. , Beechem Joseph M. , Patton Wayne F.
Published: July 2003

Ultrasensitive detection of minute amounts of phosphorylated proteins and peptides is a key requirement for unraveling many of the most important signal transduction pathways in mammalian systems. Protein microarrays are potentially useful tools for sensitive screening of global protein expression and post‐translational modifications, such as phosphorylation. However, the analysis of signaling pathways has been hampered by a lack of reagents capable of conveniently detecting the targets of protein kinases. Historically, phosphorylation detection methods have relied upon either radioisotopes ((γ‐32P)ATP(γ‐33P)ATP labeling) or phosphoamino acid‐selective antibodies. Both of these methods suffer from relatively well‐known shortcomings. In this study, a small molecule fluorophore phosphosensor technology is described, referred to as Pro‐Q Diamond dye, which is capable of ultrasensitive global detection and quantitation of phosphorylated amino acid residues in peptides and proteins displayed on microarrays. The utility of the fluorescent Pro‐Q Diamond phosphosensor dye technology is demonstrated using phosphoproteins and phosphopeptides as well as with protein kinase reactions performed in miniaturized microarray assay format. Instead of applying a phosphoamino acid‐selective antibody labeled with a fluorescent or enzymatic tag for detection, a small, fluorescent probe is employed as a universal sensor of phosphorylation status. The detection limit for phosphoproteins on a variety of different commercially available protein array substrates was found to be 312–625 fg, depending upon the number of phosphate residues. Characterization of the enzymatic phosphorylation of immobilized peptide targets with Pro‐Q Diamond dye readily permits differentiation between specific and non‐specific peptide labeling at picogram to subpicogram levels of detection sensitivity.
Profiling the progression of cancer: Separation of microsomal proteins in MCF10 breast epithelial cell lines using nonporous chromatophoresis
Authors: O’Neil Kimberly A. , Miller Fred R. , Barder Timothy J. , Lubman David M.
Published: July 2003

The heterogeneity of cellular protein expression has stimulated development of separations targeting smaller groups of related proteins rather than entire proteomes. The following work describes the development of a technique for the characterization of membrane subproteomes from five different breast epithelial cell lines. Intact membrane proteins are separated by hydrophobicity in the first dimension using nonporous reversed‐phase high‐performance liquid chromatography (RP‐HPLC) to generate unique chromatographic profiles. Fractions of eluent are further separated using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to create distinct banding patterns. This hybrid liquid phase/gel phase method circumvents issues of membrane protein precipitation and provides a simple strategy aimed at isolating and characterizing a traditionally underrepresented protein class. Membrane protein profiles are created that discriminate between microsomal fractions of breast epithelial cells in different stages of neoplastic progression. Proteins are subsequently identified using matrix‐assisted laser desorption/ionization – mass spectrometry (MALDI‐MS) mass fingerprinting and MALDI‐quadrupole time of flight – tandem mass spectrometry (QTOF‐MS/MS) peptide sequencing. Furthermore, as this strategy preserves intact protein structure, further characterization can be performed on proteins producing mass fingerprint spectra and fragmentation spectra that did not result in database protein identifications. The coupling of nonporous RP‐HPLC with SDS‐PAGE provides a useful alternative to two‐dimensional PAGE (2‐D‐PAGE) for membrane protein analysis.
High‐throughput functional affinity purification of mannose binding proteins from Oryza sativa
Authors: Andon Nancy L. , Eckert Donna , Yates John R. , Haynes Paul A.
Published: July 2003

