Pancreatic adenocarcinoma (PDAC) is a devastating cancer, leading to more than 85% mortality in the first five years after diagnosis, highlighting need for further treatment options. Human leukocyte antigen (HLA)-bound peptides at the cell surface report cellular protein expression to T lymphocytes to assist with immune surveillance and aid the detection of viral infection and cancerous cells. Immunopeptidomics, the qualitative/quantitative study of HLA-bound-peptides using tandem mass spectrometry (MS/MS), can thus enable the discovery of novel immunotherapy targets in malignancies such as PDAC. In this study, we investigated the quantitative attributes of a novel Zeno trap-enabled scanning quadrupole data-independent acquisition (ZT Scan DIA) mode on a SCIEX ZenoTOF 7600+ system.
We assessed quantitative precision using serial dilutions of a human tryptic digest in fast-micro-flow chromatographic separations. We further evaluated the accuracy of quantitation using an LFQBench approach (Human+Yeast+E.coli tryptic mixed at different ratios). The LFQBench experiments were acquired in both fast-micro-flow, and 30-min nano-flow separations. Having established the quantitative accuracy of our LC-MS/MS approaches for complex protein digest mixtures, we acquired ZT Scan DIA data from HLA-peptides isolated from the PDAC cell line Panc1 (2.5 or 25e6 cells) and analyzed the data using DIANN software and a Panc1-specific HLA peptide spectral library.
Our analyses show the superiority of ZT Scan DIA in quantitative attributes for both proteomics and immunopeptidomics purposes. Particularly, ZT Scan DIA shows versatility for rapid chromatographic separations and higher loads. LFQBench samples acquired with ZT Scan DIA demonstrate a remarkable quantitative accuracy at peptide and protein levels across different chromatographic conditions. HLA-peptide runs also show a higher quantitative precision. Moreover, ZT Scan DIA improved confidence in identifying and quantifying cancer-testis-antigen (CTA-) derived HLA-peptides, particularly a superior resolution of interfering signals in the deconvolved MS/MS spectra.
These advancements facilitate the identification of potential immunogenic targets for future immunotherapy attempts.