Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

Abstract

Leukemias are highly immunogenic but have a low mutational load, providing few mutated peptide targets. Thus, the identification of alternative neoantigens is a pressing need. Here, we identify 36 MHC class I–associated peptide antigens with O-linked β-N-acetylglucosamine (O-GlcNAc) modifications as candidate neoantigens, using three experimental approaches. Thirteen of these peptides were also detected with disaccharide units on the same residues and two contain either mono- and/or di-methylated arginine residues. A subset were linked with key cancer pathways, and these peptides were shared across all of the leukemia patient samples tested (5/5). Seven of the O-GlcNAc peptides were synthesized and five (71%) were shown to be associated with multifunctional memory T-cell responses in healthy donors. An O-GlcNAc-specific T-cell line specifically killed autologous cells pulsed with the modified peptide, but not the equivalent unmodified peptide. Therefore, these post-translationally modified neoantigens provide logical targets for cancer immunotherapy.

Identification of Post-translational Modifications on Odorant-Binding Protein Isoforms from Pig Olfactory Secretome by High-Resolution Mass Spectrometry: O-β-N-acetylglucosaminylation and Phosphorylation

“…Indeed, the study of native proteins in complex mixture could explain why we did not find the oxonium ions (204.08) for HexNAcylation, typical of the HCD method (Zhao et al, 2011). These ions are usually detected in glycoprotein samples enriched in O-GlcNAcylated peptides (Toghi Eshghi et al, 2016) or exclusively composed of synthetic O-GlcNAcylated peptides (Chalkley and Burlingame, 2001;Malaker et al, 2017). In this study, identification of modified peptides was made by a manual assignment approach instead of using software as it is done in almost all PTM identification/localization papers.…”

Mapping the tumour human leukocyte antigen (HLA) ligandome by mass spectrometry

“…Furthermore, the identification of HLA class I peptides with an O‐linked β ‐N‐acetylglucosamine (O‐GlcNAc) from leukaemia cell lines was achieved by applying an enrichment method based on esterification of alcohol groups with boronic acid coupled to POROS20 AL beads. The MS detection method was tailored to fragment ions with electron‐transfer dissociation (ETD) when CID spectra contained doubly charged ions corresponding to loss of dehydro‐N‐acetylglucosamine (Δ m = 203 Th) …”

“…The search for neoantigenic peptides, per se designated to be tumour‐specific, and HLA ligands carrying cancer‐specific PTMs by MS is described in detail above. Targeting these by cancer immunotherapies inevitably requires mutation‐ and PTM‐specific immunogenicity …”

“…The MS detection method was tailored to fragment ions with electron-transfer dissociation (ETD) when CID spectra contained doubly charged ions corresponding to loss of dehydro-N-acetylglucosamine (Dm = 203 Th). 67…”

“…Targeting these by cancer immunotherapies inevitably requires mutation-and PTM-specific immunogenicity. 4,13,17,53,67 Databases and compendia of established tumour antigens and peptides…”