Enhancing Comprehensive Analysis of Newly Synthesized Proteins Based on Cleavable Bioorthogonal Tagging.

Protein synthesis and degradation responding to environmental cues is critical for understanding the mechanisms involved. Chemical proteomics introducing bioorthogonal tagging into proteins and isolation by biotin affinity purification is applicable for enrichment of newly synthesized proteins (NSPs). Current enrichment methods based on biotin-streptavidin interaction lack efficiency to release enriched NSPs under mild conditions. Here we designed a novel method for enriching newly synthesized peptides by click chemistry followed by release of enriched peptides via tryptic digestion based on cleavable bioorthogonal tagging (CBOT). CBOT-modified peptides can further enhance identification in mass spectrometry analysis and provide a confirmation by small mass shift. Our method achieved an improvement in specificity (97.1%) and sensitivity for NSPs in cell lysate, corresponding to profiling at a depth of 4335 NSPs from 2 mg of starting materials in a single LC-MS/MS run. In addition, the CBOT strategy can quantify NSPs when coupling a pair of isotope-labeled azidohomoalanine (AHA/hAHA) with decent reproducibility. Furthermore, we applied it to analyze newly synthesized proteomes in the autophagy process after 6 h rapamycin stimulation in cells, 2910 NSPs were quantified, and 337 NSPs among them were significantly up- and down-regulated. We envision CBOT as an effective and alternative approach for bioorthogonal chemical proteomics to study stimuli-sensitive subsets.