ABSTRACT
Spliced peptides are short protein fragments spliced together in the proteasome by peptide bond formation. True estimation of the contribution of proteasome-spliced peptides (PSPs) to the global human leukocyte antigen (HLA) ligandome is critical. A recent study suggested that PSPs contribute up to 30% of the HLA ligandome. We performed a thorough reanalysis of the reported results using multiple computational tools and various validation steps and concluded that only a fraction of the proposed PSPs passes the quality filters. To better estimate the actual number of PSPs, we present an alternative workflow. We performed de novo sequencing of the HLA-peptide spectra and discarded all de novo sequences found in the UniProt database. We checked whether the remaining de novo sequences could match spliced peptides from human proteins. The spliced sequences were appended to the UniProt fasta file, which was searched by two search tools at a false discovery rate (FDR) of 1%. We find that 2-6% of the HLA ligandome could be explained as spliced protein fragments. The majority of these potential PSPs have good peptide-spectrum match properties and are predicted to bind the respective HLA molecules. However, it remains to be shown how many of these potential PSPs actually originate from proteasomal splicing events.
Subject(s)
Computational Biology/methods , HLA Antigens/metabolism , Peptides/genetics , Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Splicing , Antigen Presentation/physiology , Cell Line, Tumor , Exome , Humans , Ligands , Protein Binding , Protein Interaction Domains and Motifs , Proteome , Signal Transduction , Tandem Mass Spectrometry , Exome SequencingABSTRACT
The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P(1) position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.
Subject(s)
ATP-Binding Cassette Transporters/deficiency , HLA-A Antigens/immunology , HLA-B Antigens/immunology , HLA-C Antigens/immunology , Hybridomas/immunology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/immunology , Antigen Presentation , Cell Line, Tumor , Cytosol/immunology , Cytosol/metabolism , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Genetic Variation/immunology , HLA-A Antigens/genetics , HLA-A Antigens/metabolism , HLA-B Antigens/genetics , HLA-B Antigens/metabolism , HLA-C Antigens/genetics , HLA-C Antigens/metabolism , Humans , Hybridomas/cytology , Hybridomas/metabolism , Ligands , Mass Spectrometry , Peptides/chemical synthesis , Peptides/immunology , ProteolysisABSTRACT
The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC). The inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.
Subject(s)
Boronic Acids/pharmacology , Histocompatibility Antigens Class I/metabolism , Peptides/metabolism , Proteasome Inhibitors/pharmacology , Pyrazines/pharmacology , Bortezomib , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , MCF-7 Cells , Oligopeptides/pharmacology , ProteomeABSTRACT
The presentation of short viral peptide antigens by human leukocyte antigen (HLA) class I molecules on cell surfaces is a key step in the activation of cytotoxic T lymphocytes, which mediate the killing of pathogen-infected cells or initiate autoimmune tissue damage. HLA-B27 is a well known class I molecule that is used to study both facets of the cellular immune response. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HLA-B*2705(+) cells, we identified 200 naturally processed HLA-B*2705 ligands. Our analyses revealed that a change in the position (P) 2 anchor motif was detected in the 3% of HLA-B*2705 ligands identified. B*2705 class I molecules were able to bind these six GlnP2 peptides, which showed significant homology to pathogenic bacterial sequences, with a broad range of affinities. One of these ligands was able to bind with distinct conformations to HLA-B27 subtypes differentially associated with ankylosing spondylitis. These conformational differences could be sufficient to initiate autoimmune damage in patients with ankylosing spondylitis-associated subtypes. Therefore, these kinds of peptides (short, with GlnP2, and similar low affinity to all HLA-B27 subtypes tested but with unlike conformations in differentially ankylosing spondylitis-associated subtypes) must not be excluded from future researches involving potential arthritogenic peptides.
Subject(s)
HLA-B27 Antigen/immunology , Peptides/immunology , Spondylarthropathies/immunology , Amino Acid Motifs , Cell Line , HLA-B27 Antigen/genetics , HLA-B27 Antigen/metabolism , Humans , Peptides/genetics , Peptides/metabolism , Protein Binding/genetics , Protein Binding/immunology , Spondylarthropathies/genetics , Spondylarthropathies/metabolism , Spondylarthropathies/pathologyABSTRACT
The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.
