Stable Isotope Tagging of Epitopes

Abstract

Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes. Identification of peptides presented in major histocompatibility complex (MHC) class I molecules after viral infection is of strategic importance for vaccine development. Until recently, mass spectrometric identification of virus-induced peptides was based on comparative analysis of peptide pools isolated from uninfected and virus-infected cells. Here we report on a powerful strategy aiming at the rapid, unambiguous identification of naturally processed MHC class I-associated peptides, which are induced by viral infection. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues (i.e. residues of the peptide that have amino acid side chains that bind into pockets lining the peptide-binding groove of the MHC class I molecule) for the human leukocyte antigen allele of interest. Subsequently these cells are mixed with an equal number of non-infected cells, which are cultured in normal medium. Finally peptides are acid-eluted from immunoprecipitated MHC molecules and subjected to two-dimensional nanoscale LC-MS analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules. Using this approach we identified novel measles virus and respiratory syncytial virus epitopes as well as infection-induced self-peptides in several cell types, showing that SITE is a unique and versatile method for unequivocal identification of disease-related MHC class I epitopes. The adaptive immune response to virus infections is characterized by the generation of CD8+ cytotoxic T lymphocytes (CTLs), 1The abbreviations used are: CTL, cytotoxic T lymphocyte; FBS, fetal bovine serum; HLA, human leukocyte antigen; DC, dendritic cell; iDC, immature dendritic cell; MHC, major histocompatibility complex; m.o.i., multiplicity of infection; MV, measles virus; PBMC, peripheral blood mononuclear cell; PTM, post-translational modification; RSV, respiratory syncytial virus; RSV-F, RSV fusion protein; RSV-NS, RSV nonstructural protein; SCX, strong cation exchange; SITE, stable isotope tagging of epitopes; RRf, relative response factor; nanoscale LC-MS, nanoscale reversed phase LC and MS. 1The abbreviations used are: CTL, cytotoxic T lymphocyte; FBS, fetal bovine serum; HLA, human leukocyte antigen; DC, dendritic cell; iDC, immature dendritic cell; MHC, major histocompatibility complex; m.o.i., multiplicity of infection; MV, measles virus; PBMC, peripheral blood mononuclear cell; PTM, post-translational modification; RSV, respiratory syncytial virus; RSV-F, RSV fusion protein; RSV-NS, RSV nonstructural protein; SCX, strong cation exchange; SITE, stable isotope tagging of epitopes; RRf, relative response factor; nanoscale LC-MS, nanoscale reversed phase LC and MS. which recognize peptides presented by MHC class I molecules of infected cells. The MHC class I molecules display peptides from proteins expressed inside the cell that are usually generated in the classical proteasomal pathway. Under physiological conditions these peptides are derived from self-proteins. During viral infection, the MHC class I display of peptides is affected due to changes in host protein expression and as a result of the production of viral proteins (1York I.A. Rock K.L. Antigen processing and presentation by the class I major histocompatibility complex.Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar, 2Katze M.G. He Y. Gale Jr., M. Viruses and interferon: a fight for supremacy.Nat. Rev. Immunol. 2002; 2: 675-687Crossref PubMed Scopus (856) Google Scholar). Identification and quantification of such altered MHC-peptide expression is a goal as part of a new field of research referred to as immunoproteomics and is of strategic importance for vaccine development (1York I.A. Rock K.L. Antigen processing and presentation by the class I major histocompatibility complex.Annu. Rev. Immunol. 1996; 14: 369-396Crossref PubMed Scopus (511) Google Scholar, 3Wiesmuller K.H. Fleckenstein B. Jung G. Peptide vaccines and peptide libraries.Biol. Chem. 2001; 382: 571-579Crossref PubMed Scopus (40) Google Scholar, 4Berzofsky J.A. Ahlers J.D. Belyakov I.M. Strategies for designing and optimizing new generation vaccines.Nat. Rev. Immunol. 2001; 1: 209-219Crossref PubMed Scopus (314) Google Scholar, 5Purcell A.W. Gorman J.J. Immunoproteomics: mass spectrometry-based methods to study the targets of the immune response.Mol. Cell. Proteomics. 2004; 3: 193-208Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The analytical challenge lies in the discrimination between the few disease-related peptides among a majority of nondisease-related peptides (6de Jong A. Contribution of mass spectrometry to contemporary immunology.Mass Spectrom. Rev. 1998; 17: 311-335Crossref PubMed Google Scholar). Several strategies have been developed to explore changes in the MHC-associated peptide display on antigen-presenting cells. These analyses are generally based on the isolation of the MHC-peptide complexes, acid elution of the bound peptides from the MHC molecules followed by analysis using LC and MS as pioneered by Hunt et al. (7Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1037) Google Scholar) and adapted for various purposes by other groups (4Berzofsky J.A. Ahlers J.D. Belyakov I.M. Strategies for designing and optimizing new generation vaccines.Nat. Rev. Immunol. 