Extended Data Fig. 6: Functional assessment and validation of the identified glycolytic metabolite-target interactions belonging to the carbohydrate metabolism pathway.

(a) GO MF analysis of the TRAP-identified glycolytic targetome showing enrichment in proteins ascribed to catalytic activity. (b) KEGG pathway annotation summarized the enriched pathways for the targets ascribed to catalytic activity. (c) Illustration of how the boundary of active site is defined using the multiplexed-TRAP data. The minimal Euclidean distances between the TRPs of the TRAP-identified targets that use the assayed metabolites as substrates and the corresponding active site (retrievable from PDB) were measured, and the resultant median of the collected distances was used to represent the active site boundary that can be probed by TRAP. (d) Summarized R_{treated/control} values of the TRPs in PKM2 via the multiplexed-TRAP analysis of HCT116 cell lysates following FBP, F6P and G6P incubation, respectively. Of note, the letter before K denotes the type of the classified TRPs. (e) Relative (Rel.) PKM2 activity at different FBP concentrations normalized to the activity without FBP. (f) TRAP analysis delivering the R_{treated/control} of the TRP^{type B} carrying PKM2-K433 following 3PG incubation. (g) DrugBank and non-DrugBank fractions of the quantified proteome (n=4778) vs. the TRAP-assigned glycolytic targetome (n=913) using the multiplexed-TRAP data. For (d, e, f), data represent the mean ± SEM (n=3 biologically independent samples). For (d, f), P values were determined using an unpaired two-tailed Student’s t-test.