Benchmarking Cleavable Biotin Tags for Peptide-Centric Chemoproteomics

Click chemistry-specifically the copper-catalyzed azide-alkyne cycloaddition-has enabled the development of a wide range of chemical probes to analyze subsets of the functional proteome. The "clickable" proteome can be selectively enriched by using diverse cleavable biotin tags, but the direct identification of probe/tag-modified peptides (or peptide-centric analysis) remains challenging. Here, we evaluated the performance of five commercially available cleavable biotin tags in three most common chemoproteomic workflows, resulting in a comparative analysis of 15 methods. An acid-cleavable biotin tag with a dialkoxydiphenylsilane moiety, in combination with the protein "click", peptide "capture" workflow, outperforms all other methods in terms of enrichment efficiency, identification yield, and reproducibility, although its performance may be slightly compromised by the formation of an unwanted formate product revealed by blind search. Despite being inferior, photocleavable, and reduction-cleavable, biotin tags can also find their applicable sceneries, especially when dealing with acid-sensitive probes or probe-derived modifications. Furthermore, the systematic comparison of LC-MS/MS characteristics of tag-modified peptides provides valuable information for the future development of cleavable biotin reagents. Taken together, our data provides a much-needed practical guidance for researchers interested in developing and/or applying an ideal cleavable biotin tag to peptide-centric chemoproteomics.