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

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          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.

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          Directed evolution of APEX2 for electron microscopy and proteomics

          Stephanie Lam, Jeffrey D. Martell, Kimberli J. Kamer … (2014)
          APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
          Article 0 comments Cited 482 times – based on 0 reviews     Review now
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            Translocation of proteins into mitochondria.

            Walter Neupert, Johannes M. Herrmann, Michael Ryan (2007)
            About 10% to 15% of the nuclear genes of eukaryotic organisms encode mitochondrial proteins. These proteins are synthesized in the cytosol and recognized by receptors on the surface of mitochondria. Translocases in the outer and inner membrane of mitochondria mediate the import and intramitochondrial sorting of these proteins; ATP and the membrane potential are used as energy sources. Chaperones and auxiliary factors assist in the folding and assembly of mitochondrial proteins into their native, three-dimensional structures. This review summarizes the present knowledge on the import and sorting of mitochondrial precursor proteins, with a special emphasis on unresolved questions and topics of current research.
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              Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

              Jeffrey D. Martell, Thomas J. Deerinck, Yasemin Sancak … (2012)
              Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments 1 or require light and are difficult to use 2 . Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed 3,4 . MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.
              Article 0 comments Cited 250 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101215604
                Journal ID (pubmed-jr-id): 32338
                Journal ID (nlm-ta): Nat Methods
                Journal ID (iso-abbrev): Nat Methods
                Title: Nature methods
                ISSN (Print): 1548-7091
                ISSN (Electronic): 1548-7105
                Publication date Nihms-submitted: 19 January 2019
                Publication date (Print): December 2017
                Publication date (Electronic): 16 October 2017
                Publication date PMC-release: 25 March 2022
                Volume: 14
                Issue: 12
                Pages: 1167-1170
                Affiliations
                [1 ]Proteomics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
                [2 ]Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California, USA.
                [3 ]DRI2 LLC, Littleton, Massachusetts, USA.
                [4 ]Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
                Author notes

                AUTHOR CONTRIBUTIONS

                N.D.U. and S.A.C. conceived of the study; N.D.U., K.P., T.S., and S.F. performed experiments; N.D.U., K.P., T.S., S.F., S.A.M., O.A., K.K., K.C., D.R., T.A., V.K.M., A.Y.T., and S.A.C. contributed to experimental design, data analysis, and data interpretation. N.D.U. and S.A.C. wrote the manuscript with input from all authors.

                Correspondence should be addressed to S.A.C. ( scarr@ 123456broad.mit.edu ) or N.D.U. ( udeshi@ 123456broadinstitute.org ).
                Author information
                http://orcid.org/0000-0001-5312-1402
                http://orcid.org/0000-0001-8365-1826
                http://orcid.org/0000-0002-7203-4299
                Article
                Manuscript ID: HHMIMS1006645
                DOI: 10.1038/nmeth.4465
                PMC ID: 8954634
                PubMed ID: 29039416
                SO-VID: 39a2e796-223c-4c1e-b938-858d7460cbe8
                License:

                This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.

                Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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                ScienceOpen disciplines: Life sciences

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