2001; 1: 209-219Crossref PubMed Scopus (314) Google Scholar, 8di Marzo Veronese F. Arnott D. Barnaba V. Loftus D.J. Sakaguchi K. Thompson C.B. Salemi S. Mastroianni C. Sette A. Shabanowitz J. Hunt D.F. Appella E. Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.J. Exp. Med. 1996; 183: 2509-2516Crossref PubMed Scopus (38) Google Scholar, 9van Els C.A. Herberts C.A. van der Heeft E. Poelen M.C. van Gaans-van den Brink J.A.M. van der Kooi A. Hoogerhout P. ten Hove G.J. Meiring H.D. de Jong A.P. A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.Eur. J. Immunol. 2000; 30: 1172-1181Crossref PubMed Scopus (62) Google Scholar, 10Hickman H.D. Luis A.D. Bardet W. Buchli R. Battson C.L. Shearer M.H. Jackson K.W. Kennedy R.C. Hildebrand W.H. Cutting edge: class I presentation of host peptides following HIV infection.J. Immunol. 2003; 171: 22-26Crossref PubMed Scopus (65) Google Scholar, 11Lemmel C. Weik S. Eberle U. Dengjel J. Kratt T. Becker H.D. Rammensee H.G. Stevanovic S. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.Nat. Biotechnol. 2004; 22: 450-454Crossref PubMed Scopus (74) Google Scholar). We successfully used the mass spectrometric method to identify novel MHC class I-restricted viral epitopes by subtractive analysis of peptide pools isolated from virus-infected and control cells (9van Els C.A. Herberts C.A. van der Heeft E. Poelen M.C. van Gaans-van den Brink J.A.M. van der Kooi A. Hoogerhout P. ten Hove G.J. Meiring H.D. de Jong A.P. A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.Eur. J. Immunol. 2000; 30: 1172-1181Crossref PubMed Scopus (62) Google Scholar). This comparative method, however, needs meticulous parallel sample processing of the infected and control peptide batches, and differences in peptide recovery as well as chromatographic retention and stability between uninfected and infected peptide be This the of subtractive analysis and have to the to identify viral epitopes Marzo Veronese F. Arnott D. Barnaba V. Loftus D.J. Sakaguchi K. Thompson C.B. Salemi S. Mastroianni C. Sette A. Shabanowitz J. Hunt D.F. Appella E. Autoreactive cytotoxic T lymphocytes in human immunodeficiency virus type 1-infected subjects.J. Exp. Med. 1996; 183: 2509-2516Crossref PubMed Scopus (38) Google Scholar, 10Hickman H.D. Luis A.D. Bardet W. Buchli R. Battson C.L. Shearer M.H. Jackson K.W. Kennedy R.C. Hildebrand W.H. Cutting edge: class I presentation of host peptides following HIV infection.J. Immunol. 2003; 171: 22-26Crossref PubMed Scopus (65) Google Scholar). 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We SITE to induced epitopes for the class I and to a of epitopes as by comparative analysis (9van Els C.A. Herberts C.A. van der Heeft E. Poelen M.C. van Gaans-van den Brink J.A.M. van der Kooi A. Hoogerhout P. ten Hove G.J. Meiring H.D. de Jong A.P. A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.Eur. J. Immunol. 2000; 30: 1172-1181Crossref PubMed Scopus (62) Google we used the to identify and RSV epitopes and for and The of SITE in the identification and of several novel naturally processed viral epitopes and virus-induced which to of the and of cell was cultured in with fetal bovine in a with at to a number of cells. of this was and infected with at a multiplicity of infection of for These cells for in and which was to of and with unlabeled unlabeled from and of the unlabeled amino acids and was used for the uninfected cells. control and infected with and (i.e. cells from The cells and at peptide isolation and analysis. blood mononuclear cells from an isolated after by of cells on immature dendritic cells cultured to a by and F. A. presentation of antigen by cultured human dendritic cells is by and by Exp. Med. PubMed Scopus Google Scholar). at in in with at at in a for of the cells cultured for in human and human and on to equal of cells. was infected with human RSV number at a of for and cultured in with of and from These amino acids of the anchor residues at position and at for and and of the anchor residues at and for M. H.D. A. K. S. Hildebrand W.H. of the 2003; PubMed Scopus Google Scholar). labeling of the majority of the induced peptides bound to these was A with of the unlabeled amino acids and from was for the uninfected cells. cells cultured and in the of and after infection, of the infected dendritic cells and by analysis control and infected with and mixed (i.e. cells from which is the of that are generated from in this H.D. B. van Gaans-van den Brink J.A.M. Poelen M.C. H. G. H. van J. de Jong van Els identification of naturally processed class peptides by T Immunol. PubMed Scopus Google The cell was and at peptide isolation and analysis. cells number cultured in with to on of cells was at a of for in cultured for in with of the labeled of the unlabeled amino peptides isolated as by Hunt et al. (7Hunt D.F. Henderson R.A. Shabanowitz J. Sakaguchi K. Michel H. Sevilir N. Cox A.L. Appella E. Engelhard V.H. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.Science. 1992; 255: 1261-1263Crossref PubMed Scopus (1037) Google Scholar). the cells in a and at the was in the (i.e. the with normal (i.e. and the with for and The with acid and a mass The was to using and in acid and to a of and at analysis. of the MHC was using J. Peptide with a PubMed Scopus Google Scholar, J. E. G.J. analysis of protein using mass Biotechnol. 17: PubMed Scopus Google Scholar, D. analysis of the by protein identification Biotechnol. 2001; PubMed Scopus Google Scholar, H.D. 