Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

High-Throughput Discovery and Validation of Post-Translational Modifications on HLA-Presented Peptides from Patient Tumor Samples (#821)

Robert Salzler 1 , Darshit Shah 1 , Lisa Chen 1 , David DiLillo 1 , Olav Olsen 1 , William Olson 1
  1. Regeneron, Tarrytown, NEW YORK, United States

Introduction:  Tumor specific antigens can be processed and presented by HLA class I alleles just as normal ‘self” proteins, adding to the immunopeptidome. High Resolution Accurate Mass Mass Spectrometry (HRAM-MS) is the tool of choice to interrogate the immunopeptidome in an accurate, systematic and unbiased manner. Post-translational modifications (PTMs) are known to play a critical role in the etiology of cancer and other disease states. Cancer-associated post-translational modifications provides a potential source of tumor-specific epitopes (1,2,3,4).


Goal: The objective of this study was to use HRAM-MS to detect aberrant PTMs (methylation, phosphorylation, deamidation, citrullination, acetylation etc.) from HLA Class I presented tumor-specific peptides.


Method: We used an optimized workflow consisting of anti-HLA affinity chromatography, C18 solid phase capture and selective elution follow by LC-HRAM-MS for immunopeptidomics experiments. Data analyisis was performed using  PEAKS Studio X (BSI) database searching workflow incorporating PEAKS PTM which is specifically designed to discover hidden modifications by integrating the powerful de novo sequencing algorithm and database searching.

Results: Typically, ~7000 unique HLA-associated peptides were identified in a single 120 minutes LC-MS analysis using 1 × 108 cells or 0.5 g of fresh frozen tumor tissue. In this study, we identified a robust pattern of preferential binding of peptides with PTMs to specific HLA molecules such as methylated peptides to HLA-B*07 (Class I) from both cell lines and a variety of human tumors. There were also several HLA peptides with PTMs detected consistently in our study which were predicted binders to a wide array of HLA Class I alleles.

Conclusion: Our current workflow routinely detects HLA-peptides with specific aberrant PTMs potentially providing a unique source of disease related HLA class I presented peptides that can trigger specific immune response and hence provides additional opportunity for targeted immunotherapy.