Immunopeptidomics aims to resolve the composition and dynamic changes of endogenous peptides presented by major histocompatibility complex (MHC) proteins. This peptide display-recognition system enables the elimination of infected, cancerous, stressed, and damaged cells by cell-mediated immunity, alongside supporting antibody production and innate immune responses. The identification of disease and tumour-related HLA presented peptides in cell lines and biopsies will progress our understanding of disease pathogenesis, immune responses and aid the preparation of targeted vaccines and immunotherapies.
Existing proteomic methodologies are adapted to target tryptic peptides and are often not optimal for immunopeptidomic analyses. For example, class I peptides are present at exceptionally high dynamic ranges, they are shorter (8-14 amino acids) than tryptic peptides, and have a variable, often hydrophobic, C-termini. These characteristics result in a high number of singly charged ion species and fragmentation spectra of lower quality due to the missing dominant y-ion series observed for tryptic peptides with basic C-termini.
Here, we optimise trapped ion mobility spectrometry (TIMS) in the timsTOF SCP mass spectrometer for immunopeptidomics analysis. TIMS allows for improved signal to noise, better DDA sampling by using mobility windows, and 5-fold higher transmission of ions which boosts the identification rates particularly in low-abundant samples.
We validate our methods with a synthetic standard containing a predefined diverse repertoire of 2000 HLA peptides, observing allele dependent peptide sequencing bias. Next, we demonstrate this application on an immunopeptidomic sample generated from a leukemic T-cell lymphoblast cell line, determining sensitivity related to cell count and peptide load. Finally, we perform an intra-laboratory comparison to a HF-X Orbitrap type mass spectrometer. We observe striking improvements in lower limit of detection in the timsTOF SCP, but find that instruments are complementary when sample load is not a limiting factor.