A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom
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 of 200 spiders was extracted and fractioned into aliquots greater than or less than 10kDa. Then, each aliquot was further aliquoted and digested with trypsin (4h), trypsin (18h), Pepsin (18h), Chymotrypsin (18h), and analyzed by MudPIT on an Orbitrap XL 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 de meta-contig assemblies, and consequently de novo sequencing of practically complete proteins, culminating in a deep proteome assessment of this venom.
Sample Processing Protocol
Sample preparation: Adult L. intermedia specimens (male and female) were collected in the wild in accordance to the Brazilian Federal System for Authorization and Information on Biodiversity (SISBIO-ICMBIO, license number: 29801-1). Venom from two hundred spiders was extracted through electrostimulation method 20 and immediately diluted in ammonium bicarbonate buffer 0.4 M /urea 8 M. Protein concentration was determined through the “Coomassie blue” method using bovine serum albumin (BSA) as standard curve21. First, the venom was separated into two fractions using an ultra-filter unit (MW cut off 10 kDa) (Millipore). The two venom fractions were: Fraction A - containing venom proteins above 10 kDa (400ug) and Fraction B - containing venom proteins and peptides bellow 10 kDa (90ug). All procedures described below were performed equally for each fraction. Each fraction was further divided into four aliquots. All aliquots were reduced with dithiotreitol (DTT) to final concentration of 25mM for 3 h at room temperature. Afterwards, the samples were alkylated with iodoacetamide (IAA) to final concentration of 80 mM for 15 min at room temperature in the dark. Each aliquot was digested with one of the follow enzymes: Trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega Corporation, Madison, WI, USA), Chymotrypsin (Promega, cat. n° V1062), and Pepsin (Promega, cat. n° V1959) at the ratio of 1:50 (E:S). We note that an additional aliquot was stored and not digested. Three aliquots were incubated individually with each enzyme for 18h at 25°C for chymotrypsin and 37°C for trypsin and pepsin. One sample was also incubated only for 4h with trypsin at 37°C. Each digested fraction was divided in three aliquots and desalted with ultra micro C-18 spin columns according to the manufacturing’s instructions (Harvard Apparatus). One aliquot was stored for future use, another had its peptides desalted and directly submitted to reverse phase chromatograph coupled online with an Orbitrap XL mass spectrometry. The third aliquot of the desalted peptides were eluted with 70% acetonitrila, 0.1% formic acid, dried in a speed vacuum concentrator and suspended in buffer C i.e. 10 mM of K2HPO4, 25%ACN, pH= 3.0. Afterwards, the sample was passed through a micro strong cation exchanged spin column (SCX) according to manufacturing’s instructions. (Harvard Apparatus). Briefly, the column was equilibrated with buffer C, centrifuged one minute at 100 x g and, the sample was eluted from the SCX spin column with increasing concentration of KCL, i.e. 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM. Finally, each fraction was desalted once more with ultra-micro C-18 spin columns according to the manufactures guidelines (Harvard Apparatus). All columns were washed ten times with 0.1% of formic acid and the peptides were eluted with buffer B (70% acetonitrile, 0.1% formic acid) to proceed to next step. Mass Spectrometry Analysis: Each fraction of peptides, be it from the non-fractionated or from the SCX fractionation, was previously desalted and subjected to LC-MS/MS analysis with a nano-LC 1D plus System (Eksigent, Dublin, CA) ultra-high performance liquid chromatography (UPLC) system coupled with a LTQ-Orbitrap XL ETD (Thermo, San Jone, CA) mass spectrometer described as follows. The peptide mixtures were loaded onto a column (75 mm i.d., 15 cm long) packed in house with a 3.2 μm ReproSil-Pur C18-AQ resin (Dr. Maisch) with a flow of 500 nL/min and subsequently eluted with a flow of 250 nL/min from 5% to 40% ACN in 0.5% formic acid, in a 120 min gradient. The mass spectrometer was set in data dependent mode to automatically switch between MS and MS/MS (MS2) acquisition. Survey full scan MS spectra (from m/z 350 - 1800) were acquired in the Orbitrap analyzer with resolution R = 60,000 at m/z 400 (after accumulation to a target value of 1,000,000 in the linear trap). The three most intense ions were sequentially isolated and fragmented using CID, HCD, ETD for the same precursor. Previous target ions selected for MS/MS were dynamically excluded for 60 seconds. Total cycle time was approximately five seconds. The general mass spectrometric conditions were: spray voltage, 2.4 kV; no sheath and auxiliary gas flow; ion transfer tube temperature, 100ºC; collision gas pressure, 1.3mTorr; normalized energy collision energy using wide-band activation mode; 35% for MS2. Ion selection thresholds were of 250 counts for MS2. An activation q = 0.25 and activation time of 30 ms was applied in MS2 acquisitions.
Data Processing Protocol
The de novo sequencing approach employed in this work utilized multiple MS2 spectra from overlapping peptides, generated from multiple proteases and of precursors analyzed with CID, ETD and HCD spectrum triples. Each was then converted into prefix residue mass (PRM) spectra where MS/MS peak masses were converted to putative cumulative precursor fragment masses with intensity scores determined using likelihood models specific to each fragmentation mode; triples of PRM spectra from the same precursor were then merged into a single PRM spectrum per precursor by adding scores for matching peak masses. Spectral networks algorithms were then used to align merged PRM spectra from peptides with overlapping sequences and A-Bruijn algorithms were used to integrate these alignments into assembled ‘contigs’. Each contig was then used to construct a consensus contig spectrum capitalizing on the corroborating evidence from all of its assembled spectra to yield a high quality consensus de novo sequence22. Subsequently, the Meta-SPS algorithm was used to align the meta contigs against a FASTA sequence database16. The database contained all sequences from UniProt loxosceles, from the transcriptome of the loxosceles intermedia venom gland23, and an internal database with common mass spectrometry contaminants and proteases.
Trevisan-Silva D, Bednaski AV, Fischer JSG, Veiga SS, Bandeira N, Guthals A, Marchini FK, Leprevost FV, Barbosa VC, Senff-Ribeiro A, Carvalho PC. A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom. Sci Data. 2017 4:170090 PubMed: 28696408