Purpose
Human leukocyte antigen class I (HLA-I) molecules present short peptides from endogenous or foreign proteins to cytotoxic T cells. Mass spectrometry (MS) allows for direct identification of endogenously processed and presented peptides but further improvements in accuracy, sensitivity and accurate quantification across samples are needed. Here, we evaluate whether data independent acquisition (DIA) in combination with ion mobility can expand immunopeptidome depth and facilitate quantification of tumor specific peptides.
Methods
HLA-peptide complexes were enriched from mammalian cells using a semiautomated workflow (Agilent). Acid eluted peptides were analyzed by high-resolution LC-MS/MS on a timsTOF SCP (Bruker) coupled with a VanquishNeo (Thermo Fisher). For library generation, peptides were fractionated and analyzed with data dependent acquisition (DDA) for 67 min. For DIA experiments, peptides were eluted over 67 min without prior fractionation. Mass spectra were interpreted using Fragpipe, DIANN, or Spectronaut. A375 cells were cultured in SILAC media with Lysine-8, Arginine-10 and Leucine-6 and mixed to a total of 20e6 cells per enrichment with varying heavy to light ratios.
Results
We first optimized DIA parameters on HLA-I peptides from A375 cells and identified 11,412 unique peptides from 12.5e6 cells across 4 technical replicates. DIA analysis of peptides enriched from 5e5, 1e6, 5e6 and 10e6 cells increased immunopeptidome coverage in each condition compared to our standard DDA workflow, with up to 3,500 peptides identified from 5e5 cells. To evaluate quantitative accuracy, we enriched HLA peptides from heavy and light labeled cells and found that quantification is accurate up to five fold dilution. Next, we used this approach to identify tumor specific peptides in a C1R cell line model.
Conclusion
The sensitivity and quantitative accuracy provided by DIA can increase the peptidome coverage for lower sample amounts and enable the detection of less abundant peptide species such as neoantigens.