Time of flight mass detector

RLC-TS and RLC mutant HMMs (1–2.5 µM) were phosphorylated overnight in buffer containing 10 mM MOPS pH 7.3, 150 mM KCl, 5 mM MgCl₂, 0.2 mM CaCl₂, 0.1 mM EGTA, 1 mM DTT, 0.1 µM CaM, 0.2 mM ATP, 10 µg/mL MLCK, and 1x phosphatase inhibitor cocktail PhosSTOP (Roche, Indianapolis, IN) at 4°C. The conditions favor phosphorylation of both Threonine-20 and Serine-21 on RLC-TS whereas shorter incubation times favor phosphorylation of only Serine-21. MLCK was omitted for the unphosphorylated controls. A volume of 5–8 µl of the proteins was injected into a reverse phase HPLC (Agilent 1100 series HPLC, Agilent Technologies) with a Zorbax 300 SB-C18 (2.1×50 mm, 3.5 M, Agilent Technologies) and introduced into the mass spectrometer as described (Apffel et al., 1995 (link); Taggart et al., 2000 (link)). Positive ion Electrospray Ionization (ESI) mass spectra for intact protein were obtained with an Agilent 6224 mass spectrometer equipped with an ESI interface and a time-of-flight (TOF) mass detector (Agilent Technologies). Mass spectra were analyzed and deconvoluted using MassHunter version B.06.00 (Agilent Technologies) software.

Phosphorylation of non-muscle myosin 2A was initiated by the addition of ATP. Samples were taken at different time points and diluted with 305 acetonitrile, 0.25 TFA to stop the reaction. Proteins were injected into a reverse phase HPLC (Agilent 1100 series HPLC, Agilent Technologies) with a Zorbax 300SB-C18 (2.1×50 mm, 3.5 mm, Agilent Technologies) and introduced into the mass spectrometer as described (Apffel et al., 1995 (link); Taggart et al., 2000 (link)). Positive ion Electrospray Ionization (ESI) mass spectra for intact protein were obtained with an Agilent 6224 mass spectrometer equipped with an ESI interface and a time-of-flight (TOF) mass detector (Agilent Technologies). Mass spectra were analyzed and de-convoluted using a software, MassHunter version B.06.00 (Agilent Technologies).