Dataset Information

Antibodies to biotin enable large-scale detection of biotinylation sites on proteins.

ABSTRACT: Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.

SUBMITTER: Udeshi ND

PROVIDER: S-EPMC8954634 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Antibodies to biotin enable large-scale detection of biotinylation sites on proteins.

Udeshi Namrata D ND Pedram Kayvon K Svinkina Tanya T Fereshetian Shaunt S Myers Samuel A SA Aygun Ozan O Krug Karsten K Clauser Karl K Ryan Dominic D Ast Tslil T Mootha Vamsi K VK Ting Alice Y AY Carr Steven A SA

Nature methods 20171016 12

Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites ...[more]

PMID: 29039416

Similar Datasets

Project description:BackgroundThe total number of known three-dimensional protein structures is rapidly increasing. Consequently, the need for fast structural search against complete databases without a significant loss of accuracy is increasingly demanding. Recently, TopSearch, an ultra-fast method for finding rigid structural relationships between a query structure and the complete Protein Data Bank (PDB), at the multi-chain level, has been released. However, comparable accurate flexible structural aligners to perform efficient whole database searches of multi-domain proteins are not yet available. The availability of such a tool is critical for a sustainable boosting of biological discovery.ResultsHere we report on the development of a new method for the fast and flexible comparison of protein structure chains. The method relies on the calculation of 2D matrices containing a description of the three-dimensional arrangement of secondary structure elements (angles and distances). The comparison involves the matching of an ensemble of substructures through a nested-two-steps dynamic programming algorithm. The unique features of this new approach are the integration and trade-off balancing of the following: 1) speed, 2) accuracy and 3) global and semiglobal flexible structure alignment by integration of local substructure matching. The comparison, and matching with competitive accuracy, of one medium sized (250-aa) query structure against the complete PDB database (216,322 protein chains) takes about 8 min using an average desktop computer. The method is at least 2-3 orders of magnitude faster than other tested tools with similar accuracy. We validate the performance of the method for fold and superfamily assignment in a large benchmark set of protein structures. We finally provide a series of examples to illustrate the usefulness of this method and its application in biological discovery.ConclusionsThe method is able to detect partial structure matching, rigid body shifts, conformational changes and tolerates substantial structural variation arising from insertions, deletions and sequence divergence, as well as structural convergence of unrelated proteins.

Project description:Proteins can sample a broad landscape as they undergo conformational transition between different functional states. At the same time, as key players in almost all cellular processes, proteins are important drug targets. Considering the different conformational states of a protein is therefore central for a successful drug-design strategy. Here we introduce a novel docking protocol, termed extended-ensemble docking, pertaining to proteins that undergo large-scale (global) conformational changes during their function. In its application to multidrug ABC-transporter P-glycoprotein (Pgp), extensive non-equilibrium molecular dynamics simulations employing system-specific collective variables are first used to describe the transition cycle of the transporter. An extended set of conformations (extended ensemble) representing the full transition cycle between the inward- and the outward-facing states is then used to seed high-throughput docking calculations of known substrates, non-substrates, and modulators of the transporter. Large differences are predicted in the binding affinities to different conformations, with compounds showing stronger binding affinities to intermediate conformations compared to the starting crystal structure. Hierarchical clustering of the binding modes shows all ligands preferably bind to the large central cavity of the protein, formed at the apex of the transmembrane domain (TMD), whereas only small binding populations are observed in the previously described R and H sites present within the individual TMD leaflets. Based on the results, the central cavity is further divided into two major subsites, first preferably binding smaller substrates and high-affinity inhibitors, whereas the second one shows preference for larger substrates and low-affinity modulators. These central subsites along with the low-affinity interaction sites present within the individual TMD leaflets may respectively correspond to the proposed high- and low-affinity binding sites in Pgp. We propose further an optimization strategy for developing more potent inhibitors of Pgp, based on increasing its specificity to the extended ensemble of the protein, instead of using a single protein structure, as well as its selectivity for the high-affinity binding site. In contrast to earlier in silico studies using single static structures of Pgp, our results show better agreement with experimental studies, pointing to the importance of incorporating the global conformational flexibility of proteins in future drug-discovery endeavors.

Dataset Information

Antibodies to biotin enable large-scale detection of biotinylation sites on proteins.

Publications

Antibodies to biotin enable large-scale detection of biotinylation sites on proteins.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets