Mass spectrometry-based top-down and bottom-up
approaches for proteomic analysis of the Moroccan
Buthus occitanus scorpion venom

Khadija Daoudi, Christian Malosse, Ayoub Lafnoune, Bouchra Darkaoui, Salma Chakir, Jean-Marc Sabatier, Julia Chamot-Rooke, Rachida Cadi, Naoual Oukkache

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Khadija Daoudi, Christian Malosse, Ayoub Lafnoune, Bouchra Darkaoui, Salma Chakir, et al. Mass spectrometry-based top-down and bottom-up approaches for proteomic analysis of the Moroccan Buthus occitanus scorpion venom. FEBS Open Bio, 2021, 11 (7), pp.1867-1892. 10.1002/2211-5463.13143 . hal-03358052

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# Mass spectrometry-based top-down and bottom-up approaches for proteomic analysis of the Moroccan *Buthus occitanus* scorpion venom

Khadija Daoudi¹,², Christian Malosse³, Ayoub Lafnoune¹,², Bouchra Darkaoui¹,², Salma Chakir¹, Jean-Marc Sabatier⁴, Julia Chamot-Rooke³, Rachida Cadi² and Naoual Oukkache¹

1. Laboratory of Venoms and Toxins, Pasteur Institute of Morocco, Casablanca, Morocco

2. Laboratory of Molecular Genetics, Physiopathology and Biotechnology, Faculty of Sciences Ain Chock, Hassan II University of Casablanca, Morocco

3. Mass spectrometry for Biology Unit, Institut Pasteur, CNRS USR 2000, Paris, France

4. Laboratory INSERM UMR 1097, University of Aix Marseille, France

**Keywords**

bottom-up; *Buthus occitanus* scorpion; top-down; toxins; venom; venomic

**Correspondence**

K. Daoudi, Laboratory of Venoms and Toxins, Pasteur Institute of Morocco, 1 Place Louis Pasteur, Casablanca 20250, Morocco.
Laboratory of Molecular Genetics, Physiopathology and Biotechnology, Faculty of Sciences Ain Chock, Hassan II University of Casablanca, B.P 5366 Maarif, Casablanca, Morocco.

E-mail: khadija.daoudi1-etu@etu.univh2c.ma

(Received 29 December 2020, revised 18 February 2021, accepted 11 March 2021)

doi:10.1002/2211-5463.13143

Edited by Alberto Alape-Girón

*Buthus occitanus* (*B. occitanus*) is one of the most dangerous scorpions in the world. Despite the involvement of *B. occitanus* scorpion in severe cases of envenomation in Morocco, no study has focused yet on the proteomic composition of the Moroccan *B. occitanus* scorpion venom. Mass spectrometry-based proteomic techniques are commonly used in the study of scorpion venoms. The implementation of top-down and bottom-up approaches for proteomic analyses facilitates screening by allowing a global view of the structural aspects of such complex matrices. Here, we provide a partial overview of the venom of *B. occitanus* scorpion, in order to explore the diversity of its toxins and hereafter understand their effects. To this end, a combination of top-down and bottom-up approaches was applied using nano-high liquid chromatography coupled to nano-electrospray tandem mass spectrometry (nano-LC-ESI MS/MS). The LC-MS results showed that *B. occitanus* venom contains around 200 molecular masses ranging from 1868 to 16 720 Da, the most representative of which are those between 5000 and 8000 Da. Interestingly, combined top-down and bottom-up LC-MS/MS results allowed the identification of several toxins, which were mainly those acting on ion channels, including those targeting sodium (NaScTxs), potassium (KScTxs), chloride (ClScTxs), and calcium channels (CaScTx), as well as antimicrobial peptides (AMPs), amphipathic peptides, myotropic neuropeptides, and hypothetical secreted proteins. This study reveals the molecular diversity of *B. occitanus* scorpion venom and identifies components that may have useful pharmacological activities.

