Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

Immunopeptidomic analysis reveals that deamidated HLA-bound peptides arise predominantly from deglycosylated precursors (#68)

Shutao Mei 1 , Rochelle Ayala 1 , Sri Ramarathinam 1 , Patricia Illing 1 , Pouya Faridi 1 , Jiangning Song 1 , Anthony Purcell 1 , Nathan Croft 1
  1. Department of Biochemistry and Molecular Biology and Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia


The generation and functional recognition of post-translationally modified (PTM) peptides by T lymphocytes has attracted considerable interest in immunology. Disease associated antigen modification may generate novel PTM neo-peptides. These modified peptides presented by human leukocyte antigen (HLA) molecules have the potential to form excellent T cell targets. With the rapid advances in mass spectrometry, immunoproteomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples. However, except for S/T phosphorylation, in-depth analysis of the proportion and nature of peptides bearing one or more other PTMs has not been studied extensively.


Here, we apply a mass spectrometry-based methodology to comprehensively profile peptides with PTMs bound to a variety of HLA allotypes.


Analysis of a diverse set of HLA class I immunopeptidomes revealed that methionine oxidation was the most commonly observed PTM, with deamidation of asparagine and glutamine the next most prevalent type of modification. Detailed analysis of HLA class I deamidation motifs revealed a strong prevalence of the known N-linked glycosylation motif (NX(S/T), where X is any amino acid except proline) for asparagine deamidated peptides. Notably no such motif was observed for glutamine deamidated peptides or for asparagine deamidated peptides isolated from HLA class II molecules. Subsequent blocking of PNGase activity confirmed the role of deglycosylation of NX(S/T)-bearing asparagine-deamidated peptides as a major source of HLA-bound ligands.


These data indicate that the immunopeptidome is enriched for peptides derived from formerly glycosylated proteins and specifically those that have been retro-translocated from the ER and targeted for deglycosylation and degradation in the cytoplasm. The results not only highlight the link between glycosylation and asparagine deamidation but will help to train models to predict the presence of asparagine deamidated peptides in immunopeptidome, ultimately aiding novel vaccine design.