Proteomics. Searching for recipes for protein chips.
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Antibodies↗
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Results for “proteome mapping”
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Proteomics methods for probing molecular mechanisms in signal transduction.
mRNA splicing and various posttranslational modifications to proteins result in a larger number of proteins than genes. Assessing the dynamic nature of this proteome is the challenge of modern proteomics. Recent advances in high throughput methods greatly facilitate the analysis of proteins involved in signal transduction, their production, posttranslational modifications and interactions. Highly reproducible two dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods, coupled with matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS) allow rapid separation and identification of proteins. These methods, alone or in conjunction with other techniques such as immunoprecipitation, allow identification of various critical posttranslational modifications, such as phosphorylation. High throughput identification of important protein-protein interactions is accomplished by yeast two hybrid approaches. In vitro and in vivo pulldown assays, coupled with MALDI-TOF-MS, provide an important alternative to two hybrid approaches. Emerging advances in production of protein-based arrays promise to further increase throughput of proteomics-based approaches to signal transduction.
Animals↗
Protein identification with Teflon as matrix-assisted laser desorption/ionization sample support.
Protein identification is a critical step in proteomics, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) plays an important role in that identification. Polytetrafluoroethylene (Teflon) was tested as a new MALDI sample support to improve protein identification. The tryptic peptides obtained from a model protein were bound to the surface of a modified MALDI sample holder via the hydrophobic interactions that occur between the Teflon surface and the peptide ion-pairs, and the affinity of alpha-cyano-4-hydroxycinnamic acid for the peptides. During that surface-binding step, the peptide mixture was also desalted and concentrated. A greater number of matched peptides and a larger sequence coverage were obtained for the proteins when Teflon was used as the sample support compared with conventional sample preparation methods and a stainless-steel surface. In addition, the characterization of a small amount of protein was improved with Teflon. Nine silver-stained protein spots obtained from 2-D gel of a human cerebrospinal fluid (CSF) proteome were identified by this method. Among the nine protein spots, peptide 6:c3c fragment and procollagen c-proteinase enhancer were not annotated in any published 2-D map of human CSF. A Teflon MALDI sample support is a low-cost, simple, and effective method that can be used to improve the quality of the MALDI mass spectrum of a complex tryptic peptide mixture, and to achieve a higher level of reliability and success in protein identification.
Animals↗
Data requirements for protein identification using chemically-assisted fragmentation and tandem mass spectrometry.
Many laboratories identify proteins by searching tandem mass spectrometry data against genomic or protein sequence databases. These database searches typically use the measured peptide masses or the derived peptide sequence and, in this paper, we focus on the latter. We study the minimum peptide sequence data requirements for definitive protein identification from protein sequence databases. Accurate mass measurements are not needed for definitive protein identification, even when a limited amount of sequence data is available for searching. This information has implications for the mass spectrometry performance (and cost), data base search strategies and proteomics research.
Mass Spectrometry↗
Proteomics strategies in cardiovascular research.
Cardiovascular research of the past decades dealt with classical pathophysiological descriptions, then shifted toward the identification of relevant receptors, and then proceeded to the analysis of signal transduction pathways. Most recently, hand in hand with the achievements of the human genome project, the research has gone down the road toward molecular biological "disease gene(s) mapping". The application of proteome research will attempt to close the gap between genomic (and genetic) analysis and the physiological research. The rich source of heart surgery specimens represents an excellent starting point in data acquisition of proteomic context. Furthermore, animal models of cardiovascular diseases and deficiencies are considered, and will be explored. Examples of results from feasibility studies are given, with the emphasis on quantitative evaluation of proteomic components, hoping to discover co-regulated sets of proteins that are involved in any particular disease state. Identification of new, not yet discovered proteins will be pursued, though the emphasis of this work will be on the definition of characteristic sets of expressed proteins, which in turn might be able to delimit the state of disease and prognosis of therapy outcome. Besides the systematic issues, this paper refers to a number of methodological questions, like the comparison of the proteins detected by staining procedures and proteins detected in models in which biosynthetic labeling is applicable.
Animals↗
Using cross-correlation normalized for peptide length to optimize peptide identification in shotgun proteomics.
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Algorithms↗
High-throughput localization of organelle proteins by mass spectrometry: a quantum leap for cell biology.
Cells are the fundamental building blocks of organisms and their organization holds the key to our understanding of the processes that control Development and Physiology as well as the mechanisms that underlie disease. Traditional methods of analysis of subcellular structure have relied on the purification of organelles and the painstaking biochemical description of their components. The arrival of high-throughput genomic and, more significantly, proteomic technologies has opened hereto unforeseen possibilities for this task. Recently two reports((1,2)) show how much can be gleaned from the combination of analytical centrifugation, mass spectrometry and advanced statistical techniques focused on a high-throughput analysis of the content and organization of plant and animal cells. The results reveal intriguing possibilities for the future and the possibility of mapping much of the known proteome onto our current map of the cell.
Mass Spectrometry↗
Mapping glycosylation changes related to cancer using the Multiplexed Proteomics technology: a protein differential display approach.
