title: Direct detection of biotinylated proteins by mass spectrometry.
abstract: 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.
authors.affiliation: The Dorris Neuroscience Center, Department of Molecular and Cellular Neuroscience, ‡Department of Chemical Physiology, and §Kellogg School of Science and Technology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Lucio Matias
authors.initials: LM
authors.lastname: Schiapparelli
authors.forename: Daniel B
authors.initials: DB
authors.lastname: McClatchy
authors.forename: Han-Hsuan
authors.initials: HH
authors.lastname: Liu
authors.forename: Pranav
authors.initials: P
authors.lastname: Sharma
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.forename: Hollis T
authors.initials: HT
authors.lastname: Cline
mesh_terms: D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D013388:Succinimides / Q000737:chemistry* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011506:Proteins; D020543:Proteome; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; D001710:Biotin