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. 2023 Jun 15;14:3564. doi: 10.1038/s41467-023-39268-w

Fig. 2. LC–MS analysis on the reactions between Cys-reactive compounds and small amino acids in aqueous buffer solution.

Fig. 2

a Chemical equation showing the thiol-Michael addition of NAIA-5 with N-acetylcysteine methyl ester (N-Ac-Cys-OMe), a N- and C-terminal protected cysteine, to form the adduct product. b NAIA-5 (10 µM) in aqueous buffer solution (PBS-MeOH, 4:1, v/v) was incubated with N-Ac-Cys-OMe (250 µM). At indicated time intervals, an aliquot of the solution mixture was sent for LC–MS analysis. Selected ion chromatograms (SIC) at m/z = 325 and 502, corresponding to the molecular ion of [NAIA-5 + H]+ and [Adduct+H]+ respectively. The mass spectra (MS) at 6.20 and 5.60 min confirm the identity of NAIA-5 and adduct. c Changes in Cys-reactive compound concentration over time upon incubation with N-Ac-Cys-OMe (250 µM). d Biomolecular rate constant of reactions between cysteine-reactive compounds and N-Ac-Cys-OMe. e Changes in the level of NAIA-5 after incubation with amino acids (30 µM) for 30 min (n = 3). f Stability of NAIA-5 in the aqueous buffer solution in the absence of Cys (n = 3). Quantified data were shown on average ± SD from n = 3 different replicates/groups.