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.