High-throughput and Sensitive Immunopeptidomics Platform Reveals Profound Interferonγ-Mediated Remodeling of the Human Leukocyte Antigen (HLA) Ligandome

Abstract

Comprehensive knowledge of the human leukocyte antigen (HLA) class-I and class-II peptides presented to T-cells is crucial for designing innovative therapeutics against cancer and other diseases. However methodologies for their purification for mass-spectrometry analysis have been a major limitation. We designed a novel high-throughput, reproducible and sensitive method for sequential immuno-affinity purification of HLA-I and -II peptides from up to 96 samples in a plate format, suitable for both cell lines and tissues. Our methodology drastically reduces sample-handling and can be completed within five hours. We challenged our methodology by extracting HLA peptides from multiple replicates of tissues (n = 7) and cell lines (n = 21, 108 cells per replicate), which resulted in unprecedented depth, sensitivity and high reproducibility (Pearson correlations up to 0.98 and 0.97 for HLA-I and HLA-II). Because of the method's achieved sensitivity, even single measurements of peptides purified from 107 B-cells resulted in the identification of more than 1700 HLA-I and 2200 HLA-II peptides. We demonstrate the feasibility of performing drug-screening by using ovarian cancer cells treated with interferon gamma (IFNγ). Our analysis revealed an augmented presentation of chymotryptic-like and longer ligands associated with IFNγ induced changes of the antigen processing and presentation machinery. This straightforward method is applicable for basic and clinical applications.

Mapping the tumour human leukocyte antigen (HLA) ligandome by mass spectrometry

“…Antibodies can be either covalently coupled to sepharose or agarose resins or non‐covalently attached to Protein A or Protein G. Different commercial cross‐linking technologies are available, such as CNBr‐activated sepharose or AminoLink™ coupling resin, which employs aldehyde‐activated 4% beaded agarose . Sometimes the lysate is precleared from native antibodies before immunoaffinity chromatography with Protein A or Protein G …”

“…Peptides are frequently separated from HLA fragments through ultrafiltration, employing a molecular weight filter. Other separation methods employ C 18 fractionation through spin columns, cartridges, tips or in‐house‐generated columns . Sample concentration and purification is essential before mass spectrometry (MS), and it is usually achieved via C 18 or strong cation exchange (SCX) prefractionation …”

“…The IP method has been improved to allow for miniaturised, parallelised sample preparation. This enables higher sample throughput that is compatible with clinical settings …”

“…Commonly, HLA class I and class II peptide isolation is achieved from cell lysates by IP. Cell suspensions or solid tissues are mechanically homogenised and lysed, employing non-denaturing detergents, such as NP-40, [45][46][47] Triton X-100, 43 CHAPS, 1,48 sodium deoxycholate 49,50 or IGEPAL CA-630. 51,52 Lysis buffers contain protease inhibitors to block degradation of HLA-peptide complexes.…”

“…Optionally, phosphatase inhibitors can be added to prevent dephosphorylation of phosphopeptides. 51,53 In some cases, iodacetamide is added as well to alkylate cysteins, 45,49,50,52 thereby inhibiting disulphide bond formation.…”