A microfluidics-enabled automated workflow of sample preparation for MS-based immunopeptidomics

• DOI: 10.1016/j.crmeth.2023.100479
• Publication Date: 2023-05-19T14:51:17Z
• Authors: Xiaokang Li; Hui Song Pak; Florian Huber; Justine Michaux; Marie Taillandier...; Emma Ricart Altim...; Michal Bassani-St...
• Keywords: Proteomics; Tandem Mass Spectrometry; Microfluidics; Peptides; Workflow; Microfluidics; Proteomics/methods; Tandem Mass Spectrometry; Peptides/chemistry; automation; data-independent acquisition; immunopeptidome; mass spectrometry; microfluidics; sample preparation; Article; Workflow

Mass spectrometry (MS)-based immunopeptidomics is an attractive antigen discovery method with growing clinical implications. However, the current experimental approach to extract HLA-restricted peptides requires a bulky sample source, which remains a challenge for obtaining clinical specimens. We present an innovative workflow that requires a low sample volume, which streamlines the immunoaffinity purification (IP) and C18 peptide cleanup on a single microfluidics platform with automated liquid handling and minimal sample transfers, resulting in higher assay sensitivity. We also demonstrate how the state-of-the-art data-independent acquisition (DIA) method further enhances the depth of tandem MS spectra-based peptide sequencing. Consequently, over 4,000 and 5,000 HLA-I-restricted peptides were identified from as few as 0.2 million RA957 cells and a melanoma tissue of merely 5 mg, respectively. We also identified multiple immunogenic tumor-associated antigens and hundreds of peptides derived from non-canonical protein sources. This workflow represents a powerful tool for identifying the immunopeptidome of sparse samples.