Dataset Information

Isolation of mouse and human MHC class I- and II-associated peptides for the analysis of immunopeptidomes by mass spectrometry

ABSTRACT: Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in a MS-based immunopeptidomic workflow, sample preparationis critically important for capturing high-quality data of therapeutic relevance. Here, we describe step-by-step instructions to isolate MHC class I and II-associated peptides by immunoaffinity purification from quality control (QC) samples. Our QC samples are MHC class I and class II peptides isolated from mouse (EL4 and A20) and human (JY) cell lines. We thoroughly describe the various reagents and specfic antibodies that are used too isolate MHC-associated peptides from these cell lines, including the steps to verify the beads-binding efficiency of the antibody and the elution efficiency of the MHC-peptide complexes from the beads. QC samples generated using the described protocol can be used to establish and standardize immunopeptidomic workflow, as well as to benchmark new protocols. We anticipate that the visualized protocol provided in this report will represent a great starting point for any non-expert in addition to foster the instra- and inter-laboratory reproducibility of the sample preparation procedure in immunopeptidomics.

INSTRUMENT(S): Orbitrap Eclipse

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

TISSUE(S): T Cell, Cell Culture

SUBMITTER: isabelle sirois

LAB HEAD: Etienne Caron

PROVIDER: PXD028633 | Pride | 2021-12-07

REPOSITORIES: Pride

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Publications

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis.

Sirois Isabelle I Isabelle Maxim M Duquette Jérôme D JD Saab Frederic F Caron Etienne E

Journal of visualized experiments : JoVE 20211015 176

Immunopeptidomics is an emerging field that fuels and guides the development of vaccines and immunotherapies. More specifically, it refers to the science of investigating the composition of peptides presented by major histocompatibility complex (MHC) class I and class II molecules using mass spectrometry (MS) technology platforms. Among all the steps in an MS-based immunopeptidomics workflow, sample preparation is critically important for capturing high-quality data of therapeutic relevance. Her ...[more]

PMID: 34723952

Publication: 1/2

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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.

Dataset Information

Isolation of mouse and human MHC class I- and II-associated peptides for the analysis of immunopeptidomes by mass spectrometry

Publications

Immunopeptidomics: Isolation of Mouse and Human MHC Class I- and II-Associated Peptides for Mass Spectrometry Analysis.

Similar Datasets

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