Fig. 4: Identifying RBBP9(Dap75) conjugates.
From: Mechanism-based traps enable protease and hydrolase substrate discovery
a, The Dap-containing tryptic peptide sequence (Pept(Dap-X)) of RBBP9 with certain b and y ion masses of Pept(Dap) modified by mx. b, Pipeline to identify mx in live cells. RBBP9(Dap75-X) conjugates were affinity purified and trypsinized. The resulting peptide pool was analysed by LC–MS/MS and peptides with molecular mass no less than MS(Pept(Dap)) were individually selected for mx calculation (for example, the blue peak represents Pept(Dap), mx = 0). The experimental MS2 peaks were compared to theoretical MS2 peaks for scoring as described in Methods. In the example shown, the peaks are colour coded as b, y, b+mx or y+mx ions, using the colour scheme in a. c, mx obtained from the top-scoring spectra were plotted. Each dot represents the mass shift of the observed peptide relative to the parental peptide. +113 (Leu/Ile), +131 (Met), +147 (Phe), +163 (Tyr), +186 (Trp), +297 (pc-Dap), +42 and +77 (consistent with near cognate suppression of this amber codon in mammalian cells with Gln and Tyr, respectively). d, The entire mass of RBBP9 variants and Dap conjugates purified from human cells. Green trace (i): wild-type RBBP9 ([Ac-(RBBP9-Met)]: expected 23,995 Da, observed 23,994.5 Da); black trace (ii): RBBP9(S75A) ([Ac-(RBBP9(S75A)-Met)]: expected 23,979 Da, observed 23,978.5 Da); brown trace (iii): RBBP9(S75pc-Dap) before illumination ([Ac-(RBBP9(S75pc-Dap)-Met)]: expected 24,291 Da, observed 24,290.5 Da); purple dashed trace (iv): RBBP9(S75Dap) deprotected from RBBP9(S75pc-Dap) in vitro ([Ac-(RBBP9(S75Dap)-Met)]: expected 23,994 Da, observed 23,993.5 Da); multicolour trace (v): RBBP9(S75pc-Dap) purified from cells after illumination and substrate trapping for 3 h ([Ac-(RBBP9(S75Dapinter)-Met)]: expected 24,098 Da, observed 24,097 Da; [Ac-(RBBP9(Dap-L/I)-Met)]: expected 24,107 Da, observed 24,108 Da; [Ac-(RBBP9(Dap-F)-Met)]: expected 24,141 Da, observed 24,140.5 Da; [Ac-(RBBP9(Dap-Y)-Met)]: expected 24,157 Da, observed 24,156 Da; [Ac-(RBBP9(Dap-W)-Met)]: expected 24,180 Da, observed 24,179 Da). The experiment in c was performed in biological triplicate. In d, the entire mass acquisition of trace (v) was performed in two biological replicates with similar results, the other traces were acquired once.
