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. 2017 Jul 11;4:170090. doi: 10.1038/sdata.2017.90

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

Dilza Trevisan-Silva 1, Aline V Bednaski 1, Juliana SG Fischer 2, Silvio S Veiga 1, Nuno Bandeira 3, Adrian Guthals 3, Fabricio K Marchini 4,5, Felipe V Leprevost 2, Valmir C Barbosa 6, Andrea Senff-Ribeiro 1,a, Paulo C Carvalho 2,7,b
PMCID: PMC5505115  PMID: 28696408

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.

Keywords:

Subject terms: Proteomics, Protein analysis, Entomology

Background & Summary

Scientists have long enlisted venoms in their quest to characterize novel molecules with biotechnological applications1,2. The literature provides innumerous examples of venom-derived applications, ranging from biopesticides to medical applications. In particular, works on serpent venom are, unarguably, success stories. Some examples are: Batroxobin, a widely used thrombin-like enzyme and commonly extracted from the venom of Bothrops atrox and Bothrops moojeni, has been used as a replacement for thrombin in bleeding injuries3; Ecarin, from Echis carinatus, as the primary reagent for laboratorial tests that monitor anticoagulation4; and Captopril, developed from peptides of the Bothrops jararaca venom, as a widely adopted inhibitor of the angiotensin converting enzyme (ACE). Other examples of venom-derived drugs include: Aggrastat, for myocardial infarct and ischemia; Ancrod, for stroke; Defibrase, for acute cerebral infarction and angina pectoris; Exanta, used as an anti-coagulant; Hemocoagulase, for hemorrhage; and Integrilin, for acute coronary syndrome5. Venoms have also been used to search for inhibitors derived from other species (e.g., Didelphis marsupialis)6,7.

Motivated by all the successful research on snake venoms, efforts have been geared towards spider toxins. In particular, those from the Loxosceles genus are already being used in at least four general application fronts, viz.: as therapeutic anti-venom sera8; as tools in molecular and cellular biology research; and as aids in drug development and production of selective and environmentally friendly bioinsecticides5. Peptides originating from the venom of Thrixopelma pruriens have been used in the treatment of pain and inflammation9; the T×2–5 and T×2–6 neuropeptides from the Phoneutria nigriventer venom, for treating erectile dysfunctions10; and distinct bioactive peptides from spider venoms, in the treatment of diverse diseases, such as cancer11. Taken together, toxins have served as an endless treasure trove for biotechnological applications.

Spider venoms, in particular, comprising mainly proteins and peptides2,5,12,13 and displaying great diversity in their toxins, have drawn considerable attention. Yet, characterizing venoms poses great challenges even for state-of-the-art proteomic strategies: in fact, most species lack a reference sequence genome14 and the post-translational modifications of venoms vary greatly. Moreover, current mainstream strategies are not tailored towards performing de novo sequencing of the large (i.e., greater than tryptic), biologically active peptides that abound in venoms. Indeed, peptide-centric approaches are oblivious to whether a sequenced peptide originates from a larger peptide or a full protein, but obtaining the complete sequence of these larger molecules will undoubtedly fuel a great diversity of biotechnological applications. In this regard, it is our view that widely adopted proteomic strategies such as peptide spectrum matching (PSM)15,16 and mainstream de novo sequencing17 only reveal the tip of the iceberg in terms of what can be unveiled from venoms.

One of our goals has been to characterize the venom of the so-called brown spiders (the Loxosceles genus). Altogether, their venom is composed of a complex cocktail of biologically active compounds, with toxins ranging up to 40 kDa and over18. To the best of our knowledge, an in-depth, comprehensive proteomic profiling of the Loxosceles venom tailored towards the discovery of new molecules has so far remained elusive. Currently, there are several descriptions of enzymatic and non-enzymatic proteins from distinct Loxosceles species19,20. In 2003, a study aimed to investigate whether venoms of phylogenetically-related groups of Haplogyne spiders possess sphingomyelinase-D (SMD) toxins21. The study included 10 Loxosceles species and 2 Sicarius species, among other spider genera. The Amplex Red Phospholipase-D assay kit indicated SMD activity and these results were further supported by a Surface-Enhanced Laser Desorption/Ionization (SELDI) Time-of-Flight (TOF) analysis showing mass spectral peaks with m/z’s corresponding to those of SMD. Loxosceles SMDs, later referred to as phospholipases-D (PLDs), are known to be the major component of Loxosceles venoms and are the most well characterized toxin family in brown spider venoms. In 2005, two-dimensional protein profiles of the L. intermedia, L. laeta, and L. gaucho venoms were determined, but protein identification was focused only on the SMD toxins of the L. gaucho venom22. The identification of seven spots of interest was first attempted using data from Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) Mass Spectrometry (MS) and Electrospray Ionization (ESI) quadrupole-time-of-flight Tandem Mass Spectrometry (MS/MS) for direct search of raw data using MASCOT22. Since the searches retrieved no significant match, de novo sequencing was performed and the resulting sequences were BLASTed against the non-redundant sequences, allowing SMD identification for all analyzed spots22. Only in 2009 was a proteomic study described that targeted the total protein content of the Loxosceles venom23. Although the L. intermedia venom was analyzed using Multi-Dimensional Protein Identification Technology (MudPIT)24, only 39 proteins were identified. Of these proteins, only 14 were described as toxins generally found in animal venoms23. Thus, this proteomic study seems to have severely underestimated the great toxin diversity of the Loxosceles venom, particularly in comparison to the many publications that already described distinct molecular clones from venoms of different Loxosceles species25–32. Transcriptome analyses of the L. laeta and L. intermedia venoms revealed a huge complexity of brown-spider venoms19,20. Specifically, the analysis of the L. intermedia venom described three classes of toxins comprising most toxin-encoding transcripts, such as peptides of low molecular mass (55.9%), astacin-like proteases (22.6%), and PLDs (20.2%). Also, transcripts similar to hyaluronidases, serine proteases, serine protease inhibitors, venom allergens, and members of the translationally controlled tumor protein (TCTP) family presented low levels of expression20. Although considerable information is now available on venom gland transcripts of L. intermedia, the total protein content of this venom has remained unclear. A previous study from our group applied two-dimensional immunoblots and zymograms on the venom of L. intermedia, L. laeta, and L. gaucho, and revealed several spots with differential volume containing proteins having gelatinolytic activity corresponding to astacin-like proteases33. These results corroborate that venoms from these species present a broad astacin-like family with many isoforms22,33,34.

