Lct premier lc electrospray ionization ms instrument

Purified CrPV-1A¹⁵⁹ protein (2.35 μg) was incubated with sequencing grade trypsin (2.35 ng) (Promega) in a buffer containing 50 mM Tris pH8.0, 75 mM KCl, 5 mM beta-mercaptoethanol for 30 min at room temperature. The reaction was stopped by adding SDS sample loading buffer, and the extent of proteolysis was analyzed by Coomassie blue staining. The mass of the fragments was analyzed by electrospray ionization-time of flight (ESI-TOF) mass spectrometry using a LCT Premier LC/electrospray ionization-MS instrument (Waters Corporation) and MassLynx software.

Methionine auxotroph B834(DE3) cells (Novagen) with pHis-Gb1-II-CrPV-1A¹⁵⁹ plasmid was grown in 50 mL standard LB medium until the OD₆₀₀ = 0.6. Cells were harvested and re-suspended in 1 L fresh PASM-5052 media containing methionine, Selenomethionine, and Vitamin B12 at concentration 10 mg/L, 125 mg/L, 100 nM respectively (Studier et al., 2005). The culture was grown at 37°C till the OD₆₀₀ = 0.6, induced with IPTG to a final concentration of 0.1 mM, and grown for an additional 5 hours at 30°C. Although PASM-5052 is an auto-inducing medium, IPTG was used for the induction of the CrPV-1A159 protein. Incorporation of Selenomethionine was verified by electrospray ionization-time of flight (ESI-TOF) mass spectrometric analysis using a LCT Premier LC/electrospray ionization-MS instrument (Waters Corporation) and MassLynx software. Selenomethionine-labeled CrPV-1A¹⁵⁹ was purified as described above for the native protein.