Direct Detection of Biotinylated Proteins by Mass Spectrometry
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- 作者：Lucio Matias Schiapparelli ; Daniel B. McClatchy ; Han-Hsuan Liu ; Pranav Sharma ; John R. Yates ; III ; Hollis T. Cline
- 刊名：Journal of Proteome Research
- 出版年：2014
- 出版时间：September 5, 2014
- 年：2014
- 卷：13
- 期：9
- 页码：3966-3978
- 全文大小：640K
- ISSN：1535-3907
文摘
Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called 鈥淒irect Detection of Biotin-containing Tags鈥?(DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins 鈭?00 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h.