Dataset Information

Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics.

ABSTRACT: The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry.

SUBMITTER: Rangel DL

PROVIDER: S-EPMC9901210 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics.

Rangel Darlene Lopes DL Melani Rafael D RD Carvalho Evelise Leis EL Boldo Juliano Tomazzoni JT Gomes Dos Santos Tiago T Kelleher Neil L NL Pinto Paulo Marcos PM

Toxicon : official journal of the International Society on Toxinology 20221010

The envenomation from the Bothrops genus is characterized by systemic and local effects caused by the main toxin families in the venom. In Bothrops pubescens venom we were able to identify 89 protein groups belonging to 13 toxin families with the bottom-up proteomics approach and 40 unique proteoforms belonging to 6 toxin families with the top-down proteomics approach. We also identified multi-proteoform complexes of dimeric L-amino acid oxidase using native top-down mass spectrometry. ...[more]

PMID: 36228757

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Project description:Bottom-up proteomics relies on the use of proteases and is the method of choice for identifying thousands of protein groups in complex samples. Top-down proteomics has been shown to be robust for direct analysis of small proteins and offers a solution to the "peptide-to-protein" inference problem inherent with bottom-up approaches. Here, we describe the first large-scale integration of genomic, bottom-up and top-down proteomic data for the comparative analysis of patient-derived mouse xenograft models of basal and luminal B human breast cancer, WHIM2 and WHIM16, respectively. Using these well-characterized xenograft models established by the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium, we compared and contrasted the performance of bottom-up and top-down proteomics to detect cancer-specific aberrations at the peptide and proteoform levels and to measure differential expression of proteins and proteoforms. Bottom-up proteomic analysis of the tumor xenografts detected almost 10 times as many coding nucleotide polymorphisms and peptides resulting from novel splice junctions than top-down. For proteins in the range of 0-30 kDa, where quantitation was performed using both approaches, bottom-up proteomics quantified 3,519 protein groups from 49,185 peptides, while top-down proteomics quantified 982 proteoforms mapping to 358 proteins. Examples of both concordant and discordant quantitation were found in a ∼60:40 ratio, providing a unique opportunity for top-down to fill in missing information. The two techniques showed complementary performance, with bottom-up yielding eight times more identifications of 0-30 kDa proteins in xenograft proteomes, but failing to detect differences in certain posttranslational modifications (PTMs), such as phosphorylation pattern changes of alpha-endosulfine. This work illustrates the potency of a combined bottom-up and top-down proteomics approach to deepen our knowledge of cancer biology, especially when genomic data are available.

Project description:BackgroundBothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.ResultsOptimized conditions for two-dimensional gel electrophoresis (2-DE) of pooled venom samples were achieved using immobilized pH gradient (IPG) gels of non-linear 3-10 pH range during the isoelectric focusing step and 10-20% gradient polyacrylamide gels in the second dimension. Software-assisted analysis of the 2-DE gels images demonstrated differences in the number and intensity of spots in juvenile, sub-adult and adult venoms. Although peptide mass fingerprinting (PMF) failed to identify even a minor fraction of spots, it allowed us to group spots that displayed similar peptide maps. The spots were subjected to a combination of tandem mass spectrometry and Mascot and MS BLAST database searches that identified several classes of proteins, including metalloproteinases, serine proteinases, lectins, phospholipases A2, L-amino oxidases, nerve growth factors, vascular endothelial growth factors and cysteine-rich secretory proteins.ConclusionThe analysis of B. atrox samples from specimens of different ages by 2-DE and mass spectrometry suggested that venom proteome alters upon ontogenetic development. We identified stage specific and differentially expressed polypeptides that may be responsible for the activities of the venom in each developmental stage. The results provide insight into the molecular basis of the relation between symptomatology of snakebite accidents in humans and the venom composition. Our findings underscore the importance of the use of venoms from individual specimen at various stages of maturation for the production of antivenoms.

Dataset Information

Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics.

Publications

Venom characterization of the Brazilian Pampa snake Bothrops pubescens by top-down and bottom-up proteomics.

Similar Datasets

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