Analyst 1.5.2tm software

Experimental parameters of LC-MS/MS assay have been reported previously^{17 (link),20 (link),21 (link),37} . LC-MS/MS analysis was performed on an AB 4000 QTRAP LC-MS/MS System (AB Sciex, Foster City, CA, USA) equipped with a TurboIonSpray (electrospray ionization; ESI), and Agilent 1260 Infinity HPLC pump and autosampler (Agilent Technologies, Santa Clara, CA, USA). An Atlantis dC18 3 μm column (3.0 × 50 mm; Waters Corporation, Milford, MA, USA) was used for DS and HS analysis, and a Luna 5 μm Silica column (50 × 2.0 mm; Phenomenex Inc., CA, USA) was used for KS analysis. Data were acquired and processed using Analyst 1.5.2^TM software (AB Sciex). The intra- and inter-assay precision values of this MS/MS-based method were estimated and determined using three different known concentrations (low, medium, and high levels) of samples in triplicate over a period of 6 days regularly^{17 (link)}. Calibrations of DS, HS, and KS standards, internal standards, and pooled sample controls with known concentrations were performed with every batch analysis. The experimental parameters of mass spectrometer were checked routinely to ensure the quality of the MS measurement.

Experimental parameters of LC-MS/MS assay basically followed the report published previously by Chuang et al. [32 (link)] with some modifications specific for zebrafish embryos’ extracts. LC-MS/MS analysis was performed on an AB 4000 QTRAP LC-MS/MS System (AB Sciex, Framingham, MA, USA) equipped with a TurboIonSpray (electrospray ionization; ESI), and Agilent 1260 Infinity HPLC pump and autosampler (Agilent Technologies, Santa Clara, CA, USA). An Atlantis dC18 3 μm column (3.0 × 50 mm; Waters Corporation, Milford, MA, USA) was used for DS and HS analysis. Data were acquired and processed using Analyst 1.5.2 TM software (AB Sciex, Framingham, MA, USA). Each batch analysis of DS and HS standards was calibrated with five known concentrations mixed working standards of 12.5, 25, 50, 100, and 200 μg/mL and 6.25, 12.5, 25, 50, and 100 μg/mL for DS and HS, respectively. Total proteins prepared for each reaction were extracted from 40 zebrafish embryos at 120 hpf.