We have used affinity chromatography in combination with mass spectrometry to isolate, identify, and assign a preliminary functional annotation to a large number of both known and novel proteins from rice. Rice (Oryza sativa) leaf, root, and seed tissue extracts were fractionated by column affinity chromatography using α‐D‐mannose as the ligand. Bound fractions were eluted and subjected to one‐dimensional electrophoresis, followed by high‐performance liquid chromatography‐tandem mass spectrometric analysis of separated proteins. This multiplexed technology resulted in the isolation and identification of 136 distinct mannose binding proteins from rice. A comparative analysis demonstrates very little overlap of identified proteins between the respective tissues, and confirms the correctly compartmentalized presence of a significant number of proteins from largely tissue‐specific biochemical pathways. Over 30% of the identified proteins with a previously annotated function are directly involved in sugar metabolism, including several highly expressed known rice lectins. Direct comparison of the peptide sequences identified in this study to those peptides identified in the most comprehensive survey of the rice proteome to date indicates that our current data represents a significant enrichment of proteins unique to this dataset. Nearly 15% of the identified proteins, identified on the basis of exact peptide matching to sequences in the rice genomic database, represent proteins without a previously known functional annotation, indicating the potential of this combined chromatographic approach to assign a preliminary function to novel proteins in a high‐throughput fashion.
Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results
Authors: Masselon Christophe , Paša‐Tolić Ljiljana , Lee Sang‐Won , Li Lingjun , Anderson Gordon A. , Harkewicz Richard , Smith Richard D.
Published: July 2003

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three “y” or “b” fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform‐ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.
The human serum proteome: Display of nearly 3700 chromatographically separated protein spots on two‐dimensional electrophoresis gels and identification of 325 distinct proteins
Authors: Pieper Rembert , Gatlin Christine L. , Makusky Anthony J. , Russo Paul S. , Schatz Courtney R. , Miller Stanton S. , Su Qin , McGrath Andrew M. , Estock Marla A. , Parmar Prashanth P. , Zhao Ming , Huang Shih‐Ting , Zhou Jeff , Wang Fang , Esquer‐Blasco Ricardo , Anderson N. Leigh , Taylor John , Steiner Sandra
Published: July 2003

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme‐linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two‐dimensional electrophoresis (2‐DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion‐exchange and size‐exclusion chromatography. Serum proteins from 74 fractions were displayed on 2‐DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post‐translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brillant Blue G‐250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin‐6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2‐DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.
An efficient solubilization buffer for plant proteins focused in immobilized pH gradients
Authors: Méchin Valérie , Consoli Luciano , Le Guilloux Martine , Damerval Catherine
Published: July 2003

The solubilization of a large array of proteins before electrophoresis itself is a very critical point for proteomic analyses. We compared the efficiency of several different solubilization buffers. From this work, we defined a very efficient solubilization buffer, including two chaotropes, two reducing agents (R2), two detergents (D2), and two kinds of carrier ampholytes in combination. This so‐called R2D2 buffer (5 M urea, 2 M thiourea, 2% 3‐[(3‐cholamidopropyl) dimethyl‐ammonio]‐1‐propane‐sulfonate, 2% N‐decyl‐N,N‐dimethyl‐3‐ammonio‐1‐propane‐sulfonate, 20 mM dithiothreitol, 5 mM Tris(2‐carboxyethyl) phosphine, 0.5% carrier ampholytes 4–6.5, 0.25% carrier ampholytes 3‐10) proved to be very efficient for a large range of different samples and allowed us to obtain two‐dimensional gels of high resolution and quality.
Enhanced resolution of glycosylphosphatidylinositol‐anchored and transmembrane proteins from the lipid‐rich myelin membrane by two‐dimensional gel electrophoresis
Authors: Taylor Christopher M. , Pfeiffer Steven E.
Published: July 2003

