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

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 dea...

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Autores principales: Morales-Betanzos, Carlos A., Lee, Hyoungjoo, Gonzalez Ericsson, Paula I., Balko, Justin M., Johnson, Douglas B., Zimmerman, Lisa J., Liebler, Daniel C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The American Society for Biochemistry and Molecular Biology 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629259/
https://www.ncbi.nlm.nih.gov/pubmed/28546465
http://dx.doi.org/10.1074/mcp.RA117.000037
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author Morales-Betanzos, Carlos A.
Lee, Hyoungjoo
Gonzalez Ericsson, Paula I.
Balko, Justin M.
Johnson, Douglas B.
Zimmerman, Lisa J.
Liebler, Daniel C.
author_facet Morales-Betanzos, Carlos A.
Lee, Hyoungjoo
Gonzalez Ericsson, Paula I.
Balko, Justin M.
Johnson, Douglas B.
Zimmerman, Lisa J.
Liebler, Daniel C.
author_sort Morales-Betanzos, Carlos A.
collection PubMed
description 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(2) = 0.062, p = 0.264). PD-1 abundance was weakly correlated (r(2) = 0.3057, p = 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.
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spelling pubmed-56292592018-01-13 Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma Morales-Betanzos, Carlos A. Lee, Hyoungjoo Gonzalez Ericsson, Paula I. Balko, Justin M. Johnson, Douglas B. Zimmerman, Lisa J. Liebler, Daniel C. Mol Cell Proteomics Research 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(2) = 0.062, p = 0.264). PD-1 abundance was weakly correlated (r(2) = 0.3057, p = 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. The American Society for Biochemistry and Molecular Biology 2017-10 2017-05-25 /pmc/articles/PMC5629259/ /pubmed/28546465 http://dx.doi.org/10.1074/mcp.RA117.000037 Text en © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) .
spellingShingle Research
Morales-Betanzos, Carlos A.
Lee, Hyoungjoo
Gonzalez Ericsson, Paula I.
Balko, Justin M.
Johnson, Douglas B.
Zimmerman, Lisa J.
Liebler, Daniel C.
Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma
title Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma
title_full Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma
title_fullStr Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma
title_full_unstemmed Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma
title_short Quantitative Mass Spectrometry Analysis of PD-L1 Protein Expression, N-glycosylation and Expression Stoichiometry with PD-1 and PD-L2 in Human Melanoma
title_sort quantitative mass spectrometry analysis of pd-l1 protein expression, n-glycosylation and expression stoichiometry with pd-1 and pd-l2 in human melanoma
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629259/
https://www.ncbi.nlm.nih.gov/pubmed/28546465
http://dx.doi.org/10.1074/mcp.RA117.000037
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