Direct Detection of Biotinylated Proteins by Mass Spectrometry

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 spec...

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Bibliographic Details
Published in:Journal of proteome research 2014-09, Vol.13 (9), p.3966-3978
Main Authors: Schiapparelli, Lucio Matias, McClatchy, Daniel B, Liu, Han-Hsuan, Sharma, Pranav, Yates, John R, Cline, Hollis T
Format: Article
Language:English
Subjects:
Animals
Biotin - analogs & derivatives
Biotin - chemistry
Biotin - metabolism
HEK293 Cells
Humans
Male
Proteins - analysis
Proteins - chemistry
Proteins - metabolism
Proteome - analysis
Proteome - chemistry
Proteome - metabolism
Proteomics - methods
Rats
Rats, Wistar
Succinimides - chemistry
Succinimides - metabolism
Tandem Mass Spectrometry - methods
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Summary: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 “Direct Detection of Biotin-containing Tags” (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 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.
ISSN:1535-3893
1535-3907
DOI:10.1021/pr5002862