Dataset Information

Improved sensitivity of immunopeptidomics by leveraging available large-scale pan-HLA spectral libraries and data-independent acquisition

ABSTRACT: Mass spectrometry is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across HLA allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery-driven and data-dependent tandem mass spectrometry acquisition is commonly used, as it generates high quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that leads to lower sensitivity and reproducibility. In contrast, in data-independent acquisition, the fragmentation and acquisition of all fragment ions from a predefined list of precursor isolation windows yields a comprehensive map for a given sample. Because often the amount of HLA peptides eluted from biological samples, is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity - from sample-specific libraries to libraries combining from two to forty different immunopeptidomics samples. Matching DIA immunopeptidomics data against complex pan-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. We concluded that a comprehensive pan-HLA library for DIA approach is highly advantageous for the clinical Immunopeptidomics where low amount of biological sample is available for immunopeptidomics.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Michal Bassani-Sternberg

LAB HEAD: Michal Bassani-Sternberg

PROVIDER: PXD022950 | Pride | 2021-04-13

REPOSITORIES: Pride

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Publications

Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction.

Pak HuiSong H Michaux Justine J Huber Florian F Chong Chloe C Stevenson Brian J BJ Müller Markus M Coukos George G Bassani-Sternberg Michal M

Molecular & cellular proteomics : MCP 20210409

Mass spectrometry (MS) is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across human leukocyte antigen (HLA) allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery driven. Hence, data-dependent tandem MS (MS/MS) acquisition (DDA) is widely used, as it generates high-quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that impairs sensitivity and reprod ...[more]

PMID: 33845167

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Project description:Background: Patient derived organoids (PDOs) can be established from colorectal cancers as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFN) or MEK-inhibitors. Methods: Four PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analysed by MS. Results: We identified an average of 9,936 unique peptides per PDO which compares favourably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. Treatment of four PDOs with IFN upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFN-inducible genes. HLA class II presented peptides increased on average 16-fold with IFN treatment. MEK-inhibitor treatment showed no consistent effect on class I or II HLA expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. Conclusions: Only 3 out of 612 non-synonymous mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

Dataset Information

Improved sensitivity of immunopeptidomics by leveraging available large-scale pan-HLA spectral libraries and data-independent acquisition

Publications

Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction.

Similar Datasets

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