The lack of genomic data from this arachnid prevents employing the PSM approach in full, so most of the weightlifting must be accomplished through de novo sequencing. Mainstream de novo sequencing, however, cannot efficiently handle unanticipated post-translational modifications, being far more prone to generating sequencing errors. This is because various molecules fail to provide enough mass spectral peaks during fragmentation to enable the sequencing of full peptides. To overcome these limitations, our dataset was acquired with multiple dissociation strategies applied to the same precursor (e.g., collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), and electron-transfer dissociation (ETD)), thereby enabling the use of state-of-the-art de novo sequencing algorithms. These capitalize on complementary dissociation information and thus achieve unprecedented sequencing accuracy35,36. The use of different proteolytic enzymes on the venom aliquots unlocks the application of another very powerful paradigm, that of spectral networks37,38. These ‘specnets’ align spectra against one another, ultimately allowing the detection of unanticipated post-translational modifications. Moreover, they can assemble consensus mass spectra from overlapping peptides yielded by different proteolytic digests. A consensus spectrum thus obtained presents a better signal-to-noise ratio and allows for the de novo sequencing of amino-acid stretches far longer than those handled by the conventional approach. Once high-confidence de novo data are available, it becomes possible to employ tools, such as PepExplorer39 or Meta-SPS37, that apply pattern recognition approaches to the mapping of de novo sequencing data against sequences from homologous organisms, thereby facilitating biological interpretation.

By themselves, the meta-contig assemblies provided by spectral networks are not enough for one to conclude whether a biomolecule obtained 100% coverage. To pave the way in this direction, top-down proteomic data in combination with MS3 (i.e., product ion(s) selected from an MS/MS spectrum further fragmented and producing another tandem mass spectrum) and ETD were also acquired for a partition of the venom molecules into two sets (<~10 kDa and >~10 kDa). The top-down strategy consists of injecting intact proteins into the mass spectrometer, thus doing away with the inference limitations of the peptide-centric approach40. This provides complementary information to that of the networks and helps in the discovery of how much is required for obtaining full coverage. We anticipate that these data will be fundamental in the development of next-generation algorithms capable of bridging the gap between bottom-up, middle-down, and top-down proteomics.

Here, we present the first multi-protease, multi-dissociation, bottom-up-to-top-down proteomic dataset of the venom of L. intermedia, the ‘urban’ spider species commonly found in the city of Curitiba, Brazil41, along with an analysis using state-of-the-art tools. The approach stems from the motivation that multiple enzyme digestion increases protein coverage42, besides relying on different activation and acquisition methods.

Methods

Sample preparation

Adult L. intermedia specimens (both male and female) were collected in the wild in accordance with the Brazilian Federal System for Authorization and Information on Biodiversity (SISBIO-ICMBIO, license number 29801-1). Venom from 200 spiders was extracted through the electrostimulation method43 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 curve44. First, the venom was separated into two fractions using an ultra-filter unit (MW cutoff 10 kDa) (Millipore), one fraction containing venom proteins above ~10 kDa (400 μg) and the other containing venom proteins and peptides bellow ~10 kDa (90 μg). All procedures described next were performed equally for each fraction, after further dividing it into four aliquots, each of which was reduced with dithiothreitol (DTT) to a final concentration of 25 mM for 3 h at room temperature. Afterwards, the samples were alkylated with iodacetamide (IAA) to a 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, cat. No. V5280, WI, USA), chymotrypsin (Promega, cat. No. V1062), and pepsin (Promega, cat. No. 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 18 h, at 25 °C for chymotrypsin and 37 °C for trypsin and pepsin. The other aliquot was incubated for only 4 h with trypsin at 37 °C. Each digested fraction was divided into three aliquots and desalted with ultra-micro C-18 spin columns according to the manufacturer’s instructions (Harvard Apparatus). One of these three aliquots was stored for future use, another had its peptides desalted and directly submitted to reverse phase chromatography coupled online with an Orbitrap XL mass spectrometer. The third aliquot of the desalted peptides was eluted with 70% acetonitrile (ACN) and 0.1% formic acid, then dried in a speed vacuum concentrator, suspending 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 the manufacturer’s instructions (Harvard Apparatus). Briefly, the column was equilibrated with buffer C, centrifuged for 1 min at 100×g, and the sample was eluted from the SCX spin column with increasing concentration of KCl, i.e., 100, 170, 290, and 400 mM. Finally, each fraction was desalted once more with ultra-micro C-18 spin columns according to the manufacturer’s instructions (Harvard Apparatus). All columns were then washed ten times with 0.1% formic acid and the peptides were eluted with buffer B (i.e., 70% acetonitrile, 0.1% formic acid) to proceed to next step.

Mass spectrometry analysis

Each fraction of peptides, including the non-fractionated as well as those from the SCX fractionation, was previously desalted and subjected to an LC-MS/MS analysis on a nano-LC 1D plus System (Eksigent, Dublin, CA), an ultra-high performance liquid chromatography (UHPLC) system coupled with an LTQ-Orbitrap XL ETD (Thermo, San Jose, CA) mass spectrometer, at the Mass Spectrometry Facility RPT02H of the Carlos Chagas Institute (Fiocruz, Brazil). In these analyses, 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 to data-dependent mode to automatically switch between MS and MS/MS acquisition. Full-scan MS spectra (m/z 350–1,800) 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) using survey mode. The three most intense ions were sequentially isolated and fragmented using CID, HCD, and ETD for the same precursor. Previous target ions selected for MS/MS were dynamically excluded for 60 s. The total cycle time was approximately 5 s. The general mass spectrometric conditions were: spray voltage, 2.4 kV; no sheath or auxiliary gas flow; ion transfer tube temperature, 100 °C; collision gas pressure, 1.3 mTorr; normalized collision energy using wide-band activation mode; 35% for MS/MS. Ion selection thresholds were of 5,000 counts for MS/MS. The parameters for each fragmentation type in MS/MS acquisitions were as follows. For CID: isolation width, m/z 2.5; normalized collision energy, 35; activation, q=0.25; activation time, 30 ms. For HCD: isolation width, m/z 2.5; normalized collision energy, 35; activation time, 30 ms; full width at half maximum resolution, 15,000. For ETD: isolation width, m/z 2.5; activation time, 100 ms.

Bioinformatics analysis

The de novo sequencing approach employed in this work utilized multiple MS/MS spectra from overlapping peptides, generated from multiple proteases and of precursors analyzed with CID, HCD, and ETD spectrum triples. Each was then converted into prefix residue mass (PRM) spectra. In this conversion, MS/MS peak masses were converted into putative cumulative precursor fragment masses, with intensity scores determined from 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-network algorithms, implemented in the ProteoSAFe web platform that is freely accessible at http://proteomics.ucsd.edu/ProteoSAFe/, were then used to align merged PRM spectra from peptides with overlapping sequences. Moreover, A-Bruijn algorithms were used to integrate these alignments into assembled contigs.