**Abbreviations**

ACN, acetonitrile; AMP, antimicrobial peptides; *B. occitanus*, *Buthus occitanus*; CaScTxs, neurotoxins affecting calcium channels; ClScTxs, neurotoxins affecting chloride channels; Da, Dalton; EThcD, Electron-Transfer/Higher-Energy Collision Dissociation; FA, formic acid; HCD, higher-energy C-trap dissociation; IAA, iodoacetamide; kDa, kilodalton; KScTxs, neurotoxins affecting potassium channels; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LC-MS, liquid chromatography coupled to mass spectrometry; MS, mass spectrometry; MW, molecular weight; nano-LC-ESI MS/MS, nano-liquid chromatography coupled to electrospray tandem mass spectrometry; NaScTxs, neurotoxins affecting sodium channels; Q, quadrupole; TIC, total ion chromatogram.
Each year, scorpion stings record new cases of envenomation over the world with an incidence of more than 1.5 million and over 2600 deaths, mainly in tropical and subtropical countries of South America, Asia, and North Africa [1]. Most of these envenomation cases were caused by scorpions belonging to the Buthidae family, which contains dangerous species known by their lethal venoms [2]. The venom of these family members contains a heterogeneous cocktail of compounds, including inorganic substances, enzymes, mucopolysaccharides, allergenic compounds, and peptides with high toxicity toward ionic channels of excitable cells [3-6]. In Morocco, 26 819 cases of scorpion stings were reported in 2019 by the Poison Control and Pharmacovigilance Center of Morocco, with an incidence of 75.3 cases per 100 000 inhabitants [7]. These statistics are due to the diversified scorpion fauna represented by over 50 species, mainly widespread in the middle and southwestern provinces of the kingdom [8]. Among these species, the yellow scorpion *Buthus occitanus* (*B.occitanus*) seems to be one of the most dangerous scorpions, on account of its toxic venom causing the majority of envenomation cases [9]. Although several studies had been carried out on this venom [10-13], no study has yet focused on the proteomic composition of the Moroccan *B. occitanus* scorpion venom despite its medical importance. Moreover, there are various strategies to screen scorpion venoms, from using conventional strategies for targeting one single toxin, to applying the most throughput equipment of screening for a detailed view of all toxic components. Nowadays, mass spectrometry-based proteomic approaches are still one of the most fundamental tools to decrypt the complexity of such matrices, owing to the revolutionary advances in instrumentation and software, in addition to improvement in omics strategies (peptidomic, proteomic, transcriptionic, and genomic) [14-19]. Among the approaches that have improved significantly the proteomics workflow, there are the top-down process, which designates a rapid analytical workflow of intact proteins, and the bottom-up approach, which requires prior proteolytic digestion of proteins before mass spectrometry analysis. These approaches lead to acquiring mass fingerprints, primary structural information, and post-translational modifications [20-23]. The application of these approaches, singly or complementary, in several proteomic studies has increased the number of characterized venoms and identified toxins [24-29]. In this context, this work aimed to ensure an overview of the peptidome of *B. occitanus* scorpion (< 30 kDa), so exploring its toxins arsenal, using a combination of the top-down and bottom-up approaches applied on nano-

high liquid chromatography coupled to a nano-electro-spray tandem mass spectrometry (nano-LC-ESI MS/MS).

# Materials and methods

## Venom preparation

### Venom milking

Specimens of *B. occitanus* were collected from the region of Oualidia (32°44′N 9°01′W), in eastern Morocco. The crude venom was milked by electrical stimulation, pooled, centrifuged at 10 000 g for 20 min, freeze-dried, and stored at -20 °C until use [30].

### Venom Reduction/Alkylation

At first, 2 mg of *B. occitanus* crude venom was subjected to a 30 kDa 'cutoff' filter (Amicon® Ultra Centrifugal Filters, Merck Millipore, Tulagreen, Ireland), then centrifuged at 16 900 g for 15 min.

Disulfide-bridged half-cysteine residues of this venom filtrate were reduced by 10 mM of DTT in ammonium bicarbonate buffer (50 mM, pH 8.3), for 45 min at a temperature of 56 °C. Cysteine residues were carboxymethylated by incubation with 50 mM iodoacetamide [IAA in ammonium bicarbonate (50 mM, pH 8.3)] for 1 h in the dark. Then, these proteins/peptides were desalted by ZipTip C4 (Millipore Corporation - Billerica, USA) and concentrated on a Savant SpeedVac (Thermo Scientific, San Jose, CA, USA).

## Mass spectrometry-based proteomic approaches

### Top-down proteomics

Intact and reduced/alkylated *B. occitanus* venom filtrates were carried out on an Orbitrap Fusion™ Lumos™ mass spectrometer (Thermo Scientific™ Waltham, MA, USA), equipped with a Dionex HPLC (Fig. 1).

