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

A complementary peptide enrichment and mass spectrometric strategy for in-depth mining of immunopeptidome (#763)

Kirti Pandey 1 , Sri H Ramarathinam 1 , Nicole A Mifsud 1 , Anthony W Purcell 1
  1. Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

Background: The field of immunopeptidomics has provided critical insights into crucial aspects of antigen presentation and human leukocyte antigen (HLA) peptide interactions there by helping to deconvolute the complexity associated with immunopeptidome. HLA polymorphism also generates tremendous diversity in the processing and presentation of different peptide antigens and adds to the challenge of analysing these diverse immunopeptidomes. The use of high-resolution mass spectrometry (MS) and different peptide enrichment techniques can help in further exploring and delineating the immunopeptidome. These techniques can be exploited to unravel the immunopeptidome of cancer including leukaemia, thereby making inroads towards identifying tumour specific peptides which can be used for T cell-based vaccines or targets for CAR T cell therapies.

Methods: The eluate of 1 X 109 cells of human acute myeloid leukaemia (AML) cell line, THP-1 (HLA-A*02:01, -B*15:11 and -C*03:03) were lysed and HLA- class I bound peptides purified by immunoaffinity chromatography. The eluate from this immunoaffinity step was then further separated by two different techniques; namely off-line reverse phase HPLC (RP-HPLC) or using a molecular weight cut off filter (MWCO) followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS) on a Tribrid Orbitrap Fusion (Thermo Scientific).

Results: A total of 37675 HLA class I bound peptides were identified across the triplicate dataset with 17434 HLA A*02:01 restricted and 20241 restricted by the other two HLA class I alleles expressed on THP-1 cells. The use of the two peptide enrichment steps was shown to expand the peptide repertoire identified. Several cancer testis antigen and oncogenic peptides were identified along with post translationally modified (PTM) peptides. 

Conclusion: This study identified naturally presented peptide antigens derived from an AML cell line at unprecedented depth. This approach will aid in development of improved diagnostic and patient stratification tools along with peptide based immunotherapeutic approaches.