Each contig was then used to construct a consensus contig spectrum, or meta-contig, capitalizing on the corroborating evidence from all of its assembled spectra to yield a high-quality consensus de novo sequence36. Subsequently, the Meta-SPS algorithm was used to align the meta-contigs against a FASTA sequence database37. This database contained all Loxosceles sequences from UniProt, all from the transcriptome of the L. intermedia venom gland20, and an internal database with common mass spectrometry contaminants and proteases.

A summary of this methodology is found in Fig. 1.

Figure 1. Methodology workflow.

Figure 1

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Summary of the sequence of procedures that constitute the methodology employed, from venom extraction to the meta-contig assembling that enabled the identifications of venom proteins.

Data Records

Our bioinformatics analysis disclosed a list of 190 proteins (Table 1). As far as we know, this is the most complete comprehensive proteomic profiling of the L. intermedia venom. All mass spectrometry data are available from both the ProteomeXchange Consortium via the PRIDE45 partner repository, with dataset identifier PXD005523 (Data Citation 1), and our servers (http://proteomics.fiocruz.br/pcarvalho/lintermedia/venom/). A full list of the proteins, meta-contigs, and homologous sequences is made available in Table 1.

Table 1. Identifications resulting from the >~10 kDa and <~10 kDa venom fractions.

  >~10 kDa <~10 kDa Together
The last column, Together, eliminates redundancies (viz., by maximum parsimony53), as well as contaminants and the proteases used during sample preparation (trypsin, chymotrypsin, and pepsin).      
No. of spectral triplets 41,386 36,625 78,011
No. of contigs 642 454 1,096
No. of homologous sequences 440 228 190
No. of de novo sequences 642 454 1,096
No. of proteins 440 228 190
No. of raw files 42 46 88
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All Meta-SPS results for >~10 kDa and <~10 kDa, together with the parameter files used for running the software, are available as separate material (MetaSPS_Results.xlsx, Data Citation 2). The results are presented in six tabs, viz., for >~10 kDa grouped by contig, >~10 kDa grouped by spectrum, >~10 kDa parameter file, <~10 kDa grouped by contig, <~10 kDa grouped by spectrum, and <~10 kDa parameter file.

Technical Validation

The lack of any previous comprehensive proteomic analysis of the Loxosceles venom demonstrates that studying this venom in detail has been a challenge, one that stems from the organism being highly non-canonical and from the fact that protein sequences for it have remained scarce in databases. The present work circumvented these obstacles by using a combination of shotgun proteomic experiments and different tools to generate and analyze large proteomic datasets and de novo sequencing results.

Our results revealed 190 protein identifications, including all classes of toxins described in previous transcriptome analyses19,20 (Table 2 (available online only)). Our approach identified both high- and low-abundance toxins of the L. intermedia venom, as well as homolog sequences from distinct Loxosceles species (astacin-like proteases, PLDs, peptides, TCTPs, hyaluronidases, allergens, serine proteases, serine protease inhibitors, and housekeeping proteins) (Table 2 (available online only)). These data reinforce the holocrine nature of the Loxosceles venom gland23 and demonstrate that its venom is composed of toxins and housekeeping proteins originating from epithelial-cell content, such as the angiotensin converting enzyme, the 60S ribosomal protein, the Na-Pi co-transporter, and the myosin heavy chain (Table 2 (available online only)). Our results, therefore, validate the method used for analyzing the proteome of an organism with non-sequenced genome.

Table 2. Meta-contig assemblies output by Meta-SPS on shotgun data from the venom of Loxosceles intermedia and corresponding proteins mapped per similarity.

  de novo sequence Protein index Protein name Species
The contigs are listed according to their toxin class.        
Toxin class
        
 Metalloprotease [211.812][154.295][261.952]Y[200.869]NMIYGAG gi 116733934 gb ABK20019.1 astacin-like metalloprotease toxin precursor Loxosceles intermedia
  [168.09]PDATIVY[128.106] gi 257219865 gb ACV52010.1 astacin-like metalloprotease toxin 2 precursor Loxosceles intermedia
  DDYVI[262.263][128.148] gi 257219867 gb ACV52011.1 astacin-like metalloprotease toxin 3 precursor Loxosceles intermedia
  GVIIHE[291.835][227.31]GFY TSA 1, Contig: LIS109 putative astacin-like metalloprotease Loxosceles intermedia
  GTCICDGGSCNC[128.125]R TSA 1, Contig: LIS137 putative astacin-like metalloprotease Loxosceles intermedia
  [127.832]VRNGCNT[252.829]DVGIR TSA 1, Contig: LIS187 putative astacin-like metalloprotease Loxosceles intermedia
  RDAP[215.036][170.707][196.097]IG[153.721]F TSA 2, Contig: LIC247 putative astacin-like metalloprotease Loxosceles intermedia
  GNMFSCWSTVGMR TSA 2, Contig: LIC331 putative astacin-like metalloprotease Loxosceles intermedia
  INIIY[128.088] TSA 2, Contig: LIS63 putative astacin-like metalloprotease Loxosceles intermedia
  [128.115]PCGNNV[274.883]DP[227.119] TSA 3, Contig: LIC321 putative astacin-like metalloprotease Loxosceles intermedia
  GAAGVTIINPAAGR TSA 3, Contig: LIC369 putative astacin-like metalloprotease Loxosceles intermedia
  NAVSSD[244.341] TSA 3, Contig: LIS121 putative astacin-like metalloprotease Loxosceles intermedia
  GVIIHEIGHVIGF sp A0FKN6 VMPA LOXIN astacin-like metalloprotease toxin precursor Loxosceles intermedia
  [153.983]IDI[128.103][241.226]NTIY tr C9D7R2 C9D7R2 LOXIN astacin-like metalloprotease toxin 2 precursor Loxosceles intermedia
  GDSRIWPDGVVIY tr C9D7R3 C9D7R3 LOXIN astacin-like metalloprotease toxin 3 precursor Loxosceles intermedia
  F[242.232]D[275.039][128.008]RCVY TSA 1, Contig: LIC313 putative astacin-like metalloprotease Loxosceles intermedia
  [127.931]D[128.061][241.133]DR[184.194][174.03]PY TSA 1, Contig: LIC411 putative astacin-like metalloprotease Loxosceles intermedia
  IEAGDV[128.029]GIGCGG TSA 1, Contig: LIS117 putative astacin-like metalloprotease Loxosceles intermedia
  [349.495]IGITGF TSA 1, Contig: LIS234 putative astacin-like metalloprotease Loxosceles intermedia
  [184.051]N[257.098][216.857]I[259.978]E[291.043]SIPSN TSA 1, Contig: LIS98. putative astacin-like metalloprotease Loxosceles intermedia
  F[127.959]NHDS[204.387]D[242.74] TSA 2, Contig: LIC228 putative astacin-like metalloprotease Loxosceles intermedia
  RDAP[215.036][170.707][196.097]IG[153.721]F TSA 2, Contig: LIC247 putative astacin-like metalloprotease Loxosceles intermedia
  GIGIT[128.015]PCTCS[198.07]C[225.217] TSA 2, Contig: LIC331 putative astacin-like metalloprotease Loxosceles intermedia
  INIIY[128.088] TSA 2, Contig: LIS63 putative astacin-like metalloprotease Loxosceles intermedia
 Phospholipase-D [198.061]YITAST[234.471]D[127.861][128.324]DFA[128.058] gi 141452623 gb ABO87656.1 dermonecrotic toxin isoform 6 Loxosceles intermedia
  HYEIF[128.043]GFR gi 156067386 gb ABU43333.1 loxtox i5 Loxosceles intermedia
  [241.121]ICAIVI[202.255]G[245.305]II[326.299] gi 156067390 gb ABU43335.1 loxtox i7 Loxosceles intermedia
  [185.016]CC[215.047]DVANAEAW gi 224472025 gb ACN48894.1 sphingomyelinase D-like protein, partial Loxosceles arizonica
  VATYDDN[283.158]V[248.057][128.175][128.114] gi 224472109 gb ACN48936.1 sphingomyelinase D-like protein, partial Loxosceles spadicea
  I[128.164][185.033][320.151][199.846]V[128.057]V[257.079] gi 224472111 gb ACN48937.1 sphingomyelinase D-like protein, partial Loxosceles variegata
  [258.156]GS[215.905]C[128.131]TN[142.105] gi 224472117 gb ACN48940.1 sphingomyelinase D-like protein, partial Loxosceles arizonica
  [323.223]SIDIIAS[128.107] gi 224472133 gb ACN48948.1 sphingomyelinase D-like protein, partial Loxosceles hirsuta
  WVI[211.923][323.19][193.88]G[184.835]DWG[288.885]AGVVGGIV[168.053] gi 224472141 gb ACN48952.1 sphingomyelinase D-like protein, partial Loxosceles rufescens
  IA[277.273]D[217.017]F[286.991]V[275.199] gi 224472143 gb ACN48953.1 sphingomyelinase D-like protein, partial Loxosceles arizonica
  [127.947]IAEWFDVDVC gi 224472147 gb ACN48955.1 sphingomyelinase D-like protein, partial Loxosceles laeta
  I[172.141]NFMN[128.005]R gi 224472157 gb ACN48960.1 sphingomyelinase D-like protein, partial Loxosceles laeta
  AGADGM[212.996]DFP[127.998] gi 224472195 gb ACN48979.1 sphingomyelinase D-like protein, partial Loxosceles aff. Spinulosa GJB-2008
  [128.05][346.235]FGWEIC[128.103] gi 224472201 gb ACN48982.1 sphingomyelinase D-like protein, partial Loxosceles aff. Spinulosa GJB-2008
  I[128.005]NYWNNGDNG[230.782] gi 225008387 gb ACN48920.2 sphingomyelinase D-like protein, partial Loxosceles sabina
  [234.124]IIISI[211.97]T[220.159]Y[128.131] gi 225008389 gb ACN48924.2 sphingomyelinase D-like protein, partial Loxosceles variegata
  [217.067]VDDGS[127.933][261.153]IGGDSCC[127.973] gi 225008391 gb ACN48959.2 sphingomyelinase D-like protein, partial Loxosceles laeta
  [202.038]TYEDN[283.238]VTF[128.109]A gi 49458048 gb AAT66074.1 sphingomyelinase D-like protein 3, partial Loxosceles boneti
  WENF[229.222]FI[128.141] gi 49458050 gb AAT66075.1 sphingomyelinase D protein 1, partial Loxosceles reclusa
  IATYDDN[283.149][128.13][128.098] gi 49458052 gb AAT66076.1 sphingomyelinase D protein 2, partial Loxosceles reclusa
  WSR[225.203]IWDIAHM gi 81343346 gb ABB71184.1 dermonecrotic toxin isoform 3 Loxosceles intermedia
  [128.059]SSI[185.218]DN[127.961]AY[128.189]AGVNMATDI[275.181] gi 90192366 gb ABD91846.1 dermonecrotic toxin isoform 4 Loxosceles intermedia
  [259.057]SFADYIDYMR gi 90192368 gb ABD91847.1 dermonecrotic toxin isoform 5 Loxosceles intermedia
  AIC[262.118]N[213.308][157.942]IF[269.463]M TSA 1, Contig: LIC336 putative phospholipase-D Loxosceles intermedia
  [128.065]PI[257.302][168.016][128.103] TSA 1, Contig: LIS80 putative phospholipase-D Loxosceles intermedia
  WVI[213.173]FE[230.5]VEDWG[289.028]AGN[198.021]IV[168.053] TSA 3, Contig: LIC182 putative phospholipase-D Loxosceles intermedia
  CENISTDD[214.057]R TSA 3, Contig: LIC203 putative phospholipase-D Loxosceles intermedia
  R[169.813][231.411]ANPIGR TSA 3, Contig: LIC334 putative phospholipase-D Loxosceles intermedia
  [127.988]AE[300.164]D[128.052]IF[128.126]I[255.151] TSA 3, Contig: LIC352 putative phospholipase-D Loxosceles intermedia
  WVIGG[184.011]A[216.684][200.309]T[200.21][184.253][200.309][216.684]GVVGGIV[168.237] TSA 3, Contig: LIC395 putative phospholipase-D Loxosceles intermedia
  [255.268]TCEYIEI[245.969]DSNYSEIG[224.144] TSA 3, Contig: LIC419 putative phospholipase-D Loxosceles intermedia
  GEEYVNVFPMGIR TSA 3, Contig: LIC423 putative phospholipase-D Loxosceles intermedia
  [256.067][269.811][259.263][227.115]GHEPHC[299.732] TSA 3, Contig: LIS9 putative phospholipase-D Loxosceles intermedia
  [128.078][128.124]AGV[128.053]D[128.036]EHIW sp A4USB4 A51 LOXIN phospholipase D LiSicTox-alphaV1 Dermonecrotic toxin 6 Loxosceles intermedia
Toxin class
        
