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

• Published in: Cell reports methods Vol. 3; no. 6; p. 100479
• Main Authors: Li, Xiaokang, Pak, Hui Song, Huber, Florian, Michaux, Justine, Taillandier-Coindard, Marie, Altimiras, Emma Ricart, Bassani-Sternberg, Michal
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.06.2023; Elsevier
• Subjects: automation; data-independent acquisition; immunopeptidome; mass spectrometry; Microfluidics; Peptides - chemistry; Proteomics - methods; sample preparation; Tandem Mass Spectrometry; Workflow; CP: Immunology; automation; immunopeptidome; mass spectrometry; microfluidics; sample preparation; CP: Biotechnology; data-independent acquisition
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2023.100479

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. [Display omitted] •An automated microfluidics system with enhanced sensitivity for immunopeptidomics•DIA methods are exploited for in-depth and reliable immunopeptidomics analyses•A comprehensive immunopeptidomics spectral library for DIA analyses Mass spectrometry (MS) is to date the gold-standard method for decoding the immunopeptidome of cell lines or clinical tumor tissues. Although the recent improvements of MS instrumentation and subsequent data analytics tools have enhanced the depth of data interpretation, the overall assay sensitivity is still largely limited by the conventional sample preparation methods. To enhance the sensitivity of the traditional sample preparation of MS-based immunopeptidomics, we introduced an automated workflow to purify immunopeptidome from scarce cell and tumor samples. Recent improvements in mass spectrometry instrumentation and data analysis have advanced the field of immunopeptidomics. Nonetheless, the assay sensitivity is still largely limited, especially for low-input samples. Li et al. introduce an automated microfluidics workflow for sensitive immunopeptidomics and demonstrate the detection of tumor antigens from melanoma tissues.