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Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method
Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of...
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/PMC9820203/ https://www.ncbi.nlm.nih.gov/pubmed/36613715 http://dx.doi.org/10.3390/ijms24010273 |
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author | Langó, Tamás Kuffa, Katalin Tóth, Gábor Turiák, Lilla Drahos, László Tusnády, Gábor E. |
author_facet | Langó, Tamás Kuffa, Katalin Tóth, Gábor Turiák, Lilla Drahos, László Tusnády, Gábor E. |
author_sort | Langó, Tamás |
collection | PubMed |
description | Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000–7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research. |
format | Online Article Text |
id | pubmed-9820203 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-98202032023-01-07 Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method Langó, Tamás Kuffa, Katalin Tóth, Gábor Turiák, Lilla Drahos, László Tusnády, Gábor E. Int J Mol Sci Article Cell surface proteins, including transmembrane and other surface-anchored proteins, play a key role in several critical cellular processes and have a strong diagnostic value. The development of quick and robust experimental methods remains vital for the accurate and comprehensive characterization of the cell surface subproteome of individual cells. Here we present a high-throughput technique which relies on the biotinylation of the accessible primary amino groups in the extracellular segments of the proteins, using HL60 as a model cell line. Several steps of the method have been thoroughly optimized to capture labeled surface proteins selectively and in larger quantities. These include the following: improving the efficiency of the cell surface biotinylation; reducing the endogen protease activity; applying an optimal amount of affinity column and elution steps for labeled peptide enrichment; and examining the effect of various solid-phase extraction methods, different HPLC gradients, and various tandem mass spectrometry settings. Using the optimized workflow, we identified at least 1700 surface-associated individual labeled peptides (~6000–7000 redundant peptides) from the model cell surface in a single nanoHPLC-MS/MS run. The presented method can provide a comprehensive and specific list of the cell surface available protein segments that could be potential targets in various bioinformatics and molecular biology research. MDPI 2022-12-23 /pmc/articles/PMC9820203/ /pubmed/36613715 http://dx.doi.org/10.3390/ijms24010273 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 Langó, Tamás Kuffa, Katalin Tóth, Gábor Turiák, Lilla Drahos, László Tusnády, Gábor E. Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method |
title | Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method |
title_full | Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method |
title_fullStr | Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method |
title_full_unstemmed | Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method |
title_short | Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method |
title_sort | comprehensive discovery of the accessible primary amino group-containing segments from cell surface proteins by fine-tuning a high-throughput biotinylation method |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9820203/ https://www.ncbi.nlm.nih.gov/pubmed/36613715 http://dx.doi.org/10.3390/ijms24010273 |
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