 Phospholipase-D [278.133]A[127.914]ARDAG[128.044]V[127.924] sp B2KKW0 A22 LOXIN phospholipase D LiSicTox-alphaII2 Loxtox i5 Loxosceles intermedia
  TARDVA sp C0JAQ5 A1IA1 LOXHI phospholipase D LhSicTox-alphaIA2ai Dermonecrotic toxin Loxosceles hirsuta
  [144.086][196.168][128.016][299.803][217.693]DNGNN[142.216][128.015] sp C0JAR3 A1IA6 LOXHI phospholipase D LhSicTox-alphaIA2avi Dermonecrotic toxin Loxosceles hirsuta
  PI[128.15]TD[276.095][260.159][226.968] sp C0JAR7 A1IA7 LOXHI phospholipase D LhSicTox-alphaIA2avii Dermonecrotic toxin Loxosceles hirsuta
  [128.069][314.204][266.04]EIIE[128.136]VGY sp C0JAS6 A1I1 LOXSP phospholipase D LspaSicTox-alphaIA2i Dermonecrotic toxin Loxosceles spadicea
  [128.135]G[238.403]YEDNPW sp C0JAT4 A1H1 LOXHI phospholipase D LhSicTox-alphaIA1i Dermonecrotic toxin Loxosceles hirsuta
  [285.246]TIT[127.957][128.38][193.725]PE[244.902][128.249]F[300.312] sp C0JAU6 A1LC LOXAR phospholipase D LarSicTox-alphaIB2c Dermonecrotic toxin Loxosceles arizonica
  IISI[211.095]ID[300.088] sp C0JAV3 A1KA1 LOXAP phospholipase D LapSicTox-alphaIB1ai Dermonecrotic toxin Loxosceles apachea
  [128.046]T[261.162][127.9]SAG sp C0JAX3 A1MA1 LOXDE phospholipase D LdSicTox-alphaIB3ai Dermonecrotic toxin Loxosceles deserta
  YWTVD[128.227]Y sp C0JAY0 A1MA6 LOXDE phospholipase D LdSicTox-alphaIB3ai Dermonecrotic toxin Loxosceles deserta
  GIIISI[250.013]IAHY sp C0JAZ1 A1OA1 LOXVA phospholipase D LvSicTox-alphaIC1ai Dermonecrotic toxin Loxosceles variegata
  NAIETDVT sp C0JAZ4 A1OB1 LOXVA phospholipase D LvSicTox-alphaIC1bi Dermonecrotic toxin Loxosceles variegata
  [127.882]GI[185.036]EGC[200.002][372.207]ICA sp C0JAZ8 A1OB5 LOXVA phospholipase D LvSicTox-alphaIC1bv Dermonecrotic toxin Loxosceles variegata
  R[227.227]TT[154.041]V sp C0JB02 A1OD LOXRU phospholipase D LruSicTox-alphaIC1d Dermonecrotic toxin Loxosceles rufescens
  GEND[170.749]N[270.185]AY sp C0JB04 A1P LOXRU phospholipase D LruSicTox-alphaIC2 Dermonecrotic toxin Loxosceles rufescens
  [128.095]EVIGVTII[143.89]TCEAH[252.229][210.281]D[274.244] sp C0JB05 A21 LOXSP phospholipase D LspaSicTox-alphaII1 Dermonecrotic toxin Loxosceles spadicea
  FCGC[128.12]AWNPGHC[261.261][127.94] sp C0JB06 A21 LOXVA phospholipase D LvSicTox-alphaII1 Dermonecrotic toxin Loxosceles variegata
  Y[293.065]PCDCF sp C0JB07 A21 LOXAP phospholipase D LapSicTox-alphaII1 Dermonecrotic toxin Loxosceles apachea
  R[312.174][247.035][256.156]AVN sp C0JB09 A31 LOXAR phospholipase D LarSicTox-alphaIII1 Dermonecrotic toxin Loxosceles arizonica
  [349.464]NDGCP[314.14]CNDW sp C0JB12 A332 LOXLA phospholipase D LlSicTox-alphaIII3ii Dermonecrotic toxin Loxosceles laeta
  N[128.122]AGV[128.154]DREHVW sp C0JB14 A411 LOXHI phospholipase D LhSicTox-alphaIV1i Dermonecrotic toxin Loxosceles hirsuta
  VGGSCNDD[127.823]VCC[128.169]GG[128.002] sp C0JB18 A411 LOXAM phospholipase D LamSicTox-alphaIV1i Dermonecrotic toxin Loxosceles amazonica
  RIANYDD[345.981][289.109]F sp C0JB22 A41 LOXAR phospholipase D LarSicTox-alphaIV1 Dermonecrotic toxin Loxosceles arizonica
  WFDVDVC[128.184]GG sp C0JB23 A411 LOXLA phospholipase D LlSicTox-alphaIV1i Dermonecrotic toxin Loxosceles laeta
  [256.225]F[315.247]N[251.495]W[128.071][171.981]GI[214.973] sp C0JB25 A421 LOXLA phospholipase D LlSicTox-alphaIV2i Dermonecrotic toxin Loxosceles laeta
  [201.075]DDD[183.982]D[342.088][248.103][282.879]W[260.214] sp C0JB29 A43 LOXLA phospholipase D LlSicTox-alphaIV3 Dermonecrotic toxin Loxosceles laeta
  FI[128.05]GDYINV sp C0JB30 A71 LOXAR phospholipase D LarSicTox-alphaVII1 Dermonecrotic toxin Loxosceles arizonica
  [127.979]SY[168.058]VIVG[326.398]E[257.168][128.109]D[291.254] sp C0JB31 A611 LOXHI phospholipase D LhSicTox-alphaVI1i Dermonecrotic toxin Loxosceles hirsuta
  [243.149]R[214.248]D[228.235]I[128.113][128.353][226.9]TIY sp C0JB40 B1O LOXCS phospholipase D LcsSicTox-betaIC1 Dermonecrotic toxin Loxosceles cf. spinulosa GJB-2008
