Dataset Information

An input-controlled model system for identification of MHC bound peptides enabling laboratory comparisons of immunopeptidome experiments.

ABSTRACT: Characterization of MHC-bound peptides by mass spectrometry (MS) is an essential technique for immunologic studies. Many efforts have been made to quantify the number of MHC-presented ligands by MS and to define the limits of detection of a specific MHC ligand. However, these experiments are often complex and comparisons across different laboratories are challenging. Therefore, we compared and orthogonally validated quantitation of peptide:MHC complexes by radioimmunoassay and flow cytometry using TCR mimic antibodies in three model systems to establish a method to control the experimental input of peptide MHC:complexes for MS analysis. Following isolation of MHC-bound peptides we identified and quantified an MHC ligand of interest with high correlation to the initial input. We found that the diversity of the presented ligandome, as well as the peptide sequence itself affected the detection of the target peptide. Furthermore, results were applicable from these model systems to unmodified cell lines with a tight correlation between HLA-A*02 complex input and the number of identified HLA-A*02 ligands. Overall, this framework provides an easily accessible experimental setup that offers the opportunity to control the peptide:MHC input and in this way compare immunopeptidome experiments not only within but also between laboratories, independent of their experimental approach. SIGNIFICANCE: Although immunopeptidomics is an essential tool for the characterization of MHCbound peptides on the cell surface, there are no easily applicable established protocols available that allow comparison of immunopeptidome experiments across laboratories. Here, we demonstrate that controlling the peptide:MHC input for immunopurification and LC-MS/MS experiments by flow cytometry in pre-defined model systems allows the generation of qualitative and quantitative data that can easily be compared between investigators, independently of their methods for MHC ligand isolation for MS.

SUBMITTER: Klatt MG

PROVIDER: S-EPMC7484941 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8-12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I 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 PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5-7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry.Supplementary informationThe online version contains supplementary material available at 10.1007/s42485-021-00066-x.

Dataset Information

An input-controlled model system for identification of MHC bound peptides enabling laboratory comparisons of immunopeptidome experiments.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets