Antibodies to biotin enable large-scale detection of biotinylation sites on proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Steven A. Carr
PROVIDER: MSV000081496 | MassIVE | Thu Aug 31 14:42:00 BST 2017
REPOSITORIES: MassIVE
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Project description:Proximity-dependent biotinylation methods, such as those mediated by BioID and APEX, are being widely adopted for studying in vivo protein-protein interaction dynamics and subcellular structures. However, these along with other biotin-based technologies are severely dependent on avidin family proteins for the capture of biotinylated proteins. While the extremely high affinity of the avidin-biotin interaction nearly guarantees the capture of biotinylated proteins from a sample, it also results in an inability to recover the key species of interest: the biotin-modified peptide. To overcome this limitation, we have developed a novel strategy that instead relies on an immunoaffinity-based capture of biotinylated peptides for downstream LC-MS/MS analysis. We refer to this approach as Biotinylation Site Identification Technology (BioSITe), since it enables the direct detection of site-specific biotinylation, which substantially increases the sensitivity and specificity of data offered by proximity-dependent biotinylation methods. Using isotopically labeled biotin, we also demonstrate that this method can be used for differential analysis. Overall, we anticipate this method to replace the current methods used for detection of biotinylation sites and other applications including those employing any molecule probe for enrichment of specific classes of proteins or post-translational modifications.
2017-12-19 | PXD007862 | Pride
Project description:Background: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite the development of several enrichment methods exploiting their biochemical and biophysical properties. Methods: We report a novel method for enrichment of proteins localized to the cell surface. We developed surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. The primary amine groups of lysines on cell surface proteins in live PC3 cells in culture were first labeled with biotin with subsequent enrichment of biotinylated peptides with anti-biotin antibodies. The enriched peptides containing biotin were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Biotinylated peptides identified in triplicate were mapped to proteins. Results: By direct detection of biotinylated lysines using sBioSITe, we identified 5,851 peptides biotinylated on the cell surface derived from 1,409 proteins. 533 of these proteins have been previously shown or predicted to be localized to the cell surface, or secreted into the extracellular space. Importantly, several of the identified markers have known associations with prostate cancer and metastasis including CD59, epithelial growth factor receptor (EGFR), 4F2 cell-surface antigen heavy chain (SLC3A2) and Adhesion G protein-coupled receptor E5 (ADGRE5) thus highlighting the robustness of this method. Conclusions: Detection of biotinylation sites on cell surface proteins modified in vitro provides a reliable method for the identification of cell surface proteins. This method is an excellent tool that complements existing methods for the analysis of surface expressed proteins in qualitative as well as comparative studies.
2024-01-26 | PXD044519 | Pride
Project description:Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-beads digestion in numerous biological applications. However, the popular on-beads digestion method faces major challenges of streptavidin contamination, the lost information of biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated besides the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intra-organelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.
2021-04-21 | MSV000087256 | MassIVE
Project description:Sequencing shows that macroH2A1-emerin interaction occurs in lamina-associated domains on a genome-wide scale. We decided to verify that the biotinylated signal is indeed enriched in the chromatin domains positioned at the nuclear periphery. In this regard, we purified the biotinylated chromatin from HeLa S3 clonal cell line stably coexpressing BirA-emerin and BAP-macro-H2A1 using proximity utilizing biotinylation native chromatin immunoprecipitation (PUB-nChIP). Instead of analysing the protein fraction, we isolated DNA from the biotinylated chromatin pull down and performed high throughput sequencing in order to get insight into the genome wide distribution of the isolated DNA. Importantly, PUB-nChIP-seq of the DNA purified from HeLa S3 clonal cell line stably coexpressing BirA-emerin and BAP-macro-H2A1 showed high levels of enrichment at the lamina associated domains, which were identified previously in HeLa cells using anti-Lamin B1 and anti-Lamin A chIP-seq (Lund et al., 2015). The enrichment of BirA-emerin labelled BAP-macro-H2A at LADs is visibly clear at both the chromosome level and at individual LADs. To test if this enrichment was significant, the genome was divided into 100kb windows and monte carlo simulation using 10,000 iterations was performed to examine if probes overlapping LADs were significantly different to the genomic average. For both replicates, at both LMNA and LMNB1 LADs, the enrichment of biotinylated BAP-macroH2A1 observed was significant relative to the genome wide average.
2018-08-24 | GSE118996 | GEO
Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf
2009-12-08 | GSE18898 | GEO
Project description:Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.
2024-05-22 | PXD047979 | Pride
Project description:While protein biotinylation has been widely used in biochemistry and biotechnology with various enrichment methods, different biotin enrichment strategies have not been systematically compared. Traditional biotinylation proteomics workflows suffer from complex sample preparation steps, non-specific bindings, limited throughput, and experimental variability. To address these critical challenges, we designed a two-proteome model, where yeast proteins were biotinylated in vitro and spiked in unlabeled human proteins, allowing us to distinguish true enrichment (yeast) from non-specific bindings (human) for comprehensively benchmarking of biotinylation proteomics methods. We also significantly optimized the entire workflow and reduced the sample preparation time from the traditional 3 days to just 9 hours, enabling a fully automated 96-well format sample processing for excellent reproducibility and throughput with minimized non-specific bindings. We then applied this optimized and automated workflow to proximity labeling proteomics and investigated the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. We demonstrated that mitochondrial damage led to an increased mitochondria-lysosome membrane contact and induced broad alternations in mitophagy-related proteins. We identified and quantified biotinylated proteins and precise amino acid residues at mitochondria-lysosome contact sites and within the mitophagy pathway, revealing an elevated level of interaction between mitochondria and lysosomes and proteome-wide remodeling in response to mitochondrial damage.
2024-05-17 | MSV000094795 | MassIVE
Project description:We present a method for the tissue-specific purification of protein complexes from the nematode C. elegans, based on cell-type specific biotinylation. To purify proteins from specific C. elegans tissues, we adapted a system based on in vivo biotinylation of a protein of interest. In this approach, a protein of interest is tagged with the 15 amino acid Avi-tag, which can be biotinylated in vivo by the BirA biotin ligase from Escherichia coli. The biotinylated bait protein and any associated proteins are then purified with streptavidin-coated beads, and their identities are determined by mass spectrometry.
2016-07-18 | PXD002139 | Pride
Project description:We describe an in vivo chromatin purification system for genome-wide epigenetic profiling in C. elegans. In this system, we coexpressed the E. coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf All experiments were done using two channels per chip, comparing DNAs extracted from either salt-extracted or insoluble chromatin to whole nuclear chromatin, whole nuclear chromatin to randomly fragmented genomic DNA, streptavidin-bound biotin-tagged histone-variant-containing chromatin to salt-extracted chromatin, gel-purified mononucleosomes to whole EDTA-extracted soluble chromatin, or streptavidin-bound biotin-tagged histone-variant-containing chromatin to whole EDTA-extracted soluble chromatin to randomly fragmented DNA from embryo nuceli.
2010-05-19 | E-GEOD-18898 | biostudies-arrayexpress
Project description:Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or CRISPR-based tagging, thus prohibiting such assays in (primary) cell types that are difficult to transfect. We recently developed an ‘off the shelf’ proximity biotinylation method, which makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo-antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. Here, we present a detailed protocol for this method
2022-10-17 | PXD030963 | Pride