DOI: 10.25345/c5zp3wc2w
title: MassIVE MSV000096836 - Enhancing the efficacy in melanoma treatment: the chemosensitising role of Vipera ammodytes venom on human melanoma cell lines.
title: This study investigates the potential of Vipera ammodytes venom as a therapeutic agent for human melanoma, specifically evaluating its cytotoxicity and chemosensitizing effects. Proteomic analysis identified over 125 proteins in the venom, with phospholipases A2, C type lectins, and metalloproteinases among the most abundant components. These proteins are associated with cytotoxic, anti proliferative, and tumor inhibiting properties. Three melanoma cell lines (M001, Me501, and A375) were used to assess venom activity. The IC50 values demonstrated consistent venom sensitivity across cell lines. Combined treatment with venom and cisplatin significantly increased cytotoxicity compared to single agent treatments. Notably, venom enhanced cisplatin sensitivity in resistant cell lines (M001 and Me501), increasing cell mortality by up to 40 percentage. Protein sample preparation for mass spectrometry analysis was carried on following two protocols. In the first protocol, snake venom proteins were dissolved in 100 mM ammonium bicarbonate (AMBIC), subjected to cysteine reduction by 25mM TCEP for 10 min and alkylation by 10mM IAM for 30 min in the dark, precipitated by incubation with six volumes of an organic solution (methanol, acetone and ethanol; 25percent, 25 percent, and 50 percent v/v) at minus 20C over night. Protein pellet was centrifuged at 4C, suspended in 1M urea and 25mM AMBIC and over-night digested adding trypsin, at a substrate to enzyme ratio of 50:1 (w/w) at 37 C on a mixer heat block. In the second protocol, snake venom proteins were denatured for 10 min at 95C in 25mM AMBIC and without a protein precipitation step, directly digested after cysteine reduction and alkylation, in the previously described conditions. The peptide mixture derived from each protocol was injected four times in an Ultimate 3000 UHPLC (Thermo Fisher Scientific, CA, United States) coupled with an Orbitrap Fusion Tribrid mass spectrometer. Peptides were desalted on a trap column and then separated on a 45 cm long silica capillary .The analytical column was encased by a column oven (Sonation; 40C during data acquisition) and attached to a nanospray Flex ion source. Peptides were separated by a 120min long gradient of buffer A (95 percent water, 5 percent acetonitrile, and 0.1 percent formic acid) and buffer B (95percent acetonitrile, 5percent water, and 0,1 percent formic acid), at a flow rate of 250 nl min. The mass spectrometer was operated in positive ion mode, using a data dependent acquisition strategy. Precursor ion scanning was performed in the Orbitrap in the 350 1550 m to z range with 120K resolution. Data dependent acquisition was performed in top speed mode (3s long maximum cycle time): the most intense precursors were selected through a monoisotopic precursor selection (MIPS) filter and with charge grater than1, quadrupole isolated and fragmented by higher energy collisional dissociation (HCD) (30 percent). Product ion spectra were acquired in the ion trap with rapid scan rate. Peptide spectra were searched by the software Proteome Discoverer 2.4 using Sequest HT as search engine against two databases downloaded from UniProtKB Swiss-Prot: Vipera ammodytes ammodytes (Rev Unrev) database (Release 2024; 126 sequences) and Vipera (Rev Unrev) database (Release 2024; 1559 sequences). Spectral matches were filtered using Percolator node, with 1percent q value based false discovery rate (FDR). Only master proteins were taken into account and only specific trypsin cleavages with two miscleavages were admitted. Cysteine carbamydomethylation was set as static modification, while methionine oxidation and N acetylation on protein terminus were set as variable modifications. Precursor and fragment mass tolerance was set to 15 ppm and 0.6 Da, respectively.