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Sensitive, high-throughput single-shot HLA-I and HLA-II immunopeptidomics with improved coverage using data dependent parallel accumulation-serial fragmentation mass spectrometry

ABSTRACT: Phulphagar K, Ctortecka C, Vaca Jacome AS, Klaeger S, Verzani E, Hernandez G, Udeshi ND, Clauser KR, Abelin JG, Carr SA. 2023. 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 a powerful state-of-the-art 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 samples. Immunopeptidome depth can be increased through biochemical fractionation, which can be impeded by the 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 with up to 15,000 distinct HLA-I and HLA-II peptides from 4e7 cells. Our optimized single-shot MS acquisition method on the SCP maintains high coverage, eliminates the need for biochemical fractionation and reduces input requirements to 1e7 cells for > 800 distinct HLA-I peptides. Moreover, we find that the binding motifs of specific HLA alleles can influence peptide fragmentation patterns, and that optimized collision energies can result in complementary b/y ion sequence coverage. Our optimized single-shot SCP acquisition methods enable sensitive, high throughput and reproducible immunopeptidome profiling and the detection of peptides from non-canonical open reading frames and clinically relevant cancer-testis antigens from less than 4e7 cells or 15 mg wet weight tissue.

INSTRUMENT(S): Orbitrap Exploris 480, timsTOF SCP

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000091456 | MassIVE | Thu Mar 09 16:43:00 GMT 2023

SECONDARY ACCESSION(S): PXD040740

REPOSITORIES: MassIVE

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Project description:Treatment of cancer cells with anti-cancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumorâs gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anti-cancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited anti-tumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The transcriptomes, proteomes and HLA peptidomes of the U-87, T98G and LNT-229 GBM human cell lines were analyzed before and after treatment with Decitabine. Overall, the RNA-Seq transcriptome analyses resulted in the identification of above 26000 transcripts, the proteome analyses identified about 7500 proteins and the HLA class I peptidome analyses resulted in above 25000 identified HLA peptides. Two biological repetitions of the transcriptome, three of the proteome and three of the HLA peptidome were performed with each of the cell lines and treatment, resulting in highly reproducible datasets.

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Sensitive, high-throughput single-shot HLA-I and HLA-II immunopeptidomics with improved coverage using data dependent parallel accumulation-serial fragmentation mass spectrometry

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