Masshunter version b 06

The volatile components analysis of the saffron was carried out using gas chromatography-mass spectrometry (GC-MS) equipped with an Agilent 7890A system (A.01.01, Wilmington, DE, USA) and a mass selective detector 5975 Network MSD and coupled to a MPS automatic sampling system, as described previously by Naim et al. [62 (link)]. The chromatographic separation was performed on a HP-5MS capillary column (30 m × 0.25 mm, film thickness 0.17 mm), and the following temperature program was used: 60 °C held for 3 min, then increased to 210 °C at a rate of 4 °C/min, then held at 210 °C for 15 min, then increased to 300 °C at a rate of 10 °C/min, and finally held at 300 °C for 15 min. Helium was used as the carrier gas at a constant flow of 1 mL/min. For the quantification, the results are presented as a percentage of the peak area, considering a response factor of the fiber. Mass Hunter Version B.06.00 (Agilent Technologies) was used for the data acquisition and processing. The identification of the components was based on the comparison of the obtained mass spectrum with those from the commercial databases (NIST17 and Wiley) and by comparison with the retention index (RI) of each peak from the literature (Pherobase). The experimental retention index (RI) of the compounds were calculated following the injection of a mixture of n-alkanes C8-C20 (Sigma Aldrich, Darmstadt, Germany).

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).