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 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.