HLA presentation plays a critical role in immune surveillance. While our current understanding of the antigen presentation pathway and machinery has advanced in the past decade, there is currently a lack of small molecules capable of modulating antigen presentation. Identifying such molecules can expand our research toolbox and open the opportunity for drug development.

To identify small molecules that can modulate HLA-I and HLA-II expression in tumor cells, we screened a library of 1,500 cysteine reactive electrophilic compounds using flow cytometry. We identified molecules that regulate HLA-I, HLA-II or both HLA-I and HLA-II expression, suggesting that different molecules modulate HLA expression by distinct mechanisms. We utilized a multi-proteomic approach to uncover the mechanism by which the compounds modulate HLA expression and understand their effect on the cells. Activity-based protein profiling (ABPP) was performed to identify which cysteines are targeted by each compound, and in-depth quantitative whole proteome and phospho-proteome analyses to identify the proteome-wide changes by the compound.

Combining the information gathered by the different approaches, allowed us to identify the pathways involved in the compound's mechanisms of action. Interestingly, from the changes in cysteine availability (ABPP) results, we could infer not only the cysteine position that the compound binds to, but also changes in protein-protein interactions resulting from the compound binding.

Further, we investigated how these compounds affect the HLA immunopeptidome repertoire of the cells using immunopeptidomics. For the immunopeptidome analysis, we harnessed the advanced capabilities of the Orbitrap Astral Mass Spectrometer platform towards characterizing the HLA peptide repertoire of the treated and untreated cells. The Orbitrap Atral"s high resolution and sensitivity allow accurate peptide identification, even of low-abundance peptides. This is particularly useful for HLA immunopeptidome analysis. We explored and optimized several data acquisition parameters including mass analyzers (Orbitrap vs. Astral) and FAIMS compensation voltages (CV). Our optimized approach enabled the identification of thousands of unique HLA-I peptides in a single experiment, with peptides conforming to expected lengths and HLA allele motifs.