Fig. 5: Thiols liberated by NTF are detected by MS/MS.

NTF was added to purified Fc-tagged fragments of NOTCH3 and examined with MS/MS. The probabilities of each cysteine being labeled with NEM were calculated and compared between the samples with NTF and samples without NTF. The addition of NTF to WT NOTCH3 significantly increased the probabilities of a cysteine being labeled with NEM at positions 1, 2, 3, 4, 5, 6, 8, 9, 12, 13, 14, 15, 16, and 17 (a). The addition of NTF to R90C mutant protein significantly increased probabilities of cysteines 1, 2, 3, 4, 5, 12, 15, 16, and 17 being labeled with NEM and significantly decreased probabilities of cysteines 6, 7, and 13 being labeled with NEM (b). The addition of NTF to R141C mutant protein significantly increased probabilities of cysteines 1, 2, 3, 4, 5, 6, 8, 9, 14, 15, 17, and R141C being labeled with NEM and significantly decreased probability of cysteines 16 being labeled with NEM (c). The addition of NTF to C49Y mutant significantly increased probabilities of getting an NEM label at cysteine positions 1, 3, 4, 5, 6, 8, 9, 12, 15, 16, 17, and 18 and significantly decreased probabilities of getting an NEM label at position 7 (d). Finally, the addition of NTF to R75P mutant protein significantly increased probabilities of observing an NEM label at cysteine positions 1, 2, 3, 8, 12, 13, 15, 16, and 17 (e). The bar below each panel represents statistical significance at each cysteine position (box). All locations of mutations are marked with a red arrow, and cysteines that were not detected were labeled with “nd.” Each experiment was repeated three times using distinct samples, and the results from all three replicates are displayed in Fig. 5. Error bars represent the standard deviation. Individual data points used for analysis can be found in Fig. S4c–g.