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The an and an analytical The analytical was to a to A of the was the at a of with acid for the to The was in with the analytical and a to with acid was at a of A was used on a mass The mass was in at a of Full mass in at a of epitopes identified by analyses using chromatographic conditions and on the mass at LC-MS to the and into the mass by the and was and used for the isotope based on the stable isotope the of these stable the number of labeled amino acids in the the and the of the isotope was the mass of the MS isotope for analysis in which the unlabeled an isotope was as the The for (i.e. mass and mass to to the labeled in the using a in the The using and processed by at and using The to a M. M. identification of peptides in by peptide Chem. PubMed Scopus Google Scholar). This was to the at for peptide identification the for protein between and the and was The peptide and to and for the the epitopes these and of of the peptide was based on the equal of the and the the number of labeled amino acids (i.e. the with that in the peptide number was from the mass isotope in the MS of the of the processed and its and of the chromatographic retention of the and its response by the of the of the identified and by the of the ratios of the peptides and These used for the of the of epitopes in the cell The expression of was for viral and self-peptides as epitopes generated in the infected cell The expression was as the of the number of molecules and the number of infected self-peptides from the infected and control cells. The of of the self-peptides on the infected cells was derived from a the between the the of for a peptides by phase using a peptide The strategy of SITE is in A control of cells is cultured in normal and is in after viral infection and and anchor amino acids using the stable of This and viral proteins synthesized in the virus-infected to these residues at a in a processed in the MHC class I pathway. of cells from are to isolation of the MHC-associated the cell batches, the of expressed are the of peptides from proteins during viral infection as these peptides are in the control elution of the peptides from the class I is used in an of to the A of is subjected to nanoscale LC-MS analysis to for induced peptides H.D. van der Heeft E. ten Hove G.J. de Jong A.P. and to peptide and protein 2002; Scopus Google Scholar). This is based on the computer-assisted of the binomially distributed mass isotope of peptides labeled amino acid of the epitopes is by nanoscale analysis on the of the SITE the identification of and novel viral epitopes and virus-induced self-peptides in the MHC class I-associated peptide display of cells after infection During the infection the of the cell was with of and of and as with unlabeled as a mass isotope of a peptide showing at and which that the analysis the amino acid as which from the protein of This peptide been as a naturally processed and presented to other epitopes and identified with the SITE the (9van Els C.A. Herberts C.A. van der Heeft E. Poelen M.C. van Gaans-van den Brink J.A.M. van der Kooi A. Hoogerhout P. ten Hove G.J. Meiring H.D. de Jong A.P. A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.Eur. J. 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The strong of SITE is that the a peptide is with the infection is in single This a of in two-dimensional for a single sample strategy is based on subtractive analysis of and sample processing to differences (9van Els C.A. Herberts C.A. van der Heeft E. Poelen M.C. van Gaans-van den Brink J.A.M. van der Kooi A. Hoogerhout P. ten Hove G.J. Meiring H.D. de Jong A.P. A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.Eur. J. Immunol. 2000; 30: 1172-1181Crossref PubMed Scopus (62) Google Scholar) as changes in peptide recovery chromatographic retention stability be as altered peptide This for a method based on stable isotope labeling of the MHC-associated peptides after peptide isolation C. Weik S. Eberle U. Dengjel J. Kratt T. Becker H.D. Rammensee H.G. Stevanovic S. Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling.Nat. Biotechnol. 2004; 22: 450-454Crossref PubMed Scopus (74) Google Scholar). as this method is based on the of ratios mass viral epitopes self-peptides are as single which the of these peptides in complex to the of induced peptides, SITE the quantification of the the by of the and isotope the of of self-peptides This method of quantification as is affected by in the peptide isolation chromatographic mass spectrometric stability the stable isotope are during the viral infection. The of is at the the control and infected cell are The at an expression of (9van Els C.A. Herberts C.A. van der Heeft E. Poelen M.C. van Gaans-van den Brink J.A.M. van der Kooi A. Hoogerhout P. ten Hove G.J. Meiring H.D. de Jong A.P. A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.Eur. J. Immunol. 2000; 30: 1172-1181Crossref PubMed Scopus (62) Google the display at an of after infection as from expression of RSV epitopes differences by the RSV which was expressed the RSV on infected expression to be by the cell type as by the which was expressed on as with cells. of SITE the expression of induced epitopes and induced on class I molecules of several cell to this SITE to development of vaccine strategies that of the MHC class I We and from for blood and de and for in isolation and identification of the peptides, and for the of We Hoogerhout and for peptide with