Multiplying Identifiability of Clickable Peptides Using One-Pot Tagging of Homologous Biotinyl Azides

Chemical proteomics plays a crucial role in understanding protein functions and developing covalent drugs but faces challenges in accurately identifying probe-modified peptides due to the generation of only a single modified peptide per probe reaction and potential ambiguities in proteomic identification. This work introduces a novel Single-Sequence Identification (SSI) principle, addressing these challenges by enhancing the detectability of modified peptides in proteomic experiments. Our innovative SSI approach involves creating multiple versions of a modified peptide within the proteomic identification space, thereby increasing the probability and confidence of peptide identification. We demonstrate the efficacy of this method using a one-pot triplex tagging technique that attaches three homologous biotinyl azides with varying polyethylene glycol (PEG) linker lengths to clickable proteins. This tagging-triplication method not only enables confident identification of peptides carrying two forms of tags, but also leverages tag diagnostic ions and the dependency of elution time on linker length to further boost identification accuracy. Additionally, we identified and addressed variability in CuAAC-tagged proteins by suggesting a split-and-pool strategy. The ease of integrating our tagging-triplication method into existing chemical proteomics workflows showcases its potential in enhancing peptide analysis reliability in chemical proteomics. These advancements highlight the significant implications of the SSI principle for future proteomics research. Data are available through ProteomeXchange: identifier PXD037770.