Poster Presentation Melbourne Immunotherapy Spring Symposium 2025

Multi-dimensional Immunopeptidomic Analysis of Tumour, PBMC, and Plasma in Glioma (#141)

Erwin Tanuwidjaya 1 2 3 , Gwo Yaw Ho 2 3 , Joshua D Ooi 2 3 , Janet Chang 2 3 , Ralf B Schittenhelm 1 , Pouya Faridi 1 2 3
  1. Monash Proteomics & Metabolomics Platform, Monash University, Clayton, Victoria, Australia
  2. School of Clinical Sciences, Monash Health, Clayton, Victoria, Australia
  3. Hudson Institute of Medical Research, Clayton, Victoria, Australia

Introduction:

The human leukocyte antigen (HLA) complexes are pivotal in guiding human adaptive immune responses through their presentation of peptide ligands derived from extracellular and intracellular proteins. Characterizing this peptide repertoire, collectively known as the immunopeptidome, is critical in understanding the biology of adaptive immune defence and is central to the development of personalized cancer immunotherapies. Our recent developments in mass spectrometry (MS)-based immunopeptidomics workflow have enabled studies on smaller input amounts of cells and tissues, and more recently from biofluids. To determine whether plasma HLA peptidome could represent tumor-derived HLA peptidome repertoire, here we use our SAPrIm2.0 workflow to dissect the profiles of the immunopeptidome of tumor, plasma, and peripheral blood mononuclear cells (PBMC) derived from three glioma patient donors under the Brain on Monash Live Bio-Banking (MoLBi) initiative.

 

Method:

Immunopeptidomics profiling was performed on tumor, plasma and PBMC derived from three (1 glioma and 2 glioblastoma) patients. These samples were subjected to the SAPrIm2.0 workflow, leveraging the KingFisher instrument for automated peptide-HLA immunoaffinity purification. Liquid Chromatography/Mass Spectrometry analyses on the enriched immunopeptides were performed using Orbitrap Exploris 480 mass spectrometer operating on data-independent acquisition (DIA). RAW files were analyzed using PEAKS Studio 12.5 DeepNovo library-free workflow. Peptide identification and bioinformatics analysis was conducted using R, and peptide binding prediction analysis was done on NetMHCpan-4.1.

 

Result:

Here, we highlight the findings of the Brain on MoLBi initiative on 3 glioma patients. More than 17,000 unique HLA-I immunopeptides are identified from the 3 donors, which are predicted to bind to the donor alleles. A high proportion (~50%) of patient-derived peptidomes are shared between the tumor and the circulating compartments. Additionally, Pearson pairwise analyses reveal moderate correlation (0.2 < R < 0.7) observed across tissue compartments, suggesting that plasma soluble HLA peptidome provides a good representation of tumor HLA repertoire.

  1. Tanuwidjaya, et al. Front Oncol 2022. Soluble HLA Peptidome: A New Resource for Cancer Biomarkers.
  2. Ritz D, et al. Proteomics 2016. High Sensitivity HLA Class I Peptidome Analysis Enables a Precise Definition of Peptide Motifs and The Identification of Peptides from Cell Lines and Patients’ Sera.
  3. Shraibman, et al. Mol Cel Proteomics 2019. Identification of Tumor Antigens Among The HLA Peptidomes of Glioblastoma Tumors and Plasma.
  4. Wahle, et al. Mol Cel Proteomics 2024. IMBAS MS Discovers Organ Specific HLA Peptide Patterns in Plasma.
  5. Tanuwidjaya, et al. Front Immunol 2025. SAPrIm 2 0: A Semi-automated Protocol for Mid-throughput Soluble HLA Immunopeptidomics.