High Sensitivity Immunopeptidomics Using Mass Spectrometry

High sensitivity class I immunopeptidomics on the timsTOF SCP mass spectrometer Mass spectrometric characterization of peptide antigens presented by the major histocompatibility complex is essential to understanding infection, cancer and autoimmunity. Kristina Marx 1, Christoph Krisp 1, Gary Kruppa 1, Arun Tailor 2, Nicola Ternette 2, Robert Parker 2; 1 Bruker Daltonik GmbH & Co. KG, Bremen, Germany; 2 Centre for Immuno-Oncology, Nuffield Department of Medicine, Oxford, UK. Abstract Due to their low abundance, non-tryptic nature and high-complexity, comprehensive LC-MS analysis of Immunopeptidomes purified from surface HLA molecules remains highly challenging. Many peptides ionize as singly charged species hampering their identification. In trapped ion mobility spectrometry (TIMS), these peptides are readily discriminated from multiply charged ion signals by their Collisional Cross Section, reducing the interference from co-isolations that usually occurs in pure m/z analyses. Here, we use the timsTOF SCP system as a highly sensitive instrument with PASEF® for in depth analysis and show unprecedented coverage on low sample input of immunopeptide-like standard samples and real immunopeptidome. Introduction Immunopeptidomics aims to resolve the composition and dynamics of endogenous peptides presented by major histocompatibility complex (MHC) proteins. In humans the MHC locus encode a vital arm of adaptive immunity, where a series of highly polymorphic cell surface Human Leukocyte Antigen (HLA) proteins act to present endogenous peptides for recognition by cognate T-lymphocytes. This peptide display-recognition system enables the elimination of infected, cancerous and damaged cells by cell-mediated immunity, alongside supporting antibody production and innate immune responses. The identification of disease and tumourrelated 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 [1]. There are two distinct classes of HLA proteins, class I and class II. Class I peptide presentation is found on all nucleated cells in the vertebrate body, peptides are typically 8-14 amino acids in length and sourced from endogenous protein production and degradation machinery. Class II peptides can originate from extracellular protein sources and are processed through endosomes, to produce clusters of peptides 15-25 amino acids in length. Keywords: Immunopeptidomics, high sensitivity, timsTOF SCP, PEAKSCurrently, mass spectrometry (MS) provides the only methodology able to reveal the exact nature of the immunopeptidome. However, analytical challenges in analysis arise from sample complexity, low abundance and the distinct peptide sequence heterogeneity found in the immunopeptidome of different people. Often these factors mean that existing proteomic methodologies adapted to target tryptic peptides and ultimately quantitate a protein, not a peptide are particularly unsuitable. For example, class I peptides are often short, consist of hydrophic residues and unlike tryptic peptides do not always possess an K/R residue. This results in a large proportion of singly charged species and poorer fragmentation spectra after LC-MS analysis [2]. Trapped ion mobility spectrometry (TIMS) in the timsTOF series of time of flight (TOF) MS instruments can reduce spectrum complexity prior to MS analysis. In TIMS ions are trapped in an electric field according to their three-dimensional size and charge in the gas phase, resulting in separation by mobility. This allows for improved signal to noise and better sam