| Protein modifications (PTMs) plays critical roles in biology and medicine, thus their analyses are critical. However, it is challenging to identify protein modifications, especially unknown modifications.;Alkynes are a key component of click chemistry and used for a wide variety of applications including bioconjugation, selective tagging of protein modifications, and labeling of metabolites and drug targets. However, challenges still exist for detecting alkynes because most 1,2,3-triazole products from alkynes and azides do not possess distinct intrinsic properties that can be used for their facile detection by either fluorescence or mass spectrometry. To address this critical need, a novel brominated coumarinazide was used to tag alkynes and detect alkyne-conjugated biomolecules. This tag has several useful properties: first, it is fluorogenic and the click-chemistry products are highly fluorescent and quantifiable; second, its distinct isotopic pattern facilitates identification by mass spectrometry; and third, its click-chemistry products form a unique pair of reporter ions upon fragmentation that can be used for the quick screening of data. Using a monoclonal antibody conjugated with alkynes, a general workflow has been developed and examined comprehensively.;We foresee that this tag and workflow will be particularly powerful for more complex systems including detecting protein modifications and drug targets in the cellular milieu and systems with undefined chemistry and sites for bioconjugation or degradation, and moreover, novel chemistry and biology. |
