Dataset Information

Raising the Bar for Isobaric Amino Acid Variants in Immunopeptidome of Pancreatic Cancer: Distinguishing Ile-Leu variant Ligands by ExCID

ABSTRACT: Immunopeptidomics aims to facilitate T-cell target discovery by analyzing human leukocyte antigen (HLA)-bound peptides using tandem mass spectrometry (MS/MS). MS/MS analysis of peptides conventionally relies on collision-induced dissociation (CID) of selected precursor ions. However, CID analysis of immunopeptides, owing to their diverse termini (non-tryptic) and relatively short lengths (8- to 12-mer for class I HLA), can be challenging. It is impossible to differentiate isobaric amino acids, such as isoleucine and leucine sequence variants, which are frequent amino acids in the immunopeptidomes of many HLA allotypes. To address this, we leveraged the alternative fragmentation method, Electron-Activated Dissociation (EAD), and its CID-supplemented mode (EAD+CID, or ExCID) on the SCIEX ZenoTOF 7600+ system to characterize Ile/Leu sequence variants in the immunopeptidome. We first used a panel of seven synthetic Ile/Leu-substituted peptides and demonstrated that both EAD and ExCID efficiently generate variant-distinguishing w- and d-type satellite ions. We then determined optimal kinetic energy (KE) and EAD reaction time for utilizing hybrid ExCID ion activation in a discovery immunopeptidomics setting. We demonstrated that Ile/Leu sequence variants present in the immunopeptidome can be distinguished using ExCID but not CID alone. Using this optimized ExCID methodology, we observed improvements in fragmentation and peptide identification for both HLA class I and II studies, allowing us to benefit from EAD while minimizing the compromise to cycle time and identification depth. We further applied the optimal ExCID method to complex HLA peptide samples isolated from two model cell lines, namely a “monoallelic” HLA-A2 expressing cell line (C1R-A*02:01) and the pancreatic cancer cell line Panc1. The versatile and tuneable hybrid ExCID mode improves MS/MS fragment coverage and enriches the product spectra with diagnostic ions, enabling us to differentiate and characterize Ile/Leu sequence variants in immunopeptidomics studies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture, Permanent Cell Line Cell, Cell Culture

DISEASE(S): Pancreatic Ductal Adenocarcinoma

SUBMITTER: Mohammadreza Dorvash

LAB HEAD: Professor Anthony

PROVIDER: PXD070374 | Pride | 2026-06-16

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20231225_RD_C1RA2_3e9_ClassII_UnEnr_CID_tr1.mzML	Mzml
	20231225_RD_C1RA2_3e9_ClassII_UnEnr_CID_tr1.timeseries.data	Other
	20231225_RD_C1RA2_3e9_ClassII_UnEnr_CID_tr1.wiff	Wiff
	20231225_RD_C1RA2_3e9_ClassII_UnEnr_CID_tr1.wiff.scan	Wiff
	20231225_RD_C1RA2_3e9_ClassII_UnEnr_CID_tr1.wiff2	Wiff

Items per page:

1 - 5 of 432

Similar Datasets

Project description:Gaining a complete and unbiased understanding of the non-tryptic peptide repertoire presented by HLA-I complexes by LC-MS/MS is indispensable for therapy design for cancer, autoimmunity and infectious diseases. A serious concern in HLA peptide analysis is that the routinely used, collision-based fragmentation methods (CID/HCD) do not always render sufficiently informative MS2 spectra, whereby gaps in the fragmentation sequence coverage prevent unambiguous assignments. EThcD can be utilized to generate complementary ion series, i.e. b/y ions and c/z ions, resulting in richer, more informative MS2 spectra, thereby filling in the gaps. Here, we present data generated on a novel hybrid Orbitrap mass spectrometer, facilitating fast and efficient hybrid fragmentation due to the implementation of EThcD in the ion routing multipole. We hypothesized that this would enable more comprehensive and less error-prone analysis of immunopeptidomes at minimal costs in duty-cycle. First, we optimized ETD/EThcD methods using an elastase-digested cell lysate, as this contains peptides of similar length and charge distributions to immunopeptides. Next, we compared HCD and EThcD on immunopeptidomes originating from three cell lines with distinct HLA-I complexes that present peptides with varying physicochemical properties. We demonstrate that the new instrument not only enables efficient and fast ETD reactions, but when combined with collision-based supplemental activation, i.e. EThcD, also consistently increases the sequence coverage and identification of peptide sequences, otherwise missed by using solely HCD. We reveal several of the biochemical properties that make HLA peptides preferably identifiable by EThcD, with internal Arg residues being one of the most dominant determinants. Finally, we demonstrate the power of EThcD for the identification and localization of HLA peptides harboring post-translational modifications, focusing here on HLA Arg mono-/di-methylation. We foresee that this new instrument with efficient EThcD capabilities enhances not only immunopeptidomics analysis, but also analysis of peptides harboring post-translational modifications and de novo sequencing.

Dataset Information

Raising the Bar for Isobaric Amino Acid Variants in Immunopeptidome of Pancreatic Cancer: Distinguishing Ile-Leu variant Ligands by ExCID

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets