Dataset Information

MHC immunopeptidome of JY cells analysed by EThcD or HCD at Orbitrap Fusion after mild acid elution

ABSTRACT: To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC). Less often, mild acid elution (MAE) is used to extract MHC class I peptide ligands. MAE may provide a cheap alternative to MHC-IAC for suspension cells, but it is thought to be hampered by the high number of contaminating peptides not derived from the MHC. Here, we optimized the MAE protocol yielding MHC peptide ligand purities of more than 80%. We discovered distinct cysteinylation frequencies at individual positions of MHC peptide ligands and propose that MAE conserves the native cysteinylation pattern of MHC peptide ligands better than MHC-IAC. Key features of the observed cysteinylation pattern were independent of the applied fragmentation methods HCD or EThcD. Our improved and carefully documented MAE workflow represents a high-quality, cost-effective alternative to MHC-IAC for suspension cells and should be applicable also in laboratories not specialized in MHC peptide ligand analyses.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell

SUBMITTER: Theo Sturm

LAB HEAD: Albert J.R. Heck

PROVIDER: PXD012437 | Pride | 2020-11-11

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	F1_Agilent6_20161022_TST_C15_Probe1und2ausC13_MAE_EThcD_OT_Rep1_7B_18s-Proz3.msf	Msf
	F1_Agilent6_20161022_TST_C15_Probe1und2ausC13_MAE_EThcD_OT_Rep1_7B_18s.raw	Raw
	F1_Agilent6_20161022_TST_C15_Probe1und2ausC13_MAE_HCD_OT_Rep1_7B_18s.raw	Raw
	F1_Agilent6_20161022_TST_C15_Probe1und2ausC13_MAE_HCD_OT_Rep4_7B_18s.raw	Raw
	F1_Agilent6_20161023_TST_C15_Probe1und2ausC13_MAE_HCD_OT_Rep9_7B_18s.raw	Raw

Items per page:

1 - 5 of 6

Publications

Mild Acid Elution and MHC Immunoaffinity Chromatography Reveal Similar Albeit Not Identical Profiles of the HLA Class I Immunopeptidome.

Sturm Theo T Sautter Benedikt B Wörner Tobias P TP Stevanović Stefan S Rammensee Hans-Georg HG Planz Oliver O Heck Albert J R AJR Aebersold Ruedi R

Journal of proteome research 20201103 1

To understand and treat immunology-related diseases, a comprehensive, unbiased characterization of major histocompatibility complex (MHC) peptide ligands is of key importance. Preceding the analysis by mass spectrometry, MHC class I peptide ligands are typically isolated by MHC immunoaffinity chromatography (MHC-IAC) and less often by mild acid elution (MAE). MAE may provide a cheap alternative to MHC-IAC for suspension cells but has been hampered by the high number of contaminating, MHC-unrelat ...[more]

PMID: 33141586

Similar Datasets

Project description:The majority of peptides presented by MHC class I results from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides for MHC class I loading non-canonically. We used an im-munopeptidomics workflow combined with a prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect proteasomal processing of immune epitopes presented by A549 cells. Treatment of A549 cells with IFNγ enhanced proteasomal cleavage probability of MHC class I ligands for both, the constitutive proteasome and the im-munoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated HLA allotype-specific proteasomal cleavage probabili-ties for MHC class I ligands, peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. Peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases. The mass spectrometry raw files used in this study were originally produced for a previous publication, where they were utilized to address the influence of cigarette smoke on the antiviral T-cell immune response. Here, we have reanalyzed the data to investigate processing of MHC class I ligands by c20S or i20S, respectively. Original publication: Chen, J.; Wang, X.; Schmalen, A.; Haines, S.; Wolff, M.; Ma, H.; Zhang, H.; Stoleriu, M.G.; Nowak, J.; Nakayama, M.; et al. Antiviral CD8(+) T cell immune responses are impaired by cigarette smoke and in COPD. Eur Respir J 2023, doi:10.1183/13993003.01374-2022.