Two‐dimensional gel electrophoresis (2‐DE) has become a powerful and widely used technique for proteomic analyses. However, the limited ability of 2‐DE to resolve transmembrane and glycosylphosphatidylinositol (GPI)‐anchored proteins has slowed the identification of proteins from membrane‐rich biological samples. Myelin is an unusually lipid‐rich membrane with relatively few major proteins but many quantitatively minor proteins, most of which have an unknown identity and/or function. The goal of this study was to identify the optimal conditions of 2‐DE for the separation of myelin proteins. We have identified two detergents, the nonionic n‐dodecyl β‐D‐maltoside and the zwitterionic amidosulfobetaine ASB‐14, that are more effective in solubilizing myelin proteins than the commonly used zwitterionic detergent 3‐[(3‐cholamidopropyl)‐ dimethylammonio]‐1‐propanesulfonate (CHAPS). These detergents significantly enhance the solubility of both transmembrane (e.g., the highly hydrophobic and multiply acylated myelin proteolipid protein) and GPI‐anchored (e.g., contactin and neuronal cell adhesion molecule) myelin proteins and enable their resolution by 2‐DE. We conclude that these detergents are effective tools for the 2‐DE analysis of myelin, and that they may be more generally useful for the analysis of membrane‐rich biological samples.
Unmatched masses in peptide mass fingerprints caused by cross‐contamination: An updated statistical result
Authors: Ding Qinxue , Xiao Lin , Xiong Shaoxiang , Jia Yufeng , Que Haiping , Guo Yaojun , Liu Shaojun
Published: July 2003

Unmatched masses are often observed in the experimental peptide mass spectra when database searching is performed with the ProFound program. Comparison between theoretical and experimental mass spectra of standard proteins shows that contamination accounts for most of the unmatched masses. In this retrospective analysis, the top 100 most probable contaminating masses, as listed in order of their probability, are statistically filtered out from 118 different experimental peptide mass fingerprinting (PMF) maps. Most of the interfering masses originate from trypsin autolysis and human keratins. Subtraction of known contaminants from raw data and using cleaner masses for searching can enhance protein identification by PMF.
Innate differences in protein expression in the nucleus accumbens and hippocampus of inbred alcohol‐preferring and ‐nonpreferring rats
Authors: Witzmann Frank A. , Li Junyu , Strother Wendy N. , McBride William J. , Hunter Lawrence , Crabb David W. , Lumeng Lawrence , Li Ting‐Kai
Published: July 2003

Two‐dimensional gel electrophoresis (2‐DE) was used to separate protein samples solubilized from the nucleus accumbens and hippocampus of alcohol‐naïve, adult, male inbred alcohol‐preferring (iP) and alcohol‐nonpreferring (iNP) rats. Several protein spots were excised from the gel, destained, digested with trypsin, and analyzed by mass spectrometry. In the hippocampus, 1629 protein spots were matched to the reference pattern, and in the nucleus accumbens, 1390 protein spots were matched. Approximately 70 proteins were identified in both regions. In the hippocampus, only 8 of the 1629 matched protein spots differed in abundance between the iP and iNP rats. In the nucleus accumbens, 32 of the 1390 matched protein spots differed in abundance between the iP and iNP rats. In the hippocampus, the abundances of all 8 proteins were higher in the iNP than iP rat. In the nucleus accumbens, the abundances of 31 of 32 proteins were higher in the iNP than iP rat. In the hippocampus, only 2 of the 8 proteins that differed could be identified, whereas in the nucleus accumbens 21 of the 32 proteins that differed were identified. Higher abundances of cellular retinoic acid‐binding protein 1 and a calmodulin‐dependent protein kinase (both of which are involved in cellular signaling pathways) were found in both regions of the iNP than iP rat. In the nucleus accumbens, additional differences in the abundances of proteins involved in (i) metabolism (e.g., calpain, parkin, glucokinase, apolipoprotein E, sorbitol dehydrogenase), (ii) cyto‐skeletal and intracellular protein transport (e.g., β‐actin), (iii) molecular chaperoning (e.g., grp 78, hsc70, hsc 60, grp75, prohibitin), (iv) cellular signaling pathways (e.g., protein kinase C‐binding protein), (v) synaptic function (e.g., complexin I, γ‐enolase, syndapin IIbb), (vi) reduction of oxidative stress (thioredoxin peroxidase), and (vii) growth and differentiation (hippocampal cholinergic neurostimulating peptide) were found. The results of this study indicate that selective breeding for disparate alcohol drinking behaviors produced innate alterations in the expression of several proteins that could influence neuronal function within the nucleus accumbens and hippocampus.