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Direct detection of biotinylated proteins by mass spectrometry

Academic Article
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Overview

related to degree

  • Liu, Han-Hsuan, Ph.D. in Biology, Scripps Research 2011 - 2017

authors

  • Schiapparelli, L. M.
  • McClatchy, D. B.
  • Liu, Han-Hsuan
  • Sharma, P.
  • Yates III, John
  • Cline, Hollis

publication date

  • September 2014

journal

  • Journal of Proteome Research  Journal

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.
  • 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.

subject areas

  • Animals
  • Biotin
  • HEK293 Cells
  • Humans
  • Male
  • Proteins
  • Proteome
  • Proteomics
  • Rats
  • Rats, Wistar
  • Succinimides
  • Tandem Mass Spectrometry
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Research

keywords

  • AHA
  • DiDBiT
  • MudPIT
  • azidohomoalanine
  • biotinylated protein
  • click chemistry
  • in vivo protein synthesis
  • mass spectrometry
  • retina
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Identity

PubMed Central ID

  • PMC4156236

International Standard Serial Number (ISSN)

  • 1535-3893

Digital Object Identifier (DOI)

  • 10.1021/pr5002862

PubMed ID

  • 25117199
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Additional Document Info

start page

  • 3966

end page

  • 3978

volume

  • 13

issue

  • 9

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