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Application of Skyline for Analysis of Protein–Protein Interactions In Vivo
Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these int...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8658920/ https://www.ncbi.nlm.nih.gov/pubmed/34885753 http://dx.doi.org/10.3390/molecules26237170 |
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author | Kulyyassov, Arman |
author_facet | Kulyyassov, Arman |
author_sort | Kulyyassov, Arman |
collection | PubMed |
description | Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories. |
format | Online Article Text |
id | pubmed-8658920 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-86589202021-12-10 Application of Skyline for Analysis of Protein–Protein Interactions In Vivo Kulyyassov, Arman Molecules Article Quantitative and qualitative analyses of cell protein composition using liquid chromatography/tandem mass spectrometry are now standard techniques in biological and clinical research. However, the quantitative analysis of protein–protein interactions (PPIs) in cells is also important since these interactions are the bases of many processes, such as the cell cycle and signaling pathways. This paper describes the application of Skyline software for the identification and quantification of the biotinylated form of the biotin acceptor peptide (BAP) tag, which is a marker of in vivo PPIs. The tag was used in the Proximity Utilizing Biotinylation (PUB) method, which is based on the co-expression of BAP-X and BirA-Y in mammalian cells, where X or Y are interacting proteins of interest. A high level of biotinylation was detected in the model experiments where X and Y were pluripotency transcription factors Sox2 and Oct4, or heterochromatin protein HP1γ. MRM data processed by Skyline were normalized and recalculated. Ratios of biotinylation levels in experiment versus controls were 86 ± 6 (3 h biotinylation time) and 71 ± 5 (9 h biotinylation time) for BAP-Sox2 + BirA-Oct4 and 32 ± 3 (4 h biotinylation time) for BAP-HP1γ + BirA-HP1γ experiments. Skyline can also be applied for the analysis and identification of PPIs from shotgun proteomics data downloaded from publicly available datasets and repositories. MDPI 2021-11-26 /pmc/articles/PMC8658920/ /pubmed/34885753 http://dx.doi.org/10.3390/molecules26237170 Text en © 2021 by the author. 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 Kulyyassov, Arman Application of Skyline for Analysis of Protein–Protein Interactions In Vivo |
title | Application of Skyline for Analysis of Protein–Protein Interactions In Vivo |
title_full | Application of Skyline for Analysis of Protein–Protein Interactions In Vivo |
title_fullStr | Application of Skyline for Analysis of Protein–Protein Interactions In Vivo |
title_full_unstemmed | Application of Skyline for Analysis of Protein–Protein Interactions In Vivo |
title_short | Application of Skyline for Analysis of Protein–Protein Interactions In Vivo |
title_sort | application of skyline for analysis of protein–protein interactions in vivo |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8658920/ https://www.ncbi.nlm.nih.gov/pubmed/34885753 http://dx.doi.org/10.3390/molecules26237170 |
work_keys_str_mv | AT kulyyassovarman applicationofskylineforanalysisofproteinproteininteractionsinvivo |