Sensitive, high-throughput HLA-I and HLA-II immunopeptidomics using parallel accumulation-serial fragmentation mass spectrometry

Abstract

Comprehensive, in-depth identification of the human leukocyte antigen HLA-I and HLA-II tumor immunopeptidome can inform the development of cancer immunotherapies. Mass spectrometry (MS) is powerful technology for direct identification of HLA peptides from patient derived tumor samples or cell lines. However, achieving sufficient coverage to detect rare, clinically relevant antigens requires highly sensitive MS-based acquisition methods and large amounts of sample. While immunopeptidome depth can be increased by off-line fractionation prior to MS, its use is impractical when analyzing limited amounts of primary tissue biopsies. To address this challenge, we developed and applied a high throughput, sensitive, single-shot MS-based immunopeptidomics workflow that leverages trapped ion mobility time-of-flight mass spectrometry on the Bruker timsTOF SCP. We demonstrate >2-fold improved coverage of HLA immunopeptidomes relative to prior methods with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the timsTOF SCP maintains high coverage, eliminates the need for off-line fractionation and reduces input requirements to as few as 1e6 A375 cells for > 800 distinct HLA-I peptides. This depth is sufficient to identify HLA-I peptides derived from cancer-testis antigen, and novel/unannotated open reading frames. We also apply our optimized single-shot SCP acquisition methods to tumor derived samples, enabling sensitive, high throughput and reproducible immunopeptidome profiling with detection of clinically relevant peptides from less than 4e7 cells or 15 mg wet weight tissue.

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

“…To enable the detection of rare and clinically relevant antigens from a limited cell input, Phulphagar et al 30 developed a high-throughput single-shot MS-based immunopeptidomics workflow using the timsTOF single-cell proteomics system (SCP). This workflow was applied to sample inputs ranging from 1 million to 40 million A-375 cell equivalents, a melanoma cell line which expresses the following HLA-I genes: A*01:01, A*02:02, B*57:01, B*44:03, C*16:02, and C*06:02.…”

“…To investigate whether low input samples benefit from PSM rescoring, we rescored a timsTOF SCP dataset. For detailed information on data acquisition, please refer to the original publication by Phulphagar et al 30 . In brief, HLA-I peptides were directly enriched from 1 million to 40 million A-375 cell equivalents by single shot injections on timsTOF SCP.…”

A complete mass spectrometry-based immunopeptidomics pipeline for neoantigen identification and validation

“…Application of DeepNovo Peptidome to expand the reference HLA Ligand Atlas of benign human tissues As neoantigens mostly arise from non-canonical sources which are challenging for standard database search approaches, we built DeepNovo Peptidome, a de novo sequencing-based workflow for HLA peptide identification and neoantigen discovery. We trained the de novo sequencing model 13,14 on a very large, carefully curated dataset from 20 previous MS-based immunopeptidomics studies 5,15,16,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] . The dataset included nearly 3,000 runs on two major MS instruments Orbitrap and timsTOF, >26M PSMs and >1.1M unique HLA peptides, and covering more than 90 alleles for each HLA class.…”

Emerging potential of immunopeptidomics by mass spectrometry in cancer immunotherapy

“…Other IM-MS-based immunopeptidomics methodologies have been acknowledged by other groups as well to show their potential to expand the identification of immunopeptides. [48][49][50][51][52] Not long ago, one of the challenges in immunopeptidomics was reported to be the unfeasibility of analysis from a small amount of sample. 53 However, the situation has been improving due to the advancements of IM, improved sensitivity, and the scan speed by the mass spectrometer.…”

“…These results indicate the advantage of IM in immunopeptidomics, which requires a sensitive analysis. Other IM‐MS‐based immunopeptidomics methodologies have been acknowledged by other groups as well to show their potential to expand the identification of immunopeptides 48–52 . Not long ago, one of the challenges in immunopeptidomics was reported to be the unfeasibility of analysis from a small amount of sample 53 .…”