Lc 20adxr

The molecular size distribution of the various CPs from seaweeds and abalone viscera was determined by gel permeation chromatography (GPC) using a LC-20AD HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC20AD XR; Shimadzu, Japan), an automatic injection pump (SIL-20AC XR Prominence Autosampler; Shimadzu, Japan), a degasser, a column oven controller, and an evaporative light scattering detector (ELSD; Shimadzu, Japan). An Ultrahydrogel 500 (300 × 7.8 mm) column (Waters Co., Miliford, MA, USA) in combination with a guard column (Ultrahydrogel, 6 × 40 mm, Waters, Milford, MA, USA) was used to maximize the resolution. Standard pullulans including 1300, 6000, 12,000, 22,000, 50,000, 110,000, 200,000, 400,000, and 800,000 Da were used as molecular mass markers. The injection volume was 20 µL and eluted with distilled water at 45 °C with a flow rate of 0.5 mL/min. Each sample was analyzed in duplicate.

The amino acid sequences and molecular mass of the purified peptides were determined by Edman degradation using an automatic protein polypeptide sequencer (Shimadzu Corporation, PPSQ-30A, Kyoto, Japan) and a mass spectrometer (Shimadzu Corporation, LC-20AD XR, Kyoto, Japan).

The lyophilized crude N. atra venom was dissolved in water and centrifuged at 10,000× g for 10 min. The amount of protein in the venom was determined via a BCA Protein Assay kit (Pierce^TM, Thermo Scientific). The supernatants were diluted and further purified by size exclusion chromatography. The purified venom proteins, NTXs, were isolated from the crude venom following the procedure described by Huang et al. [18 (link)]. All the venom protein components other than the NTXs were combined and dissolved in PBS, creating the deNTXs. The crude venom and deNTXs were loaded onto a Phenomenex Jupiter^® C18 (250 × 4.6 mm, 5 µm particle size, 300 Å pore size) column with an ultraperformance liquid chromatography (UPLC) system (LC-20ADXR, Shimadzu, Kyoto, Japan) equipped with a DAD detector (SPD-M20A, Shimadzu, Kyoto, Japan) and autosampler (SIL-20ACXR, Shimadzu, Kyoto, Japan). The venom components were eluted at 1 mL/min with a linear gradient of 0.1% TFA in water (Solvent A) and 0.1% TFA in 100% ACN (Solvent B) (2% B for 5 min, followed by 2–10% B for 2 min, 10–16% B for 6 min, 16–28% B for 2 min and 28–65% B for 37 min) [18 (link)]. Protein elution was monitored at 215 nm (absorption wavelength for peptide bonds). The relative abundance (expressed as the percentage of the total venom protein) of each protein family was estimated as described by Huang et al. [18 (link)].

Several Shimadzu LC units (Tokyo, Japan), including an online vacuum degasser (DGU-20A3R), three pumps (LC-20AD_XR), an auto-sampler (SIL-20AC_XR), a column oven (0–85 °C, CTO-20 A), two electronic 2-channel/6-port valves (FCV-12AH), and a controller (CBM-20 A), were used. An ABSciex 5500 Qtrap mass spectrometer (Foster City, CA, USA) equipped with a Turbo V^TM electronic spray ionization (ESI) interface enabled quantitative measurements, while a Shimadzu IT-TOF-MS mounted ESI source was used for high-resolution mass spectrometry.

Purified FX and SX were analyzed by reversed phase HPLC using a LC-20AD HPLC system (Shimadzu, Kyoto, Japan) consisting of a binary pump (LC20AD XR; Shimadzu, Kyoto, Japan), an automatic injection pump (SIL-20AC XR Prominence Autosampler; Shimadzu, Kyoto, Japan), a degasser, a column oven controller, and a photodiode array detector (PDA; Shimadzu, Kyoto, Japan). The FX and SX were separated on a reverse-phase Sunfire C₁₈ column (5 μm particle size, 250 × 4.6 mm ID, Waters, Milford, MA, USA) coupled to a C₁₈ guard column (5 μm particle size, 15 × 4.6 mm ID), regulated at 25 °C with 20 μL sample injections. The mobile phase for FX consisted of methanol and water with a flow rate of 1 mL min⁻¹. For solvent gradient conditions, methanol/water ratio was increased from 60:40 (v/v) to 100:0 (v/v) over 20 min, 100% methanol was held for next 15 min, and then methanol/water ratio was decreased from 100:0 (v/v) to 60:40 (v/v) over 25 min. To separate SX, the isocratic mobile phase was acetonitrile, methanol, and 0.1% ammonium acetate (75:15:10, v/v/v) at a flow rate of 1 mL min⁻¹. Chromatographic peaks were identified at a wavelength of 450 nm by comparing the retention times and spectra against the known standards (Sigma-Aldrich) (St. Louis, MO, USA). Each sample was analyzed in duplicate.

Mass spectra were
obtained in the electrospray ionization (ESI) positive-
and negative-ion modes using an LC-MS-IT-TOF (Shimadzu) mass spectrometer
equipped with a binary pump (NexeraXR, LC-20ADxr), an auto-sampling
system (NexeraXR, SIL-20ADxr), and a photodiode array (SPD-M20A) detector.
CDL and heat block temperature were set at 250 °C, and the mass
screening was done in the range 100–500 m/z. A detector voltage of 1.7 kV and a flow rate of 1.5 L/min
of the nebulizing gas (N₂) were used for the purpose.
A volume of 2 μL of the sample was directly infused into the
spectrometer with an acid mobile phase (i.e., a mixture of 4-fold
v/v of methanol and 3-fold v/v of acetonitrile and 0.1% v/v of formic
acid) to improve the sensitivity of detection. The solutions were
electro-sprayed at a flow rate of 0.3 mL·min^–1. Qualitative analysis was carried out by comparing the exact masses
with the isotopic distribution, and through a study of the fragmentation
pattern of MS² when available. The LC-MS solution (V3.80.410,
Shimadzu) software was used for data acquisition and processing; the
characterization was processed with the Shimadzu LabSolutions Lite
V5.82 software package (Formula Predict and Accurate Mass Calculator).