Turboionspray

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.

The LC-MS/MS method for GAG disaccharide analysis was modified according to the literature reported by Auray-Blais et al. [19 (link)]. For GAG disaccharide LC-MS/MS analysis, a multiple reaction monitoring (MRM) experiment giving a precursor molecular ion (Q1) and a respective fragment ion (Q3) was normally applied. The instrument was operated in a positive-ion ([M + H]⁺) mode. The quadrupoles, Q1 and Q3, were tuned with unit resolution, and the MS conditions were optimized for maximum signal intensity. The LC-MS/MS analysis was performed on a 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). The experimental parameters were set and are clearly described in the following section. Data were acquired and processed using Analyst 1.5.2™ software (AB Sciex). Calibrations of GAG standards and internal standards were performed with every batch of samples.

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.