Chapter title |
BONCAT: Metabolic Labeling, Click Chemistry, and Affinity Purification of Newly Synthesized Proteomes.
|
---|---|
Chapter number | 14 |
Book title |
Site-Specific Protein Labeling
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2272-7_14 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2271-0, 978-1-4939-2272-7
|
Authors |
Peter Landgraf, Elmer R Antileo, Erin M Schuman, Daniela C Dieterich, Elmer R. Antileo, Erin M. Schuman, Daniela C. Dieterich, Landgraf, Peter, Antileo, Elmer R., Schuman, Erin M., Dieterich, Daniela C. |
Abstract |
Metabolic labeling of proteins using classical radioisotope-labeled amino acids has enabled the analysis and function of protein synthesis for many biological processes but cannot be combined with modern high-throughput mass spectrometry analysis. This chapter describes the unbiased identification of a whole de novo synthesized proteome of cultured cells or of a translationally active subcellular fraction of the mammalian brain. This technique relies on the introduction of a small bioorthogonal reactive group by metabolic labeling accomplished by replacing the amino acid methionine by the azide-bearing methionine surrogate azidohomoalanine (AHA) or the amino acid homopropargylglycine (HPG). Subsequently an alkyne- or azide-bearing affinity tag is covalently attached to the group by "click chemistry"-a copper(I)-catalyzed [3+2] azide-alkyne cycloaddition. Affinity tag-labeled proteins can be analyzed in candidate-based approaches by conventional biochemical methods or with high-throughput mass spectrometry. |
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Japan | 1 | 20% |
Unknown | 1 | 20% |
Demographic breakdown
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Members of the public | 2 | 40% |
Mendeley readers
Geographical breakdown
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Sweden | 1 | <1% |
Unknown | 124 | 97% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Ph. D. Student | 29 | 23% |
Researcher | 23 | 18% |
Student > Master | 16 | 13% |
Student > Bachelor | 14 | 11% |
Professor | 8 | 6% |
Other | 16 | 13% |
Unknown | 22 | 17% |
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Agricultural and Biological Sciences | 31 | 24% |
Neuroscience | 10 | 8% |
Immunology and Microbiology | 7 | 5% |
Chemistry | 6 | 5% |
Other | 8 | 6% |
Unknown | 30 | 23% |