Zorbax c18 column

HPLC analysis was performed on an Agilent 1100 series instrument using either a Vydac C18 column (5 μm, 4.6 × 250 mm for analytical runs) at a flow rate of 1 mL/min or an Agilent Zorbax C18 column (5 μm, 9.4 × 250 mm for semi-preparative runs) at a flow rate of 3 ml/min, using a two-component mobile phase system in which mobile phase A is 0.1% TFA in water and mobile phase B is 90% acetonitrile and 0.1% TFA in water. Absorbance was monitored at 214 and 280 nm. 400 μl of pull-down eluate were acidified with 2% TFA and injected on the above column. Peptides were separated with a 0–73% B gradient run over 40 minutes, and collected fractions were analysed by MALDI-TOF as above. For purification of Tx7335 from crude venom, 20 mg of crude venom were dissolved in 0.5 ml of 50 mM TRIS pH 7.5, 150 mM KCl, filtered, then purified on a semi-prep HPLC column using the following gradient: 0–35% B in 20 min, followed by 35–55% B in 40 min and 55–90% B in 10 min.

Samples were processed using protein precipitation method and analyzed using LC/MS/MS. A calibration curve (CC) of 5 nM to 10,000 nM range was employed to quantify the samples. 8G, 10G and 6S were eluted using a mobile phase consisting of ACN∶5 mM ammonium formate (80∶20) with 0.05% formic acid and for 6G, a mobile phase of ACN∶Milli-Q water (80∶20) with 0.1% formic acid was used. Separation was achieved using Zorbax C18 column (50×4.6 mm, 3.5 µ, Agilent) for 8G, 10G, 6S and using Hypurity C18 column (100×4.6 mm, 5 µ, Thermo) for 6G. An injection volume of 20 µL was used for all samples and a flow rate of 0.6 mL/min (6G) and 1 mL/min (8G, 10G and 6S) was used for elution. The MRM transitions (m/z) monitored were: 6G (312.3/177.1), 8G (340.0/177.1), 10G (333.0/177.1), 6S (277.2/137.1) and rolipram (276.2/208.2, Internal standard, 100 ng/mL).

The Agilent HPLC-DAD system was the analytical platform, with the same Zorbax C18 column, 150 mm 4.6 mm; 5 µm. As a mobile phase, two solutions were used: solution A: 0.1% phosphoric acid and solution B: acetonitrile, with gradient elution, at 22 min per run, with the same injection volume and flow The temperature was set at 35 °C and the detection was performed at UV 310 nm.

The levels of ITZ in the samples were evaluated using the Breeze™ 2 HPLC system (Waters, Milford, MA, USA) equipped with the Zorbax^® C₁₈ column (150 mm × 4.6 mm, 5-µm particle size; Agilent Technologies, Wilmington, DE, USA). The mobile phase was composed of acetonitrile and 20 mM potassium phosphate buffer (pH 10) at a ratio of 60:40 (v/v). The flow rate was set at 1.2 mL/min, and the UV detection wavelength was 254 nm. The column temperature was maintained at 30 °C.

The peptide mixtures were separated in a C18 column (Zorbax C18 column 150 × 2.1 mm, 1.8 μm, 300 Å (Agilent, USA)) by using Agilent HPLC-1290. The column temperature was maintained at 40°C, and the peptides were separated with a buffer system of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The flow rate was adjusted to 0.200 μL/min. The peptides were eluted with a gradient of 3%–50% mobile phase B over 130 minutes followed by 30%–90% mobile phase B over 10 minutes. An Agilent Q-TOF-MS 6530 system was used to analyze the peptides. Once separated, they passed through the electrospray ionization source (ESI) source and were ionized in the positive mode. The capillary voltage was adjusted at 4000 V with a drying temperature of 350°C. The auto MS-MS data were recorded between 300 and 1400 m/z above the 1500 count threshold. The MS-MS cycle time was 3.63s, and the maximum ion number was selected as three. The ion charge states were +2, +3, and +4, and the fragmentation energy was adjusted to 45 V. For each analysis, 80 μg protein was loaded to the LC/MS system. Three technical replicates were analyzed for the treated and untreated groups.

Glutathione was measured as previously published (18 (link), 20 (link)). Briefly, GSH and GSSG were measured using the Shimadzu Nexera X2 UHPLC system (Mandel Scientific). Quadriceps and diaphragm were homogenized in 50 mM Tris buffer with 10 mM boric acid, 2 mM l-serine, 20 μM acivicin, and 5 mM N-ethylamide and acidified using TCA (for GSH) and PCA (for GSSG). Samples were separated with a Zorbax C18 column (Agilent Technologies). GSH was assessed by UV-HPLC (265 nm wavelength) monitoring of NEM-GSH using a 0.25% glacial acetic acid mobile phase with 6% acetonitrile at 1.05 mL/min flow rate detected at 265 nm. GSSG was assessed by fluorescent HPLC (excited at 350 nm and detected at 420 nm emission) using HPLC/UHPLC fluorescence detector (Mandel Scientific). GSSG samples were diluted in 0.5 M NaOH and run using 25 mM Na₂HPO₄ in HPLC-grade water with 15% methanol mobile phase at a 0.5 mL/min flow rate by tracking o-pthalymide–tagged GSH.