  [249.996]S[320.07]C[292.123]PMI[303.312]V sp C0JB44 B1T1 LOXSN phospholipase D LspiSicTox-betaIE3i Dermonecrotic toxin Loxosceles spinulosa
  [260.294]C[274.817]N[250.295]N[229.03][250.931]INNR[248.338] sp C0JB48 B1S LOXAS phospholipase D LafSicTox-betaIE2 Dermonecrotic toxin Loxosceles cf. spinulosa GJB-2008
  [128.075]GP[260.085][128.08]FNPGNYDEEE[269.243] sp C0JB92 B31 LOXSN phospholipase D LspiSicTox-betaIII1 Dermonecrotic toxin Loxosceles spinulosa
  HGIPCDCGRSCI sp P0CE78 A1H1 LOXRE phospholipase D LrSicTox-alphaIA1i Dermonecrotic toxin Loxosceles reclusa
  [185.577]ERR[210.449]WIMG[220.232] sp P0CE79 A1H2 LOXRE phospholipase D LrSicTox-alphaIA1ii Dermonecrotic toxin Loxosceles reclusa
  ADNRG[196.202]IW sp P0CE80 A1HA LOXIN phospholipase D LiSicTox-alphaIA1a Dermonecrotic toxin 1 Loxosceles intermedia
  E[310.079]DRV[128.206][127.978] sp P0CE83 A1IA1 LOXIN phospholipase D LiSicTox-alphaIA2ai Dermonecrotic toxin LiP2 Loxosceles intermedia
  RN[172.129]A[185.125]IIMAVI sp Q1KY79 A32 LOXLA phospholipase D LlSicTox-alphaIII2 Dermonecrotic toxin Ll2 Loxosceles laeta
  [128.089]SRD[226.149]DHIW sp Q1W694 B1Q LOXIN phospholipase D LiSicTox-betaID1 Dermonecrotic toxin 5 Loxosceles intermedia
  [241.168]D[128.065][246.052]D[306.802][314.355]EF sp Q1W695 A21 LOXIN phospholipase D LiSicTox-alphaII1 Dermonecrotic toxin 4 Loxosceles intermedia
  [144.192][184.287][171.423][244.328][212.328]AFTDD[157.983][314.318] sp Q27Q54 B1H2 LOXIN phospholipase D LiSicTox-betaIA1ii Dermonecrotic toxin-like II Loxosceles intermedia
  [271.172]AI[141.933][167.905][170.252]EEI[276.104][127.878] sp Q2XQ09 B1H1 LOXIN phospholipase D LiSicTox-betaIA1i Dermonecrotic toxin-like I Loxosceles intermedia
  Y[128.246]E[128.214]IIIF sp Q5YD77 A1KA LOXBO phospholipase D LbSicTox-alphaIB1a Dermonecrotic toxin Lb1 Loxosceles boneti
  M[169.966]D[227.167]DIA[204.21][238.193]N sp Q8I912 B1H LOXLA phospholipase D LlSicTox-betaIA1 Dermonecrotic toxin LlH10 Loxosceles laeta
  [252.158][172.24]AG[128.07]IIS[346.153] sp Q8I913 A331 LOXLA phospholipase D LlSicTox-alphaIII3i Dermonecrotic toxin LlH13 Loxosceles laeta
  I[248.195][172.042]RTNCC[317.124][316.196] tr G8GZ61 G8GZ61 9ARAC Sphingomyelinase D A1 Loxosceles adelaida
  GMDIPNIRI[198.131][274.115] TSA 2, Contig: LIS181 putative phospholipase-D Loxosceles intermedia
  IADYE[245.445]RGF TSA 3, Contig: LIC316 putative phospholipase-D Loxosceles intermedia
  [229.079]VNDYDCA[278.03][197.89][257.788]N[341.945]F[188.387]GGI[173.846]E[203.936][227.312][212.04] TSA 1, Contig: LIS124 putative phospholipase-D Loxosceles intermedia
 ICK-peptide [288.103][276.948]A[228.135]PETA[217.192]E[258.063]GH[270.214] gi 118574181 sp Q27Q53.1 TX4 LOXIN U1-sicaritoxin-Li1c LiTx4 Loxosceles intermedia
  [141.867]GCTMGVC[262.034]G[127.99] gi 74786589 sp Q6B4T3.1 TX3 LOXIN U2-sicaritoxin-Li1a LiTx3 Loxosceles intermedia
  [170.014]T[257.226][128.082]CPAWSHER gi 74786590 sp Q6B4T4.1 TX2 LOXIN U1-sicaritoxin-Li1b LiTx2 Loxosceles intermedia
  [128.076][198.175]C[128.129][257.053]AWSH[285.151]ECR gi 74786591 sp Q6B4T5.1 TX1 LOXIN U1-sicaritoxin-Li1a LiTx1 Loxosceles intermedia
  [242.133]VECICSPSYYP[154.122][128.073] TSA 1, Contig: LIC275 putative LiTx3 Loxosceles intermedia
  [128.047]GI[220.146][299.366]THH[128.119]Y[196.17]E[316.185] TSA 1, Contig: LIC298 putative LiTx3 Loxosceles intermedia
  WVI[213.183]FE[230.39][229.002]DWG[288.987][127.905]VV[128.365]V[285.053] TSA 1, Contig: LIS111 putative LiTx3 Loxosceles intermedia
  [174.128]TVPVYAECGR TSA 1, Contig: LIS14 putative LiTx3 Loxosceles intermedia
  [128.108][128.122]NVMRIYVG TSA 1, Contig: LIS64 putative LiTx3 Loxosceles intermedia
  AGGASE[288.32]V[288.068]E TSA 1, Contig: LIC255 putative LiTx4 Loxosceles intermedia
         