For the online peptide fractionation, 2 µg of samples was loaded to a C4 µ-precolumn cartridge (300 µm i.d. × 5 mm, C4 PepMap 300 particles with 5 µm size and 300 Å pores); the column was equilibrated with solution A [0.1% (v/v) formic acid (FA)]. The separation was maintained over 120 min at 250 nL·min⁻¹, using a linear gradient from 5% to 60% of solution B [acetonitrile (ACN) and 0.1% (v/v) FA].

Proteins/peptides were eluted directly from the column into the mass spectrometer and operated in positive mode with a spray voltage of 1.6 kV. MS spectra were acquired at a resolution setting of 120 000.

MS/MS analysis was performed on data-dependent acquisition, the top 10 abundant precursor ions were
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Experimental workflow performed in this study. At first, *B. occitanus* venom was milked by electrical stimulation and applied to a 30 kDa filter. For the top-down venomic, the flow-through containing toxins < 30 kDa was analyzed by the Thermo Scientific™ Orbitrap Fusion Lumos Tribrid Mass Spectrometer. For the bottom-up approach, two digest methods were achieved: 1) in-solution digestion, the flow-through containing toxin < 30 kDa was directly reduced with DTT, alkylated with IAA, and digested with trypsin; and 2) in-gel digestion, the unstained gel was excised to small cubes, reduced, alkylated, and digested. The digest peptides were then desalted with ZipTip and applied to the Orbitrap Q-Exactive mass spectrometer.</figcaption></figure>

selected for an EThcD fragmentations (Electron-Transfer/
Higher-Energy Collision Dissociation) with a dynamic
exclusion time of 90 s. MS/MS spectra were acquired at a
resolution setting of 120 000, and the mass range was set
from 150 to 2000 m/z.

Bottom-up proteomics

In-solution digestion

Reduced/alkylated venom filtrate was digested overnight at
a temperature of 37 °C with 0.1 µg of trypsin (Promega,
Madison, WI, USA). Tryptic digests were analyzed on a Q-
Exactive Plus instrument (Thermo Fisher Scientific, Bre-
men, Germany) coupled to an EASY-nLC 1200 chro-
matography system (Thermo Fisher Scientific). Two
micrograms was loaded on an in-house packed 50-cm
nano-HPLC column (75 µm inner diameter) filled with C18
resin (1.9 µm particles, 100 Å pore size, Reprosil-Pur Basic
C18-HD resin; Maisch GmbH, Ammerbuch-Entringen,
Germany) and equilibrated in 97% solvent A and 3% sol-
vent B (ACN, 0.1% (v/v) FA).

Peptides were eluted at 250 nL·min⁻¹, using 3–22% gradient of

solvent B for 35 min, and finally 38–60% gradient of sol-
vent B for 15 min. The instrument method for the Q-Exac-
tive Plus was set up in the data-dependent acquisition
mode. MS and MS-MS spectra were acquired at a resolu-
tion of 60 000, 10 of the most abundant precursor ions
were selected for HCD fragmentation with collision energy
adjusted to 27. Mono-charged precursors and those with a
charge state of > 7 were excluded.

In-gel digestion

At first, 2 mg of venom filtrate was unfolded for 5 min at
95 °C in sample buffer (LDS sample buffer) and then sub-
jected to a SDS/PAGE using a 4–20% of polyacrylamide
gel (SDS Precast Gel RunBlue, 4–20%, 12 well; Expedeon,
CA, USA). The electrophoresis was performed, on a Bio-
Rad system, at a constant voltage of 140 V, and the sepa-
rated proteins were stained with Coomassie Brilliant Blue
R (InstantBlue; Expedeon, CA, USA).

Stained bands corresponding to proteins/peptides with
masses < 30 kDa (Fig. S1) were manually excised into
equal small cubes of 1 mm³, then washed with Milli-Q
water, ammonium bicarbonate 50 mM, and ACN 50%.
Subsequently, the slices were submitted to an in-gel reduction with DTT (10 mM) in ammonium bicarbonate buffer (50 mM, pH 8.3) for 45 min at a temperature of 56 °C. Reduced slices were alkylated with IAA (50 mM) in ammonium bicarbonate (50 mM, pH 8.3) buffer for 20 min in the dark, followed by an overnight digestion with 0.1 µg of trypsin (Promega) at a temperature of 37 °C [31]. The enzymatic reaction was stopped by adding 5 µL of FA 5%, and desalted by loading the peptides onto ZipTip C18. After drying, digested peptides were dissolved in 100 µL of 0.1% (v/v) FA and applied on a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) system, composed of a nano-flow HPLC pump and an Orbitrap Q-Exactive mass spectrometer (Thermo Scientific) with a nano-electrospray ion source, as described in the section above.

