Agilent 6120 msd tof system

For analysis of the intact rPrx3-TS adduct, the EE Mut of Prx3 was cycled with and without TS as described for the in vitro assay and then treated with 33 mM DTT for 30 minutes at room temperature. Samples were concentrated and re-diluted 9 times using a 0.5 ml Amicon Ultra centrifugation filter (10,000 MWC) to exchange into 25 mM ammonium bicarbonate for MS analysis. ESI-TOF MS analyses were performed on an Agilent 6120 MSD-TOF system (Agilent Technologies, Inc., Santa Clara, CA, USA) operating in positive ion mode with the following settings: capillary voltage 3500 V, nebulizer gas pressure 30 psig, drying gas flow 5 L min⁻¹, fragmentor voltage 175 V, skimmer voltage 65 V, gas temperature 325°C. Samples were introduced via direct infusion at a flow rate of 20 μL min⁻¹ using a syringe pump (KD Scientific, Holliston, MA, USA). For peptide analysis, the samples utilized for ESI-MS analysis (~30 μg protein) were digested with 1.5 μg Pierce MS-grade trypsin overnight at 37°C. Peptides were analyzed on a Bruker Autoflex MALDI-TOF mass spectrometer in positive ion and linear acquisition mode using sinapic acid as the matrix.