Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Supercharge Your Immunopeptidomics (#138)

Terry Lim Kam Sian 1 2 , Yue Ding 1 2 , Scott Blundell 1 , Ralf B Schittenhelm 1 , Pouya Faridi 1 2
  1. Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia
  2. School of Clinical Sciences, Department of Medicine, Monash University, Clayton, VIC, Australia

Human leukocyte antigen (HLA) molecules play a crucial role in the development of adaptive immune responses and therefore have been of major interest in the development of immunotherapies such as cancer vaccines and chimeric antigen receptor (CAR) T cells. Hence, a comprehensive understanding and profiling of the immunopeptidome is required to foster growth for these personalised solutions. There are however several analytical challenges associated with immunopeptidome profiling, as highlighted by the Human Immunopeptidome Project Consortium. These include (i) uncertainties around peptide isolation, (ii) the inability to analyse small amounts of biological material, (iii) low throughput, (iii) lack of sensitivity and reproducibility of mass spectrometry technologies, (iv) suboptimal identification rates, (v) lack of experimental and computational standards, and (vi) the current lack of accessibility to large-scale community generated datasets.

We herein describe a novel immunopeptidomics workflow involving the Kingfisher platform (Thermo Scientific) to isolate immunopeptidomes with anti-HLA antibodies coupled to a proprietary hyper-porous protein A microparticle (MagReSyn®), and an improved LC-MS/MS method using dimethyl sulfoxide (DMSO) as supercharging agent.

Using this workflow, we were able to identify up to ~5000 and ~7000 unique HLA class I and class II peptides, respectively, in as low as 5e6 B lymphoblastoid cell line. Moreover, addition of 5% DMSO to the LC-MS/MS buffer systems was observed to increase the number of HLA-bound peptides by ~50%. Our data strongly suggests that this increase is mostly due to the presence of higher quality ms2 spectra showing a significant increase in b and y ions intensities which result in improved signal-to-noise ratios (~1.5 and ~2 fold for HLA class I and class II bound peptides).

Altogether, this improved workflow will be very important for the future of the immunopeptidome profiling, especially for smaller sample types, and ultimately can be a useful tool for the development of immunotherapies and other precision medicine approaches.