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. 2023 May 3;22(6):100563. doi: 10.1016/j.mcpro.2023.100563

Fig. 1.

Fig. 1

Human leukocyte antigen (HLA) sample preparation overview and timsTOF single-cell proteomics (SCP) acquisition method optimization for analysis of HLA-I peptides enables inclusion of singly charged peptides.A, schematic overview of HLA sample preparation and mass spectrometry (MS) acquisition schemes. Serial HLA-I and HLA-II enrichment, acid elution of peptides from HLA complexes, and peptide desalting performed in a semiautomated 96-well format, followed by single-shot data-dependent acquisition (DDA) analysis of purified HLA-I and HLA-II peptides using our standard workflow with the Exploris + FAIMS or the timsTOF SCP. B, HLA-I peptide spectrum matches (PSMs) identified on the timsTOF SCP using the standard precursor filter (“polygon”) in pink and the extended polygon in green across m/z and ion mobility (IM) dimensions. Colors indicate precursor charge state. C, timsTOF SCP parameter overview, including IM range, polygon placement, target intensity value (“target”), collision energy (CE) slope, accumulation, and ramp time for M00–M10. D, number of unique HLA-I peptides from 1e7 cell equivalents from bulk sample preparation postfiltering in duplicates with M00–M10 on the timsTOF SCP. Median and standard deviation are shown. E, corresponding score distributions of HLA-I peptides for methods indicated in D. FAIMS, field asymmetric waveform ion mobility spectrometry.