The metastatic spread of tumor cells in malignant progression is known to be a major cause of cancer mortality. Protein glycosylation is increasingly being recognized as one of the most prominent biochemical alterations associated with malignant transformation and tumorigenesis. The Multiplexed Proteomics (MP) approach is a new technology that permits quantitative, multicolor fluorescence detection of proteins in two-dimensional (2-D) gels and on Western blots. This methodology allows the parallel determination of both altered glycosylation patterns and protein expression level changes within a single 2-D gel experiment. The linear responses of the fluorescent dyes utilized allow rigorous quantitation of changes in protein expression over a broad 3-log linear dynamic range. Global analysis of changes in protein glycosylation and total protein expression is followed by dichromatic, lectin-based profiling methods for rapidly categorizing glycan branching structures. The MP approach was applied to whole tissue extracts of normal and cancerous liver, so that altered glycosylation modification patterns and protein expression levels could be determined. One prominent glycoprotein determined to be up-regulated in the tumor tissue was haptoglobin, an acute-phase response protein. The detection methodologies associated with the MP technology radically increase the information content of 2-D gel experiments. This new information greatly enhances the applicability of these experiments in addressing fundamental questions associated with proteome-wide glycosylation changes related to cancer.
Electrophoresis, Gel, Two-Dimensional↗
Proteomics for protein expression profiling in neuroscience.
As the technology of proteomics moves from a theoretical approach to a practical reality, neuroscientists will have to determine the most appropriate applications for this technology. Neuroscientists will have to surmount difficulties particular to their research, such as limited sample amounts, heterogeneous cellular compositions in samples, and the fact that many proteins of interest are rare, hydrophobic proteins. This review examines protein isolation and protein fractionation and separation using two-dimensional electrophoresis (2-DE) and mass spectrometry proteomic methods. Methods for quantifying relative protein expression between samples (e.g., 2-DIGE, and ICAT) are also described. The coverage of the proteome, ability to detect membrane proteins, resource requirements, and quantitative reliability of different approaches is also discussed. Although there are many challenges in proteomic neuroscience, this field promises many rewards in the future.
Animals↗
Urinary proteomics of renal Fanconi syndrome.
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Chromatography, Gas↗
A Draft Map of E. coli Proteoforms.
Top-down proteomics (TDP) enables direct characterization of intact proteoforms, providing protein-level insights into molecular diversity arising from post-translational modifications and sequence variations. Despite this advantage, proteome coverage in TDP remains limited relative to bottom-up proteomics (BUP). To expand coverage, we developed an integrated multidimensional approach combining sequential protein extraction, size-exclusion chromatography (SEC) fractionation, and capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS. This approach identified 743 proteoform families and 10,613 proteoforms from E. coli cells through hundreds of MS runs. By incorporating previous E. coli TDP data sets from our group, we identified 14,932 proteoforms from 985 proteoform families, covering 43% of the E. coli proteome. The data represent the highest proteome coverage of cells by MS-based TDP, creating a draft map of E. coli proteoforms. The results offer strong evidence that MS-based TDP can reach high proteome coverage.
Escherichia coli↗
Proteomic analysis reveals novel protein targets of S-nitrosylation in mesangial cells.
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Biotinylation↗
Proteome analysis of Rickettsia conorii by two-dimensional gel electrophoresis coupled with mass spectrometry.
The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.
Animals↗
Mapping normal and cancer cell signalling networks: towards single-cell proteomics.
Oncogenesis and tumour progression are supported by alterations in cell signalling. Using flow cytometry, it is now possible to track and analyse signalling events in individual cancer cells. Data from this type of analysis can be used to create a network map of signalling in each cell and to link specific signalling profiles with clinical outcomes. This form of 'single-cell proteomics' can identify pathways that are activated in therapy-resistant cells and can provide biomarkers for cancer diagnosis and for determining patient prognosis.
Biomarkers, Tumor↗
A catalogue of human saliva proteins identified by free flow electrophoresis-based peptide separation and tandem mass spectrometry.
Human saliva has great potential for clinical disease diagnostics. Constructing a comprehensive catalogue of saliva proteins using proteomic approaches is a necessary first step to identifying potential protein biomarkers of disease. However, because of the challenge presented in cataloguing saliva proteins with widely varying abundance, new proteomic approaches are needed. To this end, we used a newly developed approach coupling peptide separation using free flow electrophoresis with linear ion trap tandem mass spectrometry to identify proteins in whole human saliva. We identified 437 proteins with high confidence (false positive rate below 1%), producing the largest catalogue of proteins from a single saliva sample to date and providing new information on the composition and potential diagnostic utility of this fluid. The statistically validated, transparently presented, and annotated dataset provides a model for presenting large scale proteomic data of this type, which should facilitate better dissemination and easier comparisons of proteomic datasets from future studies in saliva.
Electrophoresis↗
ProteomeCommons.org JAF: reference information and tools for proteomics.
SUMMARY: Analysis of proteomics data, specifically mass spectrometry data, commonly relies on libraries of known information such as atomic masses, known stable isotopes, atomic compositions of amino acids, observed modifications of known amino acids and ion masses that directly correspond to known amino acid sequences. The Java Analysis Framework (JAF) for proteomics provides a freely usable, open-source library of Java code that abstracts all of the aforementioned data, enabling more rapid development of proteomics tools. The JAF also includes several user tools that can be run directly from a web browser. AVAILABILITY: The current version and an archive of all older versions of the Java Analysis Framework for Proteomics is freely available, including complete source-code, at http://www.proteomecommons.org/current/511/.
Database Management Systems↗
Functional and quantitative proteomics using SILAC.
Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.
Amino Acids↗
Identification of chromosomally encoded membranal polypeptides of Bacillus anthracis by a proteomic analysis: prevalence of proteins containing S-layer homology domains.
Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.
Amino Acid Sequence↗