Subject(s)
ATP-Binding Cassette Transporters/immunology , Antigen Presentation , Antigens, Viral/immunology , CD8-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class I/immunology , Membrane Proteins/immunology , Vaccinia virus/immunology , Vaccinia/immunology , Viral Structural Proteins/immunology , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Motifs , Antigens, Viral/genetics , Antigens, Viral/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Line , HLA-C Antigens/genetics , HLA-C Antigens/immunology , HLA-C Antigens/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Peptides/genetics , Peptides/immunology , Peptides/metabolism , Vaccinia/genetics , Vaccinia/metabolism , Vaccinia virus/genetics , Vaccinia virus/metabolism , Viral Structural Proteins/genetics , Viral Structural Proteins/metabolism , HLA-E AntigensABSTRACT
The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.
Subject(s)
ATP-Binding Cassette Transporters/immunology , Histocompatibility Antigens Class I/immunology , Vaccinia virus/immunology , Vaccinia/immunology , ATP-Binding Cassette Transporters/genetics , Animals , Antigen Presentation , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/virology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Cell Line , Histocompatibility Antigens Class I/genetics , Humans , Ligands , Mice , Mice, Knockout , Vaccinia/genetics , Vaccinia/virology , Vaccinia virus/geneticsABSTRACT
Individuals with nonfunctional transporters associated with antigen processing (TAP) complexes are not particularly susceptible to viral infections or neoplasms. Therefore, their immune system must be reasonably efficient, and the present, though reduced, cytolytic CD8 αß T subpopulation specific for TAP-independent antigens may be sufficient to establish an immune defense protecting against viral infections in these individuals. The objective of the present study was to identify TAP-independent ligands from HIV gp160 protein. An analysis and comparison of complex human histocompatibility complex (HLA)-bound peptide pools isolated from large quantities of healthy or HIV gp160-expressing human cells was performed using mass spectrometry and bioinformatics tools. A conserved TAP-independent HLA peptide ligand endogenously processed and presented in infected human cells was identified. This ligand originates from the envelope protein bound to the HLA-Cw1 class I molecule with high affinity. It was concluded that HLA class I peptides derived from a large fraction of the N-terminal HIV envelope protein could be presented even in the absence of the TAP complex.
Subject(s)
ATP-Binding Cassette Transporters/immunology , Lymphocyte Activation/immunology , Viral Proteins/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 3 , HLA-C Antigens/immunology , Humans , Major Histocompatibility Complex , Protein BindingABSTRACT
The HLA molecules are membrane-bound transporters that carry peptides from the cytoplasm to the cell surface for surveillance by circulating T lymphocytes. Although low levels of soluble HLA molecules (sHLA) are normally released into the blood, many types of tumor cells release larger amounts of these sHLA molecules, presumably to counter immune surveillance by T cells. Here we demonstrate that these sHLA molecules are still bound with their authentic peptide repertoires, similar to those of the membranal HLA molecules (mHLA). Therefore, a single immunoaffinity purification of the plasma sHLA molecules, starting with a few milliliters of patients' blood, allows for identification of very large sHLA peptidomes by mass spectrometry, forming a foundation for development of a simple and universal blood-based cancer diagnosis. The new methodology was validated using plasma and tumor cells of multiple-myeloma and leukemia patients, plasma of healthy controls, and with cultured cancer cells. The analyses identified thousands of sHLA peptides, including some cancer-related peptides, present among the sHLA peptidomes of the cancer patients. Furthermore, because the HLA peptides are the degradation products of the cellular proteins, this sHLA peptidomics approach opens the way for investigation of the patterns of protein synthesis and degradation within the tumor cells.
Subject(s)
Antigens, Neoplasm , Biomarkers, Tumor , Neoplasms , Peptides , Proteome , Animals , Antigens, Neoplasm/blood , Antigens, Neoplasm/immunology , Biomarkers, Tumor/blood , Biomarkers, Tumor/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Neoplasms/blood , Neoplasms/diagnosis , Neoplasms/immunology , Peptides/blood , Peptides/immunology , Proteome/immunology , Proteome/metabolismABSTRACT
OBJECTIVE: The HLA-B27 allele is strongly associated with the group of inflammatory diseases known as the spondylarthritides (SpA). The aim of this study was to perform a large-scale, direct biochemical analysis of the HLA-B*2705 peptidome in order to identify candidates for mimicry between HLA-B27 peptides derived from cartilage proteins and arthritogenic bacterial sequences and to refine the consensus binding motif of this important allele. METHODS: The peptides were recovered by recombinant expression of soluble HLA-B27 molecules secreted from cultured chondrocytic cells or HeLa cells. Analysis was based on capillary chromatography and tandem mass spectrometry in combination with stable isotope labeling with amino acids in cell culture or chemical labeling with iTRAQ to enhance the validity of the data. RESULTS: Over 1,268 B27 peptides were identified, with 569 of them at high certainty, thus enabling better refinement of the B27 motif. This enabled the prediction of both short peptides and long peptides whose middle residues likely bulge out of the binding groove. Moreover, we identified a number of human B27 peptide sequences derived from human cartilage proteins, some of which are similar to common bacterial sequences. CONCLUSION: The peptides we identified may provide the missing link between bacterial infections and the resulting SpA.