Toxin class
        
 ICK-peptide [174.137]GIPNASGSIGR TSA 2, Contig: LIC315 putative LiTx3 Loxosceles intermedia
  R[217.187][200.251]FVPVG[174.181]G TSA 2, Contig: LIS1 putative LiTx3 Loxosceles intermedia
  GAD[225.773][283.154][216.215] TSA 2, Contig: LIS161 putative LiTx3 Loxosceles intermedia
  [342.104][142.018][174.19][275.359]TCHGPNWAA[342.104] TSA 2, Contig: LIS33 putative LiTx3 Loxosceles intermedia
  [371.939]WNYA[199.308][128.365]STII TSA 3, Contig: LIC212 putative LiTx3 Loxosceles intermedia
  [128.037]SSFEDF[227.173]VDCNS[261.16] TSA 3, Contig: LIS21 putative LiTx3 Loxosceles intermedia
  G[216.293]CSDG[342.128]DIPC[128.288] TSA 3, Contig: LIS35 putative LiTx3 Loxosceles intermedia
  IGISSD[320.03]PDW[128.125] TSA 3, Contig: LIS36 putative LiTx3 Loxosceles intermedia
  ECI[241.167][241.217][345.95] TSA 3, Contig: LIS43 putative LiTx3 Loxosceles intermedia
  AAGDTN[259.089][229.905][237.813][320.42]W[128.09] TSA 3, Contig: LIS44 putative LiTx3 Loxosceles intermedia
  RCE[224.918]GI[128.1]DISE TSA 3, Contig: LIS57 putative LiTx3 Loxosceles intermedia
  [226.296]PNI[128.014][185.165]T[247.019]CNN[226.997] TSA 3, Contig: LIC381 putative neurotoxin like-magi-3 Loxosceles intermedia
  [258.986]VCYC[208.258]FGV[128.123]NC[128.024] TSA 3, Contig: LIS20 putative neurotoxin like-magi-3 Loxosceles intermedia
  GIRSATTPGNA[128.185]Y sp P0CE83 A1IA1 LOXIN phospholipase D LiSicTox-alphaIA2ai Dermonecrotic toxin LiP2 Loxosceles intermedia
  [199.081]YD[225.268]DDWCCG[199.016] sp Q27Q53 TX4 LOXIN U1-sicaritoxin-Li1c LiTx4 Loxosceles intermedia
  [213.19][128.003]Y[127.926][299.318][275.069]R[244.958][183.934][277.077][128.226]G[141.885] sp Q6B4T3 TX3 LOXIN U2-sicaritoxin-Li1a LiTx3 Loxosceles intermedia
  CT[224.823]CGPYY sp Q6B4T4 TX2 LOXIN U1-sicaritoxin-Li1b LiTx2 Loxosceles intermedia
  [320.096][128.127][128.065]GTPC[128.107]CPAWSHER[289.113][174.07] sp Q6B4T5 TX1 LOXIN U1-sicaritoxin-Li1a LiTx1 Loxosceles intermedia
  TG[128.074][128.206]FI TSA 1, Contig: LIS15 putative LiTx1 Loxosceles intermedia
  [260.124][257.99][260.764]DAIESEDPV[208.163] TSA 1, Contig: LIS159 putative LiTx1 Loxosceles intermedia
  SV[127.961][200.118]GI[315.972][128.206]E[142.111] TSA 1, Contig: LIS163 putative LiTx3 Loxosceles intermedia
  M[247.954][271.519]E[198.253][128.081]D[200.22] TSA 2, Contig: LIS31 putative LiTx3 Loxosceles intermedia
  GAG[143.963]NIFA[127.947] TSA 2, Contig: LIS76 putative LiTx3 Loxosceles intermedia
  [127.704]T[128.06]FCV[128.02]NG[128.165]PICP[128.162][167.895]G TSA 3, Contig: LIC337 putative LiTx1 Loxosceles intermedia
  G[266.174]CHAFGSNCR TSA 2, Contig: LIS229 putative neurotoxin like-magi-3 Loxosceles intermedia
 Serine protease inhibitor [128.02]CP[235.787][198.175][143.863]G[241.815]DV[127.896][218.419]G TSA 1, Contig: LIS209 putative serine protease inhibitor Loxosceles intermedia
 Serine protease [269.335][262.236]S[244.714]WASFP[211.817]SA[128.235]I[269.335] TSA 1, Contig: LIC305 putative serine protease Loxosceles intermedia
 Venom allergen [346.178][257.117]N[215.219][298.78][217.218]ND[260.144]IG[256.421][372.04] TSA 2, Contig: LIC179 putative venom allergen Loxosceles intermedia
 Hyaluronidase V[207.937]SSEY[211.831]I[326.363][319.484] TSA 1, Contig: LIS222 putative hyaluronidase Loxosceles intermedia
 TCTP [256.849][275.401][210.175]I[252.242]TA[244.108][200.309][241.062]N[170.078]IIED[372.344] tr G3LU44 G3LU44 LOXIN translationally-controlled tumor protein homolog LiTCTP Loxosceles intermedia
  [269.086]FGIMAP[225.403]GEIR gi 344995179 gb AEN55462.1 translationally controlled tumor protein LiTCTP Loxosceles intermedia
Cellular processes proteins [200.059]VHEDNI[128.065]E[128.062]H[128.193][262.127] TSA 1, Contig: LIC216 putative 60S ribosomal protein L10 Loxosceles intermedia
  [199.078]CT[297.052]F[264.084][233.709]GCPSR TSA 1, Contig: LIS275 putative cysteine-rich PDZ-binding protein Loxosceles intermedia
  [269.045]D[173.858]DPDCDC[211.944]D[173.937]D[269.044] TSA 1 Contig: LIS239 putative cytochrome P450 mRNA Loxosceles intermedia
  I[204.06]CV[128.097][297.04] TSA 1, Contig: LIS199 putative histone H2B Loxosceles intermedia
  [241.241][210.11]DFYEI[170.035]SA[262.036] TSA 1, Contig: LIS189 putative myosin heavy chain Loxosceles intermedia
  [128.159]GF[218.024]D[127.978]VVIA TSA 1, Contig: LIC349 putative secreted salivary gland peptide Loxosceles intermedia
  [262.073]I[243.142]PSNPSCR TSA 2, Contig: LIC232 putative 60S ribosomal protein L27 Loxosceles intermedia
  [216.109]IVISNPDINH[260.307][226.167][128.074] TSA 2, Contig: LIC283 putative actin related protein 3 Loxosceles intermedia
  TED[128.098]V[229.139] TSA 2, Contig: LIS258 putative glycoprotein hormone alpha-2 precurso Loxosceles intermedia
  [128.037]SSFEDF[227.173]VDCNS[261.16] TSA 2, Contig: LIS217 putative myosin light chain Loxosceles intermedia
  [199.18]SIDIIAS[128.129]DVMDR TSA 2, Contig: LIS39 putative Na/Pi co-transporter Loxosceles intermedia
  [269.086]FGIMAP[225.403]GEIR TSA 2, Contig: LIS280 putative ribosomal protein L18a Loxosceles intermedia
  [247.11]G[128.088]I[183.991]N[210.738][262.174][285.2] TSA 2, Contig: LIS213 putative ribosomal protein S3 Loxosceles intermedia
  Y[127.918]GV[143.679]I[196.241]AGR TSA 3, Contig: LIC307 putative angiotensin-converting enzyme Loxosceles intermedia
  VGSIPIDI[128.058] TSA 3, Contig: LIC368 putative myosin light chain Loxosceles intermedia
  [128.071]SAI[250.284]IS[127.926]N TSA 3, Contig: LIC207 putative troponin C Loxosceles intermedia
  GTIPVT[323.12]P[128.197] TSA 3, Contig: LIS253 putative troponin T Loxosceles intermedia
  [128.057]CW[283.922]E[127.782][128.342]GA TSA 3, Contig: LIS216 putative zinc finger protein Loxosceles intermedia
  [128.066]CG[169.924]S[293.695]D[211.062]C[143.962] TSA 1, Contig: LIS257 putative cAMP-responsive element-binding protein-like 2-like Loxosceles intermedia
  HVSV[128.009] TSA 1, Contig: LIC219 putative glutamine synthetase Loxosceles intermedia
  [262.165]P[230.085]D[128.062] TSA 2, Contig: LIS220 putative 60S ribosomal protein L6 Loxosceles intermedia
  [268.215]HCTCDDVC[128.023]R[275.259]Y TSA 2, Contig: LIS231 putative calmodulin Loxosceles intermedia
  [274.04]TPIRIN[210.228]II[228.096] TSA 2, Contig: LIS27 putative Na/Pi co-transporter Loxosceles intermedia
  [372.255]DEIIN[171.15]IE[200.054]E[201.127]S[226.48]HG[293.174][372.288] TSA 2, Contig: LIS265 putative selenoprotein M Loxosceles intermedia
  NW[211.918]RIR[298.994][248.176] TSA 2, Contig: LIS205 putative ubiquitin Loxosceles intermedia
  [253.085]IG[200.301]T[251.496][171.357]ED tr Q6W974 Q6W974 LOXRE codium/potassium ATPase alpha subunit Loxosceles reclusa
  YHNN[232.454]ISII tr B8R316 B8R316 9ARAC NADH-ubiquinone oxidoreductase chain 1 Loxosceles baja
  [276.09]MISMEVG[307.07][259.494]Y tr B8R320 B8R320 LOXDE NADH-ubiquinone oxidoreductase chain 2 Loxosceles deserta
Cellular processes proteins
        
