Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, -glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma.

Morales-Betanzos CA, Lee H, Gonzalez Ericsson PI, Balko JM, Johnson DB, Zimmerman LJ, Liebler DC
Mol Cell Proteomics. 2017 16 (10): 1705-1717

PMID: 28546465 · PMCID: PMC5629259 · DOI:10.1074/mcp.RA117.000037

Quantitative assessment of key proteins that control the tumor-immune interface is one of the most formidable analytical challenges in immunotherapeutics. We developed a targeted MS platform to quantify programmed cell death-1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death 1 ligand 2 (PD-L2) at fmol/microgram protein levels in formalin fixed, paraffin-embedded sections from 22 human melanomas. PD-L1 abundance ranged 50-fold, from ∼0.03 to 1.5 fmol/microgram protein and the parallel reaction monitoring (PRM) data were largely concordant with total PD-L1-positive cell content, as analyzed by immunohistochemistry (IHC) with the E1L3N antibody. PD-1 was measured at levels up to 20-fold lower than PD-L1, but the abundances were not significantly correlated (r = 0.062, = 0.264). PD-1 abundance was weakly correlated (r = 0.3057, = 0.009) with the fraction of lymphocytes and histiocytes in sections. PD-L2 was measured from 0.03 to 1.90 fmol/microgram protein and the ratio of PD-L2 to PD-L1 abundance ranged from 0.03 to 2.58. In 10 samples, PD-L2 was present at more than half the level of PD-L1, which suggests that PD-L2, a higher affinity PD-1 ligand, is sufficiently abundant to contribute to T-cell downregulation. We also identified five branched mannose and N-acetylglucosamine glycans at PD-L1 position N192 in all 22 samples. Extent of PD-L1 glycan modification varied by ∼10-fold and the melanoma with the highest PD-L1 protein abundance and most abundant glycan modification yielded a very low PD-L1 IHC estimate, thus suggesting that N-glycosylation may affect IHC measurement and PD-L1 function. Additional PRM analyses quantified immune checkpoint/co-regulator proteins LAG3, IDO1, TIM-3, VISTA, and CD40, which all displayed distinct expression independent of PD-1, PD-L1, and PD-L2. Targeted MS can provide a next-generation analysis platform to advance cancer immuno-therapeutic research and diagnostics.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

MeSH Terms (20)

Acetylglucosamine Adult Aged B7-H1 Antigen Biopsy Cohort Studies Female Glycosylation Humans Male Mannose Mass Spectrometry Melanoma Middle Aged Polysaccharides Programmed Cell Death 1 Ligand 2 Protein Programmed Cell Death 1 Receptor Protein Processing, Post-Translational Skin Neoplasms T-Lymphocytes

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