Subject(s)
Chondrocytes/physiology , HLA-B27 Antigen , Molecular Mimicry/immunology , Spondylarthritis/immunology , Spondylarthritis/metabolism , Antigens, Bacterial/chemistry , Antigens, Bacterial/immunology , Antigens, Bacterial/metabolism , Bacterial Infections/immunology , Bacterial Infections/metabolism , Cartilage/immunology , Cartilage/metabolism , Cell Line, Transformed , Chondrocytes/cytology , Chondrosarcoma , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , HLA-B27 Antigen/chemistry , HLA-B27 Antigen/immunology , HLA-B27 Antigen/metabolism , HeLa Cells , Humans , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein Structure, Tertiary , Spondylarthritis/microbiology , T-Lymphocytes/immunologyABSTRACT
Cytotoxic T lymphocyte (CTL)-mediated death of virus-infected cells requires prior recognition of short viral peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on the surface of infected cells. The CTL response is critical for the clearance of human respiratory syncytial virus (HRSV) infection. Using mass spectrometry analysis of complex HLA-bound peptide pools isolated from large amounts of HRSV-infected cells, we identified nine naturally processed HLA-B27 ligands. The isolated peptides are derived from six internal, not envelope, proteins of the infective virus. The sequences of most of these ligands are not conserved between different HRSV strains, suggesting a mechanism to explain recurrent infection with virus of different HRSV antigenic subgroups. In addition, these nine ligands represent a significant fraction of the proteome of this virus, which is monitored by the same HLA class I allele. These data have implications for vaccine development as well as for analysis of the CTL response.
Subject(s)
Antigen Presentation/immunology , Antigens, Viral/immunology , Ligands , Respiratory Syncytial Virus Infections/immunology , Respiratory Syncytial Virus, Human/immunology , Amino Acid Sequence , Animals , Antigens, Viral/chemistry , Antigens, Viral/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Humans , Molecular Sequence Data , Peptides/chemistry , Peptides/genetics , Peptides/immunology , Spectrometry, Mass, Electrospray Ionization/methods , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Small cell lung carcinoma (SCLC) is an aggressive, highly metastatic cancer with a strong tendency for chemotherapy resistance. Identification of proteins uniquely expressed in SCLC cells, can facilitate the development of new diagnostic tools, improve immunotherapy, and deepen our understanding of the underlying mechanisms of the disease. Here we describe a comparative proteomics analysis of ten SCLC cell lines and three controls lines, while searching for proteins preferentially expressed in SCLC cells as potential disease markers. Total protein extracts were compared by two-dimensional gel electrophoresis and by two-dimensional liquid chromatography resulting in the identification of 1093 proteins, 202 of which were detected only in the SCLC cells. These include proteins of different cellular functions, including cellular proliferation and known tumor antigens. Since SCLC has a neuroendocrine origin, of major interest are the identified proteins involved in nerve and brain embryonic development. These proteins are potentially valuable as both tumor markers and as antigens for immunotherapy.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Small Cell/metabolism , Lung Neoplasms/metabolism , Neoplasm Proteins/metabolism , Proteomics , Biomarkers, Tumor/analysis , Cell Line, Tumor , Humans , Neoplasm Proteins/analysisABSTRACT
Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.
Subject(s)
Antigens, Neoplasm/metabolism , Histocompatibility Antigens/metabolism , Peptides/metabolism , Amino Acid Sequence , Humans , Kinetics , Molecular Sequence Data , Time FactorsABSTRACT
HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.
Subject(s)
Blood Proteins/chemistry , Databases, Protein , Proteomics/methods , Algorithms , Anticoagulants/pharmacology , Citric Acid/pharmacology , Computational Biology , DNA/chemistry , Edetic Acid/chemistry , Edetic Acid/pharmacology , Heparin/chemistry , Humans , Immunoassay , Mass Spectrometry/methods , Open Reading Frames , Pilot Projects , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The human Plasma Proteome Project (PPP) is a large-scale collaboration between many laboratories. One of the most demanding tasks in the PPP involved the analysis of very large amounts of raw MS/MS data produced by the participants. The main approach for managing this task was letting the participants analyze their own data and submit the results to the central PPP repository as lists of identified proteins and peptides. To complement this distributed approach, we also performed centralized analysis of the raw MS/MS data provided by the participants. Due to the data redundancy inherent in such a project, centralized analysis has the potential to reduce the computational effort by reducing redundancy before the analysis. Centralized analysis can also unify the process and take advantage of data sharing among laboratories to improve protein identification and validation. The process we employed included removing low-quality spectra, clustering spectra by mutual similarity, and applying uniform peptide and protein identification procedures. To demonstrate the process, we analyzed 5.28 million MS/MS spectra derived by eight laboratories from tryptic peptides of serum and plasma proteins.
