Zorbax 300sb c18 column

Edman degradation was carried out by the Australian Proteome Analysis Facility (APAF, Sydney, Australia). Purified native calliotoxin was solubilised in ammonium bicarbonate (25 mM)/10% ACN and reduced using DTT (25 mM) at 56 °C for 0.5 h, followed by alkylation using iodoacetamide (55 mM) at room temperature for 0.5 h. The reaction mix was then desalted/purified by RP-HPLC using a Zorbax 300SB-C18 column (3 × 150 mm, Agilent, Santa Clara, CA, USA). The volume was reduced under vacuum and loaded onto a precycled, Biobrene-treated disc and subjected to 60 cycles of Edman N-terminal sequencing using an Applied Biosystems 494 Procise Protein Sequencing System (Applied Biosystems, Foster City, CA, USA), resulting in unambiguous identification of 47 amino acid residues. Venom gland transcriptomics were conducted by the IMB Sequencing Facility (Institute for Molecular Bioscience, The University of Queensland, St Lucia, Qld, Australia). Libraries were prepared with the TruSeq Stranded mRNA kit (Illumina, San Diego, CA, USA), and were sequenced on the Illumina NextSeq (Illumina) 500 using 2 × 150 bp reads and V2 chemistry. To identify the full sequence of δ-elapitoxin-Cb1a, forward and reverse sequences were merged using MacQIIME (Werner Lab, SUNY Cortland, NY, USA) join_paired_end.py and matched to the sequence determined by Edman degradation using standalone BLAST.

The content of SV in NLCs was determined by high-performance liquid chromatography system (HPLC), equipped with an Agilent ZORBAX 300SB-C18 column (4.6 mm × 250 mm, 5 μm) with a constant flow rate of 1 mL/min and ultraviolet detection at a wavelength of 238 nm. To test the encapsulation efficiency (EE%) and drug loading (DL%), the formulated SV/NLCs dispersions with the original carriers concentration were flocculated by adding 1 M of hydrochloric acid until the pH value was adjusted to 1.2. The SV/NLCs aggregation was separated via centrifugation (3K30, Sigma Labrorzentrifugen GmbH, Germany) at 20 000 rpm for 30 min. The SV concentration in the supernatant was measured. The EE% and DL% of SV in the NLCs were calculated by the following equations:
$\mathrm{EE}\hspace{1em}\%=({\mathrm{M}}_{\mathrm{a}}-{\mathrm{M}}_{\mathrm{s}})/{\mathrm{M}}_{\mathrm{a}}\times 100\%$
$\mathrm{DL}\mathrm{\hspace{1em}}\%=({\mathrm{M}}_{\mathrm{a}}-{\mathrm{M}}_{\mathrm{s}})/({\mathrm{M}}_{\mathrm{a}}+{\mathrm{M}}_{\mathrm{c}}-{\mathrm{M}}_{\mathrm{s}})\times 100\%$
where M_s indicates the mass of SV in supernatant. M_a indicates the mass of SV added in the system. M_c indicates the mass of lipid added in the system.

Protein identification was performed using nano LC-ESI-MS/MS. The MS system consisted of an Agilent 1100 nanoLC system (Agilent), PicoTip electrospray emitter (New Objective) and an Orbitrap XL mass spectrometer (Thermo-Fisher). Protein spots from the membranes were in-gel digested by trypsin (Promega) (with and without citraconic anhydride treatment) and applied to nanoLC-ESI-MS/MS. Peptides were trapped and desalted on the enrichment column (Zorbax SB C18; 0.3x5 mm; Agilent) for five minutes using 2.5% acetonitrile/0.5% formic acid as eluent, then peptides were separated on a Zorbax 300 SB C18 column (75µmx150mm; Agilent) using an acetonitrile/0.1% formic acid gradient from 5 to 35% acetonitril within 40 minutes. MS/MS spectra were recorded data-dependently by the mass spectrometer, according to manufacturer's recommendations.

Chromatographic analysis was performed using an Agilent 1260 liquid chromatography system (Agilent Technology, Inc., Santa Clara, CA, USA), which was equipped with a G1311C quat pump, a G1329B autosampler, a G1316A column compartment, and a G1315D diode array detection (DAD) system. The obtained data were analyzed on an Agilent open LAB CDS ChemStation C.01.04. The analysis of fingerprint was carried out at 25 °C on an Agilent ZORBAX 300SB-C18 column (4.6 × 250 mm, 5 μm). The elution gradient of eluents A (acetonitrile with 0.1% TFA) and B (water with 0.1% TFA) was used for the separation of the target analytes. The gradient program was as follows: 0–40 min, 5–77% A; 40–58 min, 77–95% A. Each run was followed by an equilibration time of 5 min. The UV absorbance was monitored at 215 nm, and the solvent flow rate was kept at 1.0 mL/min. All injection volumes of both the samples were 5 μL.