A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

Abstract

Venoms are a rich source for the discovery of molecules with biotechnological applications, but their analysis is challenging even for state-of-the-art proteomics. Here we report on a large-scale proteomic assessment of the venom of Loxosceles intermedia, the so-called brown spider. Venom was extracted from 200 spiders and fractioned into two aliquots relative to a 10 kDa cutoff mass. Each of these was further fractioned and digested with trypsin (4 h), trypsin (18 h), pepsin (18 h), and chymotrypsin (18 h), then analyzed by MudPIT on an LTQ-Orbitrap XL ETD mass spectrometer fragmenting precursors by CID, HCD, and ETD. Aliquots of undigested samples were also analyzed. Our experimental design allowed us to apply spectral networks, thus enabling us to obtain meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of the venom. Data are available via ProteomeXchange, with identifier PXD005523.

Forty Years of the Description of Brown Spider Venom Phospholipases-D

“…In addition, a comprehensive profile of Loxosceles intermedia crude venom toxins was obtained by the digestion of the venom followed by the separation of fragments using UHPLC liquid chromatography and analysis by mass spectrometry (ORBITRAP). The final data showed the presence of peptides compatible with different members of the PLD family in the venom, confirming the existence of a large intra-species family of these molecules [77].…”

TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism

“…The effects of L. intermedia crude venom on RBL-2H3 degranulation ( Figure 2C) are related to the histaminergic effects of Loxoscelism. The report of TCTP-related toxins in the Loxosceles venoms were previously done by transcriptomic studies using venom gland transcripts [1], by cloning and recombinant expression of LiRecTCTP [2], and recently, TCTP was identified in the proteomic study of the L. intermedia venom [32]. Additionally, TCTP was immunodetected in the whole venom of Loxosceles species (L. intermedia, L. gaucho, and L. laeta) [3], pointing to conservation and biological relevance.…”

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms

“…LC-MS/MS can be conducted with a variety of analyzers including the lower-massresolving ion trap and triple quadrupole instruments, intermediate resolving power quadrupole-quadrupole-TOF (QqTOF) instruments, and higher-resolving-power triple-quadropole-TOF (QqQ-TOF), Orbitrap or FT-ICR instruments. LC-MS/MS using DDA is the most common method for obtaining venom peptide fragmentation data in many laboratories, [22,25,[48][49][50][51][52]57,[65][66][67][68][69][70][71][72][73][74][75][76][77][78][79] including ours. [29,42,43,53,61,[80][81][82][83] These data are typically searched using an automated algorithm against a database of protein sequences to identify peptide primary structures.…”

“…Taken together, CID, ETD, and ECD provide complementary fragmentation data that can be valuable for de novo sequencing, [67][68][69][70] particularly in hybrid mass spectrometers, such as the LTQ-Orbitrap family, that have parallel scanning capability allowing simultaneous collection of high-resolution MS data and lower resolution ion trap MS/MS data. For example, Ueberheide et al [84] developed an ETD-based de novo sequencing method specifically tailored to disulfide-rich venom peptides.…”

“…MuDPIT has also been used to resolve the proteomes of the tarantula P. nigriventer [131] and the jewel wasp A. compressa, yielding 194 and 264 polypeptide sequences from these complex venoms, respectively. Trevisan-Silva et al [69] combined MuDPIT separation with multi-enzyme digests and a combined top-down and bottom-up approach to resolve the proteome of the necrosis-causing venom of the brown recluse spider Loxosceles intermedia.…”