Finely tuned DDA-PASEF-based immunopeptidomics workflow enhances HLA class I coverage
- 1Laboratoire de Spectrométrie de Masse Bio-Organique (LSMBO), IPHC, UMR 7178, Université de Strasbourg, CNRS, 67087 Strasbourg, France
Abstract
Mass spectrometry-based immunopeptidomics is essential for the direct identification of HLA-presented peptides, but its application to low-input and clinical samples remains challenging. In this study, we present an optimized DDA-PASEF-based workflow to improve HLA class I peptide coverage. First, we optimized a magnetic bead-based sample preparation protocol to support analysis from reduced starting material and increase compatibility with automation. We then evaluated LC gradient length and column dimensions to define complementary configurations for high-throughput and high-coverage analysis. For MS acquisition, we fine-tuned key parameters, including ion accumulation time, number of PASEF scans, ion mobility range, and polygon filtering, to improve sensitivity while maintaining a suitable cycle time and limiting pre-CID truncations. The customized polygon filter improved the detection of singly charged HLA class I peptides, thereby broadening the identified immunopeptide repertoire. Finally, TIMS2Rescore was integrated to improve confidence in peptide identification. Overall, this optimized workflow improves HLA class I immunopeptide coverage across different sample types, even from low injected sample amounts, supporting its future applicability to clinically relevant samples.
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BibTeX
@misc{rijal2026finely,
title = {{Finely tuned DDA-PASEF-based immunopeptidomics workflow enhances HLA class I coverage}},
author = {J. B. Rijal and A. Hirschler and A. Declercq and M. Sommer and C. Keller and A. Jung and A. Burel and L.-M. Fornecker and D. Rolland and R. Gabriels and L. Martens and C. Carapito},
howpublished = {bioRxiv},
year = {2026},
month = nov,
doi = {10.64898/2026.11.03.761330},
url = {https://www.biorxiv.org/content/10.64898/2026.11.03.761330v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Finely tuned DDA-PASEF-based immunopeptidomics workflow enhances HLA class I coverage
AU - J. B. Rijal
AU - A. Hirschler
AU - A. Declercq
AU - M. Sommer
AU - C. Keller
AU - A. Jung
AU - A. Burel
AU - L.-M. Fornecker
AU - D. Rolland
AU - R. Gabriels
AU - L. Martens
AU - C. Carapito
PY - 2026
DA - 2026/11/03
PB - bioRxiv
DO - 10.64898/2026.11.03.761330
UR - https://www.biorxiv.org/content/10.64898/2026.11.03.761330v1
AB - Mass spectrometry-based immunopeptidomics is essential for the direct identification of HLA-presented peptides, but its application to low-input and clinical samples remains challenging. In this study, we present an optimized DDA-PASEF-based workflow to improve HLA class I peptide coverage. First, we optimized a magnetic bead-based sample preparation protocol to support analysis from reduced starting material and increase compatibility with automation. We then evaluated LC gradient length and column dimensions to define complementary configurations for high-throughput and high-coverage analysis. For MS acquisition, we fine-tuned key parameters, including ion accumulation time, number of PASEF scans, ion mobility range, and polygon filtering, to improve sensitivity while maintaining a suitable cycle time and limiting pre-CID truncations. The customized polygon filter improved the detection of singly charged HLA class I peptides, thereby broadening the identified immunopeptide repertoire. Finally, TIMS2Rescore was integrated to improve confidence in peptide identification. Overall, this optimized workflow improves HLA class I immunopeptide coverage across different sample types, even from low injected sample amounts, supporting its future applicability to clinically relevant samples.
ER -