Highly Efficient Peptide-Based Click Chemistry for Proteomic Profiling of Nascent Proteins

Copper-catalyzed
azide–alkyne cycloaddition (CuAAC) has
been widely used in a variety of scientific research, including dynamic
proteomics. The current well-established protocols of CuAAC for proteomics
analysis introduce labeling tags (azide- or alkyne-containing reagents)
at the protein level, followed by downstream analysis by mass spectrometry.
In the present study, a new method for proteomic profiling of nascent
proteins relying on highly efficient peptide-based click chemistry
is proposed, in which the CuAAC reaction was performed at the peptide
level, leading to a significant increase in efficiency of the click
conjugation reaction. A remarkable improvement in identification rate
for spectrum, distinct peptide, and protein was achieved when proteins
to be analyzed were proteolytically cleaved into peptides prior to
the click conjugation reaction, which would be beneficial to downstream
proteomics analysis, especially for the detection of AHA-tagged proteins
in very low amounts.