Table 2.

Fraction of Cysteic Acid for M471C TβM Proteolytic Fragment 459-483.

	0 mina	30 min	60 min
50 mM Ascb	0.28 ± 0.09	0.66 ± 0.11	0.71 ± 0.07
1 mM Asc	0.28 ± 0.09	0.28 ± 0.05	0.40 ± 0.07
50 mM Asc, 500 μM Tyrc	0.28 ± 0.09	0.56 ± 0.05	0.72 ± 0.03

^aFor the ‘0 min’ timepoint Met471Cys TβM (15 μM) was denatured in urea (8 M) and Tris buffer, pH 8.5 (100 mM), reduced with TCEP (5 mM), and capped with IAA (10 mM). Proteolysis was subsequently initiated by TPCK-trypsin (0.5 μg/mL) and allowed to react overnight at 37 °C. Neat formic acid (1 μL) was added to each reaction before mass spectral analysis. The fraction of cysteic acid detected at 0 min is attributed to the inherent instability at this position during protein purification and work up for the analyses.

^bMet471Cys TβM (15 μM) was incubated for 30 and 60 min with the following: KPi, pH 6.0 (0.1 M), KCl (0.1 M), ascorbate (50 mM), catalase (20 μg/mL), and CuSO₄ (30 μM). These reactions were subjected to the same proteolysis conditions as described above.

^cThese are the conditions of the earlier experiment (27).