Assessment of active-site labeling can be accomplished by Western blot or mass spectrometry (MS)
A. Nucleotide analogues bind to the active sites of ATPases and the biotin affinity tag is irreversibly transferred to highly conserved lysine residues in the active site. B. Structures of desthiobiotin nucleotide analogues. Desthiobiotin binding to streptavidin is easily reversible under acidic elution conditions, allowing high recovery of labeled proteins and peptides. Desthiobiotin is attached to the nucleotide through a labile acyl phosphate linkage, allowing efficient desthiobiotin label transfer to amines near the active site. ATP and ADP nucleotide analogues label a complementary set of ATPases, which is likely due to differences in the proximity of the acyl phosphate linkage to conserved lysines near the active site.
Comparison of desthiobiotin ATP and ADP probe labeling of kinases using the Western blot workflow