# Dimethyl Sulfoxide Enhances HLA Peptide Identification

## TL;DR

Adding dimethyl sulfoxide (DMSO) to mass spectrometry workflows improves detection of HLA-bound peptides, enhancing sensitivity and reliability in immunopeptidomics.

## Contribution

Demonstrates that 5% DMSO in mobile-phase buffers systematically improves peptide identification and fragmentation in immunopeptidomics.

## Key findings

- DMSO increased peptide identifications by ~1.33-fold for HLA class I, ~1.55-fold for HLA class II, and ~1.24-fold for elastase digests.
- DMSO improved ionisation efficiency, charge states, and MS2 spectral quality with ~2-fold higher b- and y-ion intensities.
- DMSO did not introduce sequence bias, preserving motif integrity and binding characteristics of identified peptides.

## Abstract

Background: Mass spectrometry (MS)-based immunopeptidomics has emerged as the gold standard for profiling HLA-bound peptides, yet detection remains challenging due to their non-tryptic nature, variable lengths, and lack of basic residues, which limit ionisation and fragmentation efficiency. Methods: To address these limitations, we investigated the impact of incorporating 5% dimethyl sulfoxide (DMSO) into LC-MS/MS mobile-phase buffers on immunopeptidomic workflows. Using B-lymphoblastoid cell lines expressing HLA class I and II alleles and elastase-digested HeLa lysates as a surrogate for non-tryptic peptides, we assessed peptide identification, ionisation efficiency, charge state distribution, and fragmentation quality. Results: DMSO significantly increased peptide identifications across all sample types, with gains of ~1.33 folds for HLA class I, ~1.55 folds for HLA class II, and ~1.24 folds for elastase digests. Improvements were systematic and reproducible, driven by enhanced electrospray ionisation, higher charge states, and superior MS2 spectral quality, evidenced by ~2-fold increase in b- and y-ion intensities. Importantly, DMSO did not introduce major sequence bias, preserving motif integrity and predicted binding characteristics. Conclusions: Overall, these findings establish DMSO as a robust additive for improving sensitivity and reliability in immunopeptidomics, particularly for low-input or clinically derived samples.