In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Kulyyassov, Arman; Ogryzko, Vasily

doi:10.3390/biom10010142

Cited by 7 publications

(12 citation statements)

References 40 publications

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“…Since the PUB is LC-MS/MS oriented method, experiments were also performed to identify and analyze the new peptide BAP1108 using mass spectrometry. As previously shown [38], the use of on-bead digestion in sample preparation containing BAP1070 reduced the time and number of steps in the protocol. For example, the steps of imidazole elution, polyacrylamide gel electrophoresis, staining and destaining, gel cutting, and subsequent washing of gel pieces were eliminated.…”

Section: Design Generation and Properties Of Bap Peptidesmentioning

confidence: 74%

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Kulyyassov

Ramankulov

Ogryzko

2022

Life

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Protein tags are peptide sequences genetically embedded into a recombinant protein for various purposes, such as affinity purification, Western blotting, and immunofluorescence. Another recent application of peptide tags is in vivo labeling and analysis of protein–protein interactions (PPI) by proteomics methods. One of the common workflows involves site-specific in vivo biotinylation of an AviTag-fused protein in the presence of the biotin ligase BirA. However, due to the rapid kinetics of labeling, this tag is not ideal for analysis of PPI. Here we describe the rationale, design, and protocol for the new biotin acceptor peptides BAP1070 and BAP1108 using modular assembling of biotin acceptor fragments, DNA sequencing, transient expression of proteins in cells, and Western blotting methods. These tags were used in the Proximity Utilizing Biotinylation (PUB) method, which is based on coexpression of BAP-X and BirA-Y in mammalian cells, where X or Y are candidate interacting proteins of interest. By changing the sequence of these peptides, a low level of background biotinylation is achieved, which occurs due to random collisions of proteins in cells. Over 100 plasmid constructs, containing genes of transcription factors, histones, gene repressors, and other nuclear proteins were obtained during implementation of projects related to this method.

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Section: Design Generation and Properties Of Bap Peptidesmentioning

confidence: 74%

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Kulyyassov

Ramankulov

Ogryzko

2022

Life

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“…The vendors’ default method (Bruker Company) was used for the creation of the MRM method, as described earlier [ 34 ]. Precursor ions: m / z 563.2 (ILEAQK(Prop)IVR) propionylated form of BAP, and m / z 648.8 (ILEAQK(Biot)IVR) biotinylated form of BAP.…”

Section: Resultsmentioning

confidence: 99%

“…Contrary to Sox2, GFP lacks DNA binding domains, and in a control experiment with the coexpression of BAP-GFP and BirA-Oct4, very low biotinylation levels were observed (sample 0–9 on Figure 3 A). Recombinant proteins BAP-GFP and BAP-Sox2 from HEK293T cell nuclear lysates were purified on Ni sepharose beads and propionylated before trypsin digest, as described earlier [ 34 ].…”

Section: Resultsmentioning

confidence: 99%

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Application of Skyline for Analysis of Protein–Protein Interactions In Vivo

Kulyyassov

2021

Molecules

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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.

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“…In our laboratory, we also studied protein-protein interactions using MRM. For in vivo detection and quantitative analysis of the interactions of transcription factors Sox2 and Oct4 [169], we used the proximity utilizing biotinylation method (PUB). This method is based on coexpression of two recombinant proteins, one of which is fused with the biotin acceptor peptide (BAP), and the second is fused with wild type biotin ligase BirA [170].…”

Section: Biological Researchmentioning

confidence: 99%

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

2021

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Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is now the main analytical method for the identification and quantification of peptides and proteins in biological samples. In modern research, identification of biomarkers and their quantitative comparison between samples are becoming increasingly important for discovery, validation, and monitoring. Such data can be obtained following specific signals after fragmentation of peptides using multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM) methods, with high specificity, accuracy, and reproducibility. In addition, these methods allow measurement of the amount of posttranslationally modified forms and isoforms of proteins. This review article describes the basic principles of MRM assays, guidelines for sample preparation, recent advanced MRM-based strategies, applications and illustrative perspectives of MRM/PRM methods in clinical research and molecular biology.

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In Vivo Quantitative Estimation of DNA-Dependent Interaction of Sox2 and Oct4 Using BirA-Catalyzed Site-Specific Biotinylation

Cited by 7 publications

References 40 publications

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Generation of Peptides for Highly Efficient Proximity Utilizing Site-Specific Biotinylation in Cells

Application of Skyline for Analysis of Protein–Protein Interactions In Vivo

Targeted liquid chromatography‐tandem mass spectrometry analysis of proteins: Basic principles, applications, and perspectives

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