Finely tuned DDA-PASEF-based immunopeptidomics workflow enhances HLA class I coverage

A finely tuned DDA-PASEF immunopeptidomics workflow increases detection of singly charged HLA class I peptides, broadening the identified immunopeptide repertoire.

• Why it matters: Immunopeptidomics is needed to directly identify HLA-presented peptides, but low-input and clinical samples are hard to analyze with sufficient coverage and sensitivity.
• What they did: The authors optimized magnetic bead sample prep for reduced starting material and automation, tuned LC gradient and column dimensions, and adjusted DDA-PASEF acquisition settings (including ion accumulation time, PASEF scans, ion mobility range, and polygon filtering).
• The result: A customized polygon filter improved singly charged HLA class I peptide detection, and integrating TIMS2Rescore increased identification confidence, enabling higher HLA class I coverage across multiple sample types, including low injected amounts.

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