## Data analysis

The top-down liquid chromatography coupled to mass spectrometry (LC-MS) data analysis of native *B. occitanus* venom filtrate was deconvoluted using the Xtract algorithm within Thermo Scientific XCALIBUR 2.2 software (Thermo Fisher Scientific).

For protein identification, data from both of the venomic nano-LC-MS/MS approaches were processed using the PROTEOME DISCOVER 2.2 software (Thermo Fisher Scientific), against the UniProtKB database, downloaded in 2016 10 11, taxon identifier: 6855 and 4309 entries.

Parameters of processing were as follows: a mass tolerance of MS set at 50 p.p.m. and 0.3 Da for MS/MS. One unique peptide was required for protein identification, minimum peptide length was required at five amino acids, and the false discovery rate cutoff was 1%. Trypsin was chosen

as the specific enzyme, with a maximum number of two missed cleavages for the bottom-up analysis. Variable modifications included oxidation of methionine and carbamidomethylation, while no fixed modification was set.

## Results

### Mass spectrometry-based proteomic approaches

The whole proteomic approaches are based only on the UniProtKB database-dependent analysis without any manually *de novo* sequence annotation; therefore, the majority of reported peptide annotations are still an approximation. Also, it is important to stress that the relative abundances and the percentages of the described peptides are purely based on total number counts and not concentrations as long as no quantitative analysis was performed.

### Top-down proteomics

The total ion chromatogram (TIC) generated from the top-down LC-MS analysis of native *B. occitanus* venom filtrate (Fig. 2) gave a partial picture of the venom complexity, with around 60 peaks, most of them detected with high relative abundance.