  [128.205]MFTIIM[235.906]GV[215.128] tr B8R326 B8R326 9ARAC NADH-ubiquinone oxidoreductase chain 1 Loxosceles chinateca
  [245.288]RA[260.078]NTSTIGTA[212.046]R tr B8R332 B8R332 9ARAC NADH-ubiquinone oxidoreductase chain 1 Loxosceles kaiba
  S[246.297][301.019]IVS[170.255]H tr B8R341 B8R341 9ARAC NADH-ubiquinone oxidoreductase chain 1 Sicarius aff. patagonicus
  RT[255.953][231.083][299.111]CVG tr C1ITN3 C1ITN3 LOXAS cytochrome c oxidase subunit 1 Loxosceles aff. spinulosa
  I[211.14][268.32]I[187.685]IV[246.077] tr C1ITP7 C1ITP7 9ARAC cytochrome c oxidase subunit 1 Sicarius dolichocephalus
  FF[238.18]IITAG[273.235] tr C1ITR2 C1ITR2 9ARAC cytochrome c oxidase subunit 1 Drymusa serrana
  YEDRIVVR[230.004] tr C1ITR3 C1ITR3 9ARAC cytochrome c oxidase subunit 1 Drymusa dinora
  [316.142]TVYCMSIEITA[226.21]IEDI[241.221] tr C1IZB2 C1IZB2 LOXLA cytochrome c oxidase subunit 1 Loxosceles laeta
  GVI[248.157]NITGYR tr C5J3X9 C5J3X9 LOXRU cytochrome c oxidase subunit 1 Loxosceles rufescens
  IA[128.08]MFTIIE[216.039]GV[215.166] tr B8R317 B8R317 LOXAP NADH-ubiquinone oxidoreductase chain 1 Loxosceles apachea
  [278.338]A[234.386]WHVEN[210.973]VNI[210.062] tr B8R335 B8R335 LOXS4 NADH-ubiquinone oxidoreductase chain 1 Loxosceles sp.
  [265.105]V[213.051][172.098]A[283.126]Y[201.071][210.087][278.104][269.011]RGIVMV[283.117] tr C1ITR3 C1ITR3 9ARAC cytochrome c oxidase subunit 1 Drymusa dinora
  [127.983]AG[153.948]ACTGEMGSCG tr C5J3X7 C5J3X7 LOXGA cytochrome c oxidase subunit 1 Loxosceles gaucho
  YSVFCMM[128.12]V tr C5J3X9 C5J3X9 LOXRU cytochrome c oxidase subunit 1 Loxosceles rufescens
  [225.309]SWD[217.039][271.219]C[258.226][218.143][302.82]GMIGSEN tr Q6W974 Q6W974 LOXRE sodium/potassium ATPase alpha subunit Loxosceles reclusa
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Taken together, the identified toxins in the L. intermedia venom include representatives from all toxin groups, even if in low abundances (as in the case of, e.g., hyaluronidases and serine proteases). We also find it noteworthy that we obtained significant coverage of the three major families present in the venom, viz., PLDs, astacin-like metalloproteases, and ICK peptides. These families are of great importance for studies of the brown-spider envenomation features and of biotechnological and medical applications.

Many of the aligned contigs mapped to distinct PLD isoforms from a variety of Loxosceles species. In fact, these toxins are the most studied and well-characterized components of the Loxosceles venom5,20,26,31,46–48. PLDs are able to reproduce the deleterious effects observed in loxoscelism and represent a great target for drug discovery against brown-spider envenomation2,5.

As for the astacin-like metalloproteases identified, we note that astacins were first described as an animal-venom component in 2007 (ref. 28) and only later recognized as a family of toxins present in the Loxosceles venom33. These toxins present proteolytic activity on distinct extracellular matrix proteins and are related to the hemostatic effects in loxoscelism43,49.

ICK peptides, the major components of the L. intermedia venom-gland transcriptome (54,9% of the expressed sequence tags), were identified with correspondence to all four different ICK peptides described for L. intermedia (LiTx1, LiTx2, LiTx3, and LiTx4)50,51. These ICK peptides, also called knottins, are characterized by the neurotoxic properties they exhibit on ion channels and receptors expressed in the nervous systems of insects and mammals52. The high expression of LiTx transcripts, which correlates with the proteomic results found herein, are consistent with the venom’s effects of paralyzing and killing both preys and predators1,20,51.

Additional Information

How to cite this article: Trevisan-Silva, D. et al. A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom. Sci. Data 4:170090 doi: 10.1038/sdata.2017.90 (2017).

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Material

sdata201790-isa1.zip (4.4KB, zip)

Acknowledgments

The authors thank CNPq and CAPES for financial support. They also thank Fiocruz for use of Mass Spectrometry Facility RPT02H at the Carlos Chagas Institute, as well as Dr Michel Batista for aiding in the mass spectrometry procedures. V.C.B. acknowledges support from a FAPERJ BBP grant. N.B. is an Alfred P. Sloan Research Fellow and was partially supported by the US National Institutes of Health Grant 2 P41 GM103484-06A1 from the National Institute of General Medical Sciences. The authors thank Wagner Nagib from Carlos Chagas Instituto, Fiocruz—Paraná for generating the final cover art.

Footnotes

N.B. has an equity interest in Digital Proteomics, LLC, a company that may potentially benefit from the research results; Digital Proteomics, LLC was not involved in any aspects of this research. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. The remaining authors declares no competing financial interests.

Data Citations

  1. Trevisan-Silva D. 2016. Dummy. PXD005523
  2. Trevisan-Silva D. 2017. Figshare. https://doi.org/10.6084/m9.figshare.c.3709168

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Trevisan-Silva D. 2016. Dummy. PXD005523
  2. Trevisan-Silva D. 2017. Figshare. https://doi.org/10.6084/m9.figshare.c.3709168

Supplementary Materials

sdata201790-isa1.zip (4.4KB, zip)

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