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Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma
We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunor...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100080/ https://www.ncbi.nlm.nih.gov/pubmed/35563452 http://dx.doi.org/10.3390/ijms23095061 |
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author | Zeneyedpour, Lona Stingl, Christoph Kros, Johan M. Sillevis Smitt, Peter A. E. Luider, Theo M. |
author_facet | Zeneyedpour, Lona Stingl, Christoph Kros, Johan M. Sillevis Smitt, Peter A. E. Luider, Theo M. |
author_sort | Zeneyedpour, Lona |
collection | PubMed |
description | We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed. |
format | Online Article Text |
id | pubmed-9100080 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91000802022-05-14 Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma Zeneyedpour, Lona Stingl, Christoph Kros, Johan M. Sillevis Smitt, Peter A. E. Luider, Theo M. Int J Mol Sci Article We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed. MDPI 2022-05-02 /pmc/articles/PMC9100080/ /pubmed/35563452 http://dx.doi.org/10.3390/ijms23095061 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Zeneyedpour, Lona Stingl, Christoph Kros, Johan M. Sillevis Smitt, Peter A. E. Luider, Theo M. Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma |
title | Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma |
title_full | Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma |
title_fullStr | Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma |
title_full_unstemmed | Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma |
title_short | Novel Antibody–Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma |
title_sort | novel antibody–peptide binding assay indicates presence of immunoglobulins against egfr phospho-site s1166 in high-grade glioma |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100080/ https://www.ncbi.nlm.nih.gov/pubmed/35563452 http://dx.doi.org/10.3390/ijms23095061 |
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