The mass fingerprint of *B. occitanus* venom was generated from a manual deconvolution of spectra gained by top-down LC-MS approach, thus detecting a total of 197 monoisotopic masses ranging from 1868 to 16 720 Da (Table 1). We get one mass less than 2000 Da, 28 molecular masses ranging between 2000 and 5000 Da, 147 mass values from 5000 to 8000 Da, and 21 masses for those over 8000 Da.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** TIC of native *B. occitanus* venom filtrate, generated from top-down mass spectrometry analysis (MS1). The x-axis represents the relative abundance (%), and the y-axis, the retention time (min). Spectra were deconvoluted, and generated monoisotopic masses were distributed according to their MW.</figcaption></figure>
<table><caption>**Table 1.** List of the 197 monoisotopic masses detected by the top-down LC-MS analysis.</caption><thead><tr><th>Retention time (min)</th><th>MW (Da)</th></tr></thead><tbody><tr><td>0–10</td><td>N.D</td></tr><tr><td>10–20</td><td>1868.0157</td></tr><tr><td>20–30</td><td>2208.2634; 2506.4634</td></tr><tr><td>30–40</td><td>2813.4212; 2851.4287; 2966.3848; 3124.4545; 3219.5691; 3233.4756; 3461.4966; 3486.7774; 3538.283; 3550.4334; 3670.8935; 3718.7023; 3823.4412; 3937.8078; 4093.8732; 4321.8654; 4366.9752; 4366.986; 5731.6152; 5919.5155.</td></tr><tr><td>40–50</td><td>3522.2898; 3614.8741; 3807.4466; 3937.7725; 4333.933; 4366.9856; 4568.7172; 4572.9253; 5185.3781; 6148.8879; 6423.7104; 6439.6786; 6527.7246; 6539.6502; 6541.7326; 6595.7719; 6606.8166; 6610.768; 6611.7946; 6635.0442; 6744.712; 6829.8098; 6831.8926; 6832.876; 6860.9183; 6861.9012; 6872.9404; 6876.9037; 6877.9284; 6893.9821; 6940.948; 6952.1809; 6974.2357; 6979.0052; 6995.0399; 6997.024; 7014.2508; 7016.0204; 7022.0148; 7024.0653; 7107.2902; 7152.0763; 7162.3796; 7177.1647; 7218.3026; 7220.0387; 7220.2052; 7243.2414; 7297.2395; 7393.2604.</td></tr><tr><td>50–60</td><td>6488.9021; 6609.8127; 6611.7977; 6629.8447; 6677.8651; 6749.8876; 6765.9533; 6779.2433; 6807.922; 6823.1194; 6836.974; 6837.8837; 6862.9698; 6879.9966; 6907.3347; 6919.9628; 6972.7789; 7007.0404; 7011.1444; 7012.1231; 7020.055; 7028.0976; 7035.2491; 7024.1049; 7051.0799; 7061.1245; 7062.1114; 7069.1111; 7079.1299; 7082.3444; 7115.0302; 7115.2113; 7122.274; 7130.9674; 7143.0368; 7250.1077; 7262.1172; 7266.1721; 7268.152; 7283.1496; 7307.2070; 7328.1353; 7394.3224; 7394.5252; 7400.289; 7416.5358; 7435.2763; 7449.3831; 7468.4297; 7491.1348; 7506.1972; 7534.4067; 7607.5077; 7681.4621; 7777.5363; 7840.6401; 7894.5677; 7912.5297; 7924.5736; 7943.5256; 8174.6428; 8344.5958; 9875.9204; 6896.9694; 6880.9937; 7016.998; 7056.1905; 7074.1478; 7104.0354; 7122.2913; 7115.9848; 7175.0715; 7309.2612; 7414.4224; 7600.5; 7654.5083; 7798.6334; 7817.6424; 7832.6366; 7833.6635; 8140.6441; 8159.4822; 8345.5484; 9959.0054; 11068.3376; 11243.5823; 16720.7335.</td></tr><tr><td>60–70</td><td>6896.9694; 6880.9937; 7016.998; 7056.1905; 7074.1478; 7104.0354; 7122.2913; 7115.9848; 7175.0715; 7309.2612; 7414.4224; 7600.5; 7654.5083; 7798.6334; 7817.6424; 7832.6366; 7833.6635; 8140.6441; 8159.4822; 8345.5484; 9959.0054; 11068.3376; 16720.7335.</td></tr><tr><td>70–80</td><td>6809.9307; 6857.9428; 6859.9368; 6865.9432; 6875.9565; 6880.9796; 6982.0159; 6913.9378; 7009.0523; 7104.9914; 7172.1987; 7200.1528; 7214.1558; 7316.2804; 7377.2599; 7300.0933; 7394.5084.</td></tr><tr><td>80–90</td><td>7377.2678; 7301.1747; 9140.1069; 11377.1636; 12971.6074; 13004.7435.</td></tr><tr><td>90–100</td><td>7390.4025; 7466.4483; 7482.4543; 7500.4753; 7704.4655; 7791.5128; 7792.5813; 8672.6993; 8882.0067; 8978.0645; 14577.4253.</td></tr><tr><td>100–110</td><td>9302.1043; 12990.2825; 12985.6009.</td></tr><tr><td>110–120</td><td>N.D</td></tr></tbody></table>

N.D: not determined.

**Fig. 3.** Molecular mass distribution of the monoisotopic masses from MS1 spectra deconvolution. 197 components were detected, with their MW ranging from 1868 to 16 720 Da. These peptides distributed from 1000 to 17 000 Da with 1000 Da mass range windows. The x-axis represents the MW in Da, and the y-axis represents the percentage (%) based on total number counts.

<figure><img src="image_6.png" /></figure>

The most representative molecular masses were those from 5000 to 8000 Da, followed by those between 2000 and 5000 Da, which represents respectively 74% and 10% of the total number of measured molecular masses (Fig. 3).

