Mass spectrometry data and size exclusion chromatography profiles of Australian taipan venom toxins

The compositions of paradoxin and taipoxin (PDx and TPx, respectively) were investigated using size exclusion chromatography (SEC) and nano-electrospray ionization mass spectrometry (nano-ESI-MS). The elution profiles from size exclusion chromatography of the venoms from Oxyuranus microlepidotus and Oxyuranus scutellatus were similar. Fractions corresponding to the trimeric toxins were treated with guanidinium hydrochloride and the individual subunits were separated by HPLC. In this report we present the size exclusion chromatography profiles for these toxins, and the nano-ESI mass spectra of the subunits after separation by HPLC: the first such comparative study of these toxins at the protein level. Data in this article are associated with the research article published in Toxicon: “Insight into the subunit arrangement and diversity of paradoxin and taipoxin” (J.A. Harrison, J.A. Aquilina, 2016) [1].


Subject area
Chemistry and Biology More specific subject area

Structural proteomics
Type of data Table, figure How data was acquired Size exclusion chromatography, HPLC and mass spectrometry

Data format Analyzed Experimental factors
Amino acid sequences of toxin subunits were sourced from uniprot.org

Experimental features
Toxin oligomers and subunits were separated by chromatographic techniques for analysis by mass spectrometry Data source location University of Wollongong, NSW, Australia Data accessibility Data are within this article

Value of the data
Outlines the purification steps required to separate oligomeric PLA 2 . Presents size exclusion chromatography elution profiles for comparison with oligomeric toxins from other snake species.
Describes isolation of individual toxin subunits for further characterization such as for sequence and post-translational modification analysis.
Describes the instrumentation and conditions required for acquiring ESI mass spectra of the oligomeric toxins and subunits.
Presents nano-ESI mass spectra of oligomeric toxins and subunits from Oxyuranus microlepidotus and Oxyuranus scutellatus. These spectra can serve as a comparison for those from other species.

Data
Three figures showing the size exclusion chromatograms for whole venom analysis and the nano-ESI mass spectra of the peaks from the HPLC of PDx and TPx subunits. Three tables, two containing the experimental subunit masses determined using nano-ESI-MS after HPLC separation, and a third showing the sequence data for the previously characterized subunits of TPx and PDx.

Size exclusion chromatography (SEC)
For each of the chromatographic runs, lyophilized whole venom from O. microlepidotus or O. scutellatus was dissolved in 200 mM ammonium acetate (NH 4 OAc) and loaded onto a Superdex 100 10/30 size exclusion column (GE Healthcare, Uppsala, Sweden). Fig. 1 shows the elution profiles. The second fractions (labeled 2, primarily containing the trimeric PLA 2 , PDx or TPx) were analyzed by SDS-PAGE ( Fig. 1, insets). Each gel consisted of a 4% stacking gel and 15% resolving gel. Electrophoresis was performed using a Mini Protean 3 system (Bio-Rad, California, U.S.A), which was powered by a Bio-Rad power pack 300 power supply (Bio-Rad, California, U.S.A). Protein samples were mixed with a cracking buffer solution (0.5 M Tris-HCl, pH 6.8: 14 mL, 0.02 g Coomassie Blue, glycerol: 16 mL, 10% SDS: 16 mL) at a ratio of 1:1, then heated to 100°C for 5 min prior to loading 10 mL of each sample into individual wells. Molecular weight standards were 5 mL of Precision Plus Protein™ Dual Color Standards (Bio-Rad, California, U.S.A.). Separation was achieved at 120 V after 1 h. Gels were stained with Coomassie Blue for at least 2 h, followed by destaining until protein bands were visible. Gels were scanned using a GS-800 calibrated densitometer (Bio-Rad, California, U.S.A).

Mass spectrometry analysis of HPLC samples
All spectra were acquired using a Synapt (G1) HDMS time-of-flight mass spectrometer (Waters, Manchester, UK) and calibrated against a spectrum of CsI solution (10 mg/mL) acquired on the same day. Samples from HPLC separations were dissolved in 200 mM NH 4 OAc and introduced into the vacuum region of the instrument by electrospraying 2 mL of solution from a gold-coated borosilicate nano-ESI capillary (prepared in-house). Conditions for the acquisition of mass spectra were as follows: capillary voltage, 1.5 kV; sample cone, 140 V; extraction cone, 4 V; trap collision 12.5 V; transfer collision 25 V; collision cell gas pressure, 1.16 Â 10 À 2 mbar; backing gas, 4.02 Â 10°mbar. Mass spectra of the subunits of the toxins are shown in Figs. 2 and 3; the masses determined from the spectra are shown in Tables 1 and 2. These data were then compared to the existing sequence data for these toxins (Table 3). Fig. 3. Mass spectra of TPx subunits separated by HPLC [1]. For HPLC peaks one to four (Fig. 5 in [1]), charge state series were evident between 1800 and 2750 m/z, while peak 5 ( Figure 5 in [1]) contained components with charge states arising within a higher m/z window. Each inset shows the mass distribution of β TPx isoforms for relevant HPLC peaks. The subunit identified by each series is shown on the top of each spectra. Table 1 Nano-ESI-MS analysis of PDx components separated using HPLC. The number of each HPLC peak (Fig. 5 in [1] and Fig. 2 above) is shown in the left column of the table. The right column shows the observed mass of each protein detected in the nano-ESI-MS analyses, with the proposed identity in brackets.