Subject(s)
Blood Proteins/chemistry , Mass Spectrometry/methods , Proteomics/methods , Statistics as Topic/methods , Cluster Analysis , Computational Biology/methods , Databases, Protein , Feasibility Studies , Humans , Peptides/chemistry , Pilot Projects , Proteins/chemistry , Proteome , Trypsin/pharmacologyABSTRACT
Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using Pep-Miner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.
Subject(s)
Blood Proteins/chemistry , Chromatography/methods , Proteomics/methods , Blood Proteins/isolation & purification , Cations , Chromatography, Ion Exchange , Cluster Analysis , Databases, Protein , Electrophoresis, Polyacrylamide Gel , Humans , Isoelectric Focusing/methods , Mass Spectrometry , Peptides/chemistry , Proteins/chemistryABSTRACT
Tandem mass spectrometry (MS/MS), coupled with liquid chromatography (LC), is a powerful tool for the analysis and comparison of complex protein and peptide mixtures. However, the extremely large amounts of data that result from the process are very complex and difficult to analyze. We show how the clustering of similar spectra from multiple LC-MS/MS runs can help in data management and improve the analysis of complex peptide mixtures. The major effect of spectrum clustering is the reduction of the huge amounts of data to a manageable size. As a result, analysis time is shorter and more data can be stored for further analysis. Furthermore, spectrum quality improvement allows the identification of more peptides with greater confidence, the comparison of complex peptide mixtures is facilitated, and the entire proteomics project is presented in concise form. Pep-Miner is an advanced software tool that implements these clustering-based applications. It proved useful in several comparative proteomics projects involving lung cancer cells and various other cell types. In one of these projects, Pep-Miner reduced 517 000 spectra to 20 900 clusters and identified 2518 peptides derived from 830 proteins. Clustering and identification lasted less than two hours on an IBM Thinkpad T23 computer (laptop). Pep-Miner's unique properties make it a very useful tool for large-scale shotgun proteomics projects.
Subject(s)
Algorithms , Models, Statistical , Proteome , Software , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Chromatography, Liquid , Cluster Analysis , Computational Biology , Peptides/analysis , Proteins/analysisABSTRACT
A large number of HLA-Cw4 (Cw *0402) peptides were purified, sequenced, and identified from breast and ovarian carcinoma cell lines. HLA-Cw4 molecules were expressed in these cells as soluble, secreted HLA (sHLA) and recovered from the growth medium. The peptides were separated by capillary reversed-phase HPLC and analyzed by tandem mass-spectrometry. The resulting peptides fit to some extent, but not completely, the known consensus of the Cw4 peptide-binding motif. Among the identified peptides, there are a few that originate from proteins of possible interest for cancer immunotherapy or diagnostics, including mucin-5B, ART-1, fatty acid synthase, putative prostate cancer tumor suppressor, DNA topoisomerase-1, and Rac1. This work demonstrates that large-scale identification of HLA peptides recovered from sHLA is an advantageous approach for establishing the HLA peptide consensus of different haplotypes and the identification of useful peptides for treatment of diseases such as cancer, viral, and autoimmune diseases.
Subject(s)
HLA-C Antigens/chemistry , Peptides/chemistry , HumansABSTRACT
The Human MHC Project aims at comprehensive cataloging of peptides presented within the context of different human leukocyte antigens (HLA) expressed by cells of various tissue origins, both in health and in disease. Of major interest are peptides presented on cancer cells, which include peptides derived from tumor antigens that are of interest for immunotherapy. Here, HLA-restricted tumor-specific antigens were identified by transfecting human breast, ovarian and prostate tumor cell lines with truncated genes of HLA-A2 and HLA-B7. Soluble HLA secreted by these cell lines were purified by affinity chromatography and analyzed by nano-capillary electrospray ionization-tandem mass spectrometry. Typically, a large peptide pool was recovered and sequenced including peptides derived from MAGE-B2 and mucin and other new tumor-derived antigens that may serve as potential candidates for immunotherapy.