Dataset Information

Dimethyl Sulfoxide Enhances HLA Peptide Identification

ABSTRACT: Immunopeptidomics relies on LC-MS/MS for comprehensive profiling of 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. 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. DMSO significantly increased peptide identifications across all sample types, with gains of ~1.33-fold for HLA class I, ~1.55-fold for HLA class II, and ~1.24-fold for elastase digests. Improvements were systematic and reproducible, driven by enhanced electrospray ionisation, higher charge states, and superior MS2 spectral quality, evidenced by a two-fold increase in b- and y-ion intensities. Importantly, DMSO did not introduce major sequence bias, preserving motif integrity and predicted binding characteristics. Overall, these findings establish DMSO as a robust additive for improving sensitivity and reliability in immunopeptidomics, particularly for low-input or clinically derived samples.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell

SUBMITTER: Terry Lim

LAB HEAD: Pouya Faridi

PROVIDER: PXD072997 | Pride | 2026-03-30

REPOSITORIES: Pride

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Dimethyl Sulfoxide Enhances HLA Peptide Identification.

Lim Kam Sian Terry C C TCC Ding Yue Y Blundell Scott A SA Schittenhelm Ralf B RB Faridi Pouya P

Proteomes 20260313 1

<b>Background:</b> 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. <b>Methods:</b> 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-lymphobl ...[more]

PMID: 41893728

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Project description:Gaining a complete and unbiased understanding of the non-tryptic peptide repertoire presented by HLA-I complexes by LC-MS/MS is indispensable for therapy design for cancer, autoimmunity and infectious diseases. A serious concern in HLA peptide analysis is that the routinely used, collision-based fragmentation methods (CID/HCD) do not always render sufficiently informative MS2 spectra, whereby gaps in the fragmentation sequence coverage prevent unambiguous assignments. EThcD can be utilized to generate complementary ion series, i.e. b/y ions and c/z ions, resulting in richer, more informative MS2 spectra, thereby filling in the gaps. Here, we present data generated on a novel hybrid Orbitrap mass spectrometer, facilitating fast and efficient hybrid fragmentation due to the implementation of EThcD in the ion routing multipole. We hypothesized that this would enable more comprehensive and less error-prone analysis of immunopeptidomes at minimal costs in duty-cycle. First, we optimized ETD/EThcD methods using an elastase-digested cell lysate, as this contains peptides of similar length and charge distributions to immunopeptides. Next, we compared HCD and EThcD on immunopeptidomes originating from three cell lines with distinct HLA-I complexes that present peptides with varying physicochemical properties. We demonstrate that the new instrument not only enables efficient and fast ETD reactions, but when combined with collision-based supplemental activation, i.e. EThcD, also consistently increases the sequence coverage and identification of peptide sequences, otherwise missed by using solely HCD. We reveal several of the biochemical properties that make HLA peptides preferably identifiable by EThcD, with internal Arg residues being one of the most dominant determinants. Finally, we demonstrate the power of EThcD for the identification and localization of HLA peptides harboring post-translational modifications, focusing here on HLA Arg mono-/di-methylation. We foresee that this new instrument with efficient EThcD capabilities enhances not only immunopeptidomics analysis, but also analysis of peptides harboring post-translational modifications and de novo sequencing.

Project description:Background: Patient derived organoids (PDOs) can be established from colorectal cancers as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFN) or MEK-inhibitors. Methods: Four PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analysed by MS. Results: We identified an average of 9,936 unique peptides per PDO which compares favourably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. Treatment of four PDOs with IFN upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFN-inducible genes. HLA class II presented peptides increased on average 16-fold with IFN treatment. MEK-inhibitor treatment showed no consistent effect on class I or II HLA expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. Conclusions: Only 3 out of 612 non-synonymous mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

			Action	DRS
	ClassII_peptides.csv	Csv
	ClassI_peptides.csv	Csv
	Elastase_DDA_CTRL_Rep1.raw	Raw
	Elastase_DDA_CTRL_Rep2.raw	Raw
	Elastase_DDA_CTRL_Rep3.raw	Raw