BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.
Dae In Kim,Jevon Cutler,Chan Hyun Na,Sina Reckel,Santosh Renuse,Anil K. Madugundu,Raiha Tahir,Hana L. Goldschmidt,Karen L. Reddy,Richard L. Huganir,Xinyan Wu,Natasha E. Zachara,Oliver Hantschel,Akhilesh Pandey +13 more
Reads0
Chats0
TLDR
The utility of BioSITe is demonstrated when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites and the use of isotopically labeled biotin for quantitative BioSITS experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC.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-specifi...read more
Citations
More filters
Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging
Hyun-Woo Rhee,Peng Zou,Namrata D. Udeshi,Jeffrey D. Martell,Vamsi K. Mootha,Vamsi K. Mootha,Steven A. Carr,Alice Y. Ting,Alice Y. Ting +8 more
TL;DR: In this article, a peroxidase-based method was used to identify 495 proteins within the human mitochondrial matrix, including 31 proteins not previously linked to mitochondria, and the labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space.
Journal ArticleDOI
Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles.
TL;DR: PDB can help to identify interactions with and amongst membrane proteins, and other polypeptide classes that are less amenable to study by standard pulldown techniques, and provide deep insight into the organization of membrane-less organelles and other subcellular structures that cannot be easily isolated or purified.
Journal ArticleDOI
Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches.
TL;DR: The structural diversity and mechanisms of the two main classes of PDB enzymes are reviewed, the biotin protein ligases (BioID) and the peroxidases (APEX) are described, and the engineering of these enzymes for PDB is described and emerging applications, including the development of P DB for coincidence detection (split-PDB).
Journal ArticleDOI
Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems.
Deepanksha Arora,Nikolaj B. Abel,Chen Liu,Petra Van Damme,Klaas Yperman,Dominique Eeckhout,Lam Dai Vu,Jie Wang,Anna Tornkvist,Francis Impens,Barbara Korbei,Jelle Van Leene,Alain Goossens,Geert De Jaeger,Thomas Ott,Panagiotis N. Moschou,Panagiotis N. Moschou,Panagiotis N. Moschou,Daniël Van Damme +18 more
TL;DR: It is shown that TurboID is the most promiscuous variant in several plant model systems and protocols that combine mass spectrometry-based analysis with harsh extraction and washing conditions are established, providing initial evidence that the approach has the potential to suggest structural information of protein complexes.
Journal ArticleDOI
Analytical and Biochemical Perspectives of Protein O-GlcNAcylation.
Junfeng Ma,Ci Wu,Gerald W. Hart +2 more
TL;DR: A comprehensive review of protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification from analytical and biochemical perspectives is presented in this article.
References
More filters
Journal ArticleDOI
A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes.
Journal ArticleDOI
Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
Shao En Ong,Blagoy Blagoev,Irina Kratchmarova,Dan B. Kristensen,Hanno Steen,Akhilesh Pandey,Matthias Mann +6 more
TL;DR: SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system and is applied to the relative quantitation of changes in protein expression during the process of muscle cell differentiation.
Journal ArticleDOI
A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells
TL;DR: Proximity-dependent biotin identification is a new approach making use of biotin ligase fusion proteins for the identification of both interacting and neighboring proteins in their native cellular environment.
Journal ArticleDOI
Cycling of O-linked β- N -acetylglucosamine on nucleocytoplasmic proteins
TL;DR: Emerging data indicate that O-GlcNAc glycosylation has a role in the aetiology of diabetes and neurodegeneration.
Journal ArticleDOI
MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins.
TL;DR: The improved MitoCarta 2.0 inventory provides a molecular framework for system-level analysis of mammalian mitochondria and helps to understand mitochondrial pathways in health and disease.