ATP Affinity Probe Labeling and Enrichment

The detailed procedures for ATP affinity
probe labeling, tryptic
digestion, and affinity purification of desthiobiotin-labeled peptides
were reported previously.^{13 (link)} Briefly, after
incubation with the ATP affinity probe for 2.5 h,^{11 (link),23 (link)} the labeled cell lysate mixture was reduced with dithiothreitol,
alkylated with iodoacetamide, and digested with sequencing grade trypsin
(Roche Applied Science, Indianapolis, IN). The resultant desthiobiotin-labeled
peptides were enriched by using avidin–agarose resin and eluted
with a buffer containing 1% TFA in CH₃CN/H₂O
(7:3, v/v). The resulting enriched peptide samples were desalted by
employing OMIX C₁₈ pipet tips (Agilent Technologies, Santa
Clara, CA).