posted on 2024-01-12, 11:03authored byAndreas Wiest, Pavel Kielkowski
We report here a Cu-catalyzed azide–alkyne–thiol
reaction forming thiotriazoles as the major byproduct under widely
used bio-orthogonal protein labeling “click” conditions.
The development of Cu(I)-catalyzed azide–alkyne cycloaddition
(CuAAC) had a tremendous impact on many biological discoveries. However,
the considered chemoselectivity of CuAAC is hampered by the high reactivity
of cysteine free thiols, yielding thiotriazole protein conjugates.
The reaction byproducts generate false-positive protein hits in functional
proteomic studies. The reported detail investigation of conjugates
between chemical probes containing terminal alkynes, azide tags, and
cell lysates reveals the formation of thiotriazoles, which can be
readily detected by in-gel fluorescence scanning or after peptide
and protein enrichment by mass spectrometry-based proteomics. In protein
level identification and quantification experiments, the produced
fluorescent bands or enriched proteins may not result from the important
enzymatically driven reaction and can be falsely assigned as hits.
This study provides a complete list of the most common background
proteins. The knowledge of this previously overlooked reactivity now
leads to the introduction of modified CuAAC conditions, which avoids
the undesired product formation, diminishes the background, and hence
improves the signal-to-noise ratio.