Biorthogonal noncanonical amino acid tagging is a labeling strategy that covalently adds a biotin-alkyne (BA) to methionine analogs via a click reaction. When methionine analogs are incorporated into a proteome, enrichment of the BA-labeled proteins allows the detection of newly synthesized proteins (NSP) by mass spectrometry. We previously reported that our direct detection of biotin-containing tags strategy increased protein identifications by enriching for BA-peptides instead of BA-proteins. We compared the performance of cleavable BA (DADPS) and uncleavable BA in the identification and TMT quantification of the NSP. DADPS identified and quantified more than double the number of peptides than uncleavable BA. Interrogation of the data revealed that multiple factors are responsible for the superior performance of DADPS.
Keywords: BONCAT; DADPS; DidBiT; TMT; azidohomoalanine; azidonorleucine; mass spectrometry.