timsTOF mass spectrometry-based immunopeptidomics refines tumor antigen identification

Abstract

T cell recognition of human leukocyte antigen (HLA)-presented tumor-associated peptides is central for cancer immune surveillance. Mass spectrometry (MS)-based immunopeptidomics represents the only unbiased method for the direct identification and characterization of naturally presented tumor-associated peptides, a key prerequisite for the development of T cell-based immunotherapies. This study reports on the de novo implementation of ion mobility separation-based timsTOF MS for next-generation immunopeptidomics, enabling high-speed and sensitive detection of HLA-presented peptides. A direct comparison of timsTOF-based with state-of-the-art immunopeptidomics using orbitrap technology showed significantly increased HLA peptide identifications from benign and malignant primary samples of solid tissue and hematological origin. First application of timsTOF-based immunopeptidomics for tumor antigen discovery enabled (i) the expansion of benign reference immunopeptidome databases with > 150,000 HLA-presented peptides from 94 primary benign tissue samples, (ii) the refinement of previously described tumor antigens, and (iii) the identification of a vast array of novel tumor antigens, comprising low abundant neoepitopes, that might serve as targets for future cancer immunotherapy development.

Fragment ion intensity prediction improves the identification rate of non-tryptic peptides in timsTOF

“…We hypothesized that integrating fragment ion intensity predictions into the database searching process would improve the identification rate of HLA peptides measured on a timsTOF, similar to what was previously observed for tryptic and non-tryptic peptides measured on other instruments 11 . To investigate this, we reanalyzed data from a recently published benchmarking study on timsTOF-based immunopeptidomics for tumor antigen discovery 14 .…”

“…To compare the rescoring performance on Orbitrap versus timsTOF data, we utilized a comparison dataset comprising both HLA-I and HLA-II peptides measured on an Orbitrap and on a timsTOF. For detailed information on data acquisition, please refer to the original publication by Gravel et al 14 . In brief, 10 samples were measured in technical triplicate (two technical replicates for the HNSCC sample) on the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientic, Waltham, USA) and on the timsTOF Pro (Bruker Daltonik, Germany).…”

“…In the context of immunopeptidomics, it is critical to use highly sensitive instrumentation due to the relatively low abundance of immunopeptides. A timsTOF-based approach has been shown to significantly increase HLA peptide identifications compared to immunopeptidomics using an Orbitrap mass spectrometer 14 . Furthermore, optimization of the timsTOF acquisition method has been recently demonstrated to further improve HLA peptide identification rates 15 .…”