SAPrIm, a semi-automated protocol for mid-throughput immunopeptidomics

Terry C C Lim Kam Sian; Gabriel Goncalves; Joel R Steele; Tima Shamekhi; Liesl Bramberger; Dongbin Jin; Mohammad Shahbazy; Anthony W Purcell; Sri Ramarathinam; Stoyan Stoychev; Pouya Faridi

doi:10.3389/fimmu.2023.1107576

SAPrIm, a semi-automated protocol for mid-throughput immunopeptidomics

Front Immunol. 2023 Jun 2:14:1107576. doi: 10.3389/fimmu.2023.1107576. eCollection 2023.

Authors

Terry C C Lim Kam Sian^{1

2}, Gabriel Goncalves^{1

3}, Joel R Steele², Tima Shamekhi¹, Liesl Bramberger¹, Dongbin Jin¹, Mohammad Shahbazy^{1

3}, Anthony W Purcell³, Sri Ramarathinam³, Stoyan Stoychev⁴, Pouya Faridi^{1

2}

Affiliations

¹ Department of Medicine, School of Clinical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Clayton, VIC, Australia.
² Monash Proteomics and Metabolomics Platform, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
³ Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
⁴ ReSyn Biosciences (Pty) Ltd., Pretoria, South Africa.

Abstract

Human leukocyte antigen (HLA) molecules play a crucial role in directing adaptive immune responses based on the nature of their peptide ligands, collectively coined the immunopeptidome. As such, the study of HLA molecules has been of major interest in the development of cancer immunotherapies such as vaccines and T-cell therapies. Hence, a comprehensive understanding and profiling of the immunopeptidome is required to foster the growth of these personalised solutions. We herein describe SAPrIm, an Immunopeptidomics tool for the Mid-Throughput era. This is a semi-automated workflow involving the KingFisher platform to isolate immunopeptidomes using anti-HLA antibodies coupled to a hyper-porous magnetic protein A microbead, a variable window data independent acquisition (DIA) method and the ability to run up to 12 samples in parallel. Using this workflow, we were able to concordantly identify and quantify ~400 - 13000 unique peptides from 5e5 - 5e7 cells, respectively. Overall, we propose that the application of this workflow will be crucial for the future of immunopeptidome profiling, especially for mid-size cohorts and comparative immunopeptidomics studies.

Keywords: DIA; HLA-bound peptides; cancer antigen; human leukocyte antigen; immunopeptidomics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

HLA Antigens
Histocompatibility Antigens Class I*
Histocompatibility Antigens Class II
Humans
Immunotherapy
Peptides*

Substances

Histocompatibility Antigens Class I
Peptides
HLA Antigens
Histocompatibility Antigens Class II

Grants and funding

PF was supported by the Victorian Department of Health and Human Services acting through the Victorian Cancer Agency. This project was supported by grant 2001870 awarded through the 2020 Priority-driven Collaborative Cancer Research Scheme and co-funded by Cancer Australia and the Australian Lions Childhood Cancer Research Foundation and My Room Children’s Cancer Charity Ltd.