Dataset Information

Extending the Mass Spectrometry-Detectable Landscape of the Immunopeptidome by use of Restricted access Material

ABSTRACT: Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from cell culture as well as from tissue or tumor samples and is increasingly applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally accounted as an unbiased method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. For instance, highly hydrophobic peptides get easily lost already during sample preparation when C18 solid phase extraction (SPE) is used for separating MHC peptides from proteins. To overcome this limitation, we established an optimized protocol for this sample preparation step applying restricted access material (RAM). Compared to C18-SPE, RAM-SPE improves the overall MHC peptide recovery and extends the landscape of mass spectrometry-detectable MHC peptides towards more hydrophobic peptides.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Melissa Bernhardt

LAB HEAD: Andreas Schlosser

PROVIDER: PXD034017 | Pride | 2022-11-07

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	2020-11-02_Fusion_23778.raw	Raw
	2020-11-02_Fusion_23779.raw	Raw
	2020-11-02_Fusion_23781.raw	Raw
	2020-11-02_Fusion_23782.raw	Raw
	2020-11-04_Fusion_23785.raw	Raw

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Publications

Extending the Mass Spectrometry-Detectable Landscape of MHC Peptides by Use of Restricted Access Material.

Bernhardt Melissa M Cruz-Garcia Yiliam Y Rech Anne A Meierjohann Svenja S Erhard Florian F Schilling Bastian B Schlosser Andreas A

Analytical chemistry 20221004 41

Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from a cell culture as well as from tissue or tumor samples and is applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally considered an "unbiased" method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. Here, we demonstrate that hig ...[more]

PMID: 36194871

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Project description:Peptides displayed by MHC molecules on a cell’s surface, referred to as its immunopeptidome, play an important role in the adaptive the immune response. Antigen processing for MHC class I presentation is a ubiquitous pathway present in all nucleated cells which generate and present peptides of both self and non-self origin. Peptides with post-translational modifications (PTMs) are one of the classes of peptides presented by MHC class I molecules. However, due to the high background of self-peptides presented by the cells, the diversity of peptides with post-translational modifications is not well reported. In this study, we have carried out MHC Class I immunopeptidomics analysis on Jurkat and A375 cell lines to characterize the diversity of post-translational modifications among MHC class I peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both the cell lines using Bolt and Sequest search engines. High specificity of the enrichment method is demonstrated by identifying ~90% of the peptides with typical length distribution of 8-12 aa and enriched motifs within those peptides similar to the binding motifs of MHC alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these post-translational modifications compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that account for ~5-7% of MHC-bound peptides across the two cell lines. These peptides share similar features with respect to normal MHC-bound peptides such as peptide length distribution and binding motifs. We validated the identification of several post-translationally modified peptides and spliced peptides using synthetic peptide sequences. In conclusion, our study demonstrates unbiased identification of these low stoichiometric PTMs and unusual spliced peptides using high resolution mass spectrometry.