Quantification of snake venom proteomes by mass spectrometry‐considerations and perspectives

Abstract

The advent of soft ionization mass spectrometry‐based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global‐integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of “an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available.” This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label‐free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term “non‐model” organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review‐essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof‐of‐concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Venomics and Peptidomics of Palearctic vipers: Clade-wide analysis of seven taxa of the generaVipera,Montivipera,MacroviperaandDaboiaacross Türkiye

“…The proteomic landscapes of snake venoms are intensively investigated and reviewed 16,81,144 . However, the knowledge about their lower molecular weight, peptidic compounds more restricted.…”

“…The used quantification protocol is based and adapted to the common three-step ‘snake venomics’ approach as summarised in Calvete et al 2023 81 . The comparable approach determine a toxin family abundance in the venom as the sum of all its normalized toxin abundances T : The normalized toxin abundance within a single protein band T band is calculated with the normalized values of the RP-HPLC peak integral P measured at 214 nm, the densitometric gel band intensity D and if necessary the relative MS ion intensity M of the most abundant and identified peptides: For the peak quantification after blank run subtraction, the HPLC separation chromatogram fractions were integrated as area under the curve P peak in ratio to the total sum of all peaks: For the densitometric quantification of a single SDS band, the non-highly compressed gel scan (here in PNG format) was processed by Fiji 82 .…”

“…The band area A band and the corresponding integrated band densities D band were measured for each band, as well as a corresponding background areas A bg and integrated band densities D bg . By removing the proportion of background, we calculated the normalized gel band intensity D for each toxin band in the gel: In case of multiple toxin identification within a single band, single normalized toxin abundances M were estimated based on the ion intensity sum of the three most intensive peptide ions of one toxin from M 3 in relation to the sum of all top3 toxin ions from the other co-migrated toxins families within this MS sample, as summarised in Calvete et al 2023 81 : In total, band identification based on the BU, TD and peptidomics results, in comparison to the IMP and the apparent masses of the SDS bands.…”

“…The used quantification protocol is based and adapted to the common three-step 'snake venomics' approach as summarised in Calvete et al 2023 81 . The comparable approach determine a toxin family abundance in the venom as the sum of all its normalized toxin abundances T:…”

Knowledge about Snake Venoms and Toxins from Colombia: A Systematic Review

“…In addition to those shown in Figure 2, other proteomic studies have been performed using a "bottom-up whole venom shotgun profiling" approach, which can identify the proteins present in the venoms, but does not generate a reliable quantification of their relative abundance discussed in [26]. Nevertheless, the qualitative information provided by such analyses is valuable.…”