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J Proteome Res. 2018 Feb 2;17(2):759-769. doi: 10.1021/acs.jproteome.7b00775. Epub 2017 Dec 28.

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.

Author information

1
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
2
Pre-Doctoral Training Program in Human Genetics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
3
Department of Biological Chemistry, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
4
Center for Proteomics Discovery, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
5
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, École polytechnique fédérale de Lausanne (EPFL) , CH-1015 Lausanne, Switzerland.
6
Institute of Bioinformatics , International Technology Park, Bangalore 560 066, India.
7
Biochemistry, Cellular and Molecular Biology Graduate Program, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
8
Solomon H. Snyder Department of Neuroscience, Kavli Neuroscience Discovery Institute, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
9
Center for Epigenetics, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
10
Departments of Pathology and Oncology, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.

Abstract

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.

KEYWORDS:

APEX; BioID; biotinylation; peptide; protein−protein interactions; proximity-dependent biotinylation; subcellular proteome

PMID:
29249144
PMCID:
PMC6092923
DOI:
10.1021/acs.jproteome.7b00775
[Indexed for MEDLINE]
Free PMC Article

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