Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani

Abstract

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-μHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-μHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Keywords: Walterinnesia aegyptia; Walterinnesia morgani; absolute quantification of venom proteome; combined top-down and bottom-up venomics; desert black cobra; hybrid elemental and molecular mass spectrometry; snake venomics.

Figure 1

Schematic of the combined molecular and elemental mass spectrometry methodology applied in this work for the absolute quantification of the venom proteomes of black desert cobras, W. aegyptia and W. morgani. The workflow comprises four RP-HPLC venom protein separations and downstream analysis through bottom-up (2a–c) and top-down (3a–c) venomics and combined parallel mass profiling (4a,b and 6) and absolute sulfur determination by ICPQQQ-MS/MS (5a–e). Continuous sulfur quantification along the chromatographic run was correlated with the molecular masses measured in the parallel RP-capHPLC-ESI-QToF run for the parent venom toxins (6) and assigned to amino acid sequences gathered from bottom-up and top-down venomics (7). Molar ratios sulfur/protein [μmol S/n(Cys + Met)] computed throughout the chromatogram were translated into the corresponding absolute protein amounts (8) using the equation [μmol S/n(Cys + Met)] × M_Ti = μg Ti, where n(Cys + Met) is the number (n) of cysteine and methionine residues in the amino acid sequence of toxin “i” (T_i) and M_Ti is the ESI-MS determined monoisotopic molecular mass of toxin i.

Figure 2

Bottom-up venomics analysis of the toxin arsenal of desert black cobras, W. aegyptia and W. morgani. Panels (A–C) display, respectively, reverse-phase chromatographic separations of the venom proteins of two W. aegyptia specimens (Sinai Peninsula, Egypt, and Riyadh, Saudi Arabia) and a venom sample from a W. morgani specimen original from Çörten village (Turkey). For venomics analyses, chromatographic fractions were collected manually and analyzed by SDS-PAGE (inset) under nonreduced (upper panels) and reduced (lower panels) conditions. Protein bands were excised, in-gel digested with trypsin, and the resulting proteolytic peptides were fragmented through LC-nESI-MS/MS. Parent proteins were identified by database searching (against the last update of the NCBI nonredundant database, including the W. aegyptia venom gland transcriptomic data deposited with the SRA and TSA databases, Supporting Information Table S1) and de novo sequencing followed by BLAST analysis (Supporting Information Tables S2–S4). Picture of W. aegyptia specimens displayed in panels (A) and (B) were taken by Salvador Carranza. Picture of W. morgani, Bayram Göçmen.

Figure 3

Total ion current (TIC) profiles of reduced venom proteins of Egyptian and Saudi Arabian W. aegyptia (panels A and B, respectively) and Turkish W. morgani (panel C) separated by reverse-phase HPLC. Peak numbering same as in the homologous UV-monitored chromatographic traces displayed in Figure 2. Top-down MS identifications of proteins in the proteomes of W. aegyptia and W. morgani venoms are listed in the Supporting Information Table S5 and integrated with the homologous bottom-up datasets in the Supporting Information Tables S2–S4.

Figure 4

Pie charts displaying the BUV quantified relative occurrence (in the percentage of total venom proteins) of the different protein families in the venom proteome of desert black cobras, W. aegyptia (Sinai Peninsula, Egypt) (panel A), W. aegyptia (Riyadh, Saudi Arabia) (panel B), and W. morgani (Çörten village, Turkey) (panel C). Major TDV-identified family member components are highlighted in each pie chart. Acronyms: 3FTx, three-finger toxin; KUN, Kunitz-type serine proteinase inhibitor-like protein; CRISP, cysteine-rich secretory protein; PLA₂, phospholipase A₂; 5′NT, 5′ nucleotidase; svNGF, snake venom nerve growth factor; PIII-SVMP, snake venom metalloproteinase of class PIII; PDE, phosphodiesterase; LAO, l-amino acid oxidase; VEGF, vascular endothelial growth factor; AcChol, acetylcholinesterase; Endo, endonucleotidase.

Figure 5

Overlay of the ICP-MS mass flow chromatograms (red) and the ESI-MS chromatograms (black) of the venoms of (A) W. aegyptia (Sinai Peninsula, Egypt), (B) W. aegyptia (Riyadh, Saudi Arabia), and (C) W. morgani (Çörten village, Turkey). Peak matching displayed in the Supporting Information Tables S6–S8 enabled correlating molecular peak identity and elemental S quantitation.