The analysis of reduced/alkylated *B. occitanus* venom filtrate by tandem mass spectrometry allowed the identification of 68 peptides with a molecular weight (MW) from 1959.13 to 7943.53 Da. The detected experimental sequences are shown in Table 2;
<table><caption>**Table 2.** List of the identified peptides by top-down analysis of the reduced/alkylated *B. occitanus* venom filtrate. Data sets generated from the mass spectrometer were analyzed by the PROTEOME DISCOVER 2.2 software, against UniProtKB/Swiss-Prot database. The amino acids sequences colored in black were those detected by the analysis. Peptide entries in bold were identified by both top-down and bottom-up approaches.</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Identified Sequence</th></tr></thead><tbody><tr><td rowspan="11">NaScTx</td><td>P59356</td><td>Alpha-like toxin Lqh6</td><td>MVRDGYIAQPENCVYHCIPDCDTLCKDNGGTGGHCGFKLGHGIAWCNALPDNVGIIV DGVKCHK</td></tr><tr><td>P13488</td><td>Alpha-like toxin Bom3</td><td>MGRDGYIAQPENCVYHCFPGSSGCDTLCKEKGATSGHCGFLPGSGVACWCDNLPNK VPIVVGGEKCH</td></tr><tr><td>P56678</td><td>Alpha-like toxin Lqh3</td><td>MVRDGYIAQPENCVYHCFPGSSGCDTLCKEKGGTSGHCGFKVGHGLACWCNALPDNV GIIVEGEKCHS</td></tr><tr><td>Q9NJC4</td><td>Chain (toxin BmKaTx17) [10–73] in toxin BmKaTx17</td><td>MLLMTGVESGRDAYIAKNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGNACWC INLPDDKPIRIPGKCHRR</td></tr><tr><td>Q4TUA4</td><td>Chain (alpha-toxin 4) [20–85] in alpha-toxin 4</td><td>MNYLVFFALLLLMTGVESVRDGYIADDKNCAYFCGRNAYCDDECKKKGAESGYCQWA GYGNACWCYKLPDKVPIRVPGRCNGG</td></tr><tr><td>P59863</td><td>Beta-toxin BotlT2</td><td>MDGYIKGYKGCKITCVINDDYCDTECKAEGGTYGYCWKWGLACWCECDLPDEKRWKSE TNTC</td></tr><tr><td>P60163</td><td>Toxin Cg2</td><td>MKDGYLVNKSTGCKYSCIENINDSHCNEECISSIRKGSYGYCYKFYCIGMPDSTQVYP IPGKTCSTE</td></tr><tr><td>P60256</td><td>Toxin Boma6b</td><td>MVRDAYIAQNYNCVYDCARDAYCNEЛТКNGAKSHGCEWFGPHGDACWCIDLPNNVPI KVEGKCHRK</td></tr><tr><td>O77091</td><td>Chain/beta-insect excitatory toxin BmK IT-AP) [19–90] in beta-insect excitatory toxin BmK IT-AP</td><td>MKFFLIFLVIFFIMGVLGKKNGYAVDSSGKVAECLFNNYCNNECTKVYYADKGYCCLLKCYCFGLADDKPVLDIWDSTKNYCDVQIIDLS</td></tr><tr><td>P21150</td><td>Toxin AaHIT4</td><td>MEHGYLLNKKYTGCKVWVCVINNEECGYLCNKRRGGYYGYCYFWKLACYCQGARKSELW NYKTNKCDL</td></tr><tr><td>P80962</td><td>Beta-insect depressant toxin BalT2</td><td>MDGYIERRDGCKVSCLFGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDDKTWKS ETNTCG</td></tr><tr><td>P01485</td><td>Alpha-mammal toxin Bot3; chain (alpha-mammal toxin Bot3) [10–73] in alpha-mammal toxin Bot3</td><td>MLVMAGVESVKDGYIVDDRNCITYFCGRNAYCNEECTLKGEKGSYCQWASPYGNACYCYKVPDHVRTKGPGRCN</td></tr><tr><td>Q86BW9</td><td>Chain (Makatoxin-2) [20–83] in Makatoxin-2</td><td>MNYLIVISFALLLLMTSVESGRDAYIADSENCTYFCGSNPYCNDLCTENGAKSGYCQWAG RYGNACWCIDLPDKVPIRIPGPCRGR</td></tr><tr><td>G4V3T9</td><td>Neurotoxin BmK AGAP-SYPU2</td><td>MVKDGYIVDDKNCAYFCGRNAYCDDECEKNGAESGYCQWAGVYGNACWCYKLPDKV PIRVPGRCNG</td></tr><tr><td>P84614</td><td>Alpha-toxin Bs-Tx28</td><td>MGVRDAYIADDKNCVYTCGSNSYCNTECТKNGAESGYCQWFGRGWNGWCWIKLPDKV PIRIPGKCR</td></tr><tr><td>Q9BLM4</td><td>Toxin AahP1005; Chain (toxin AahP1005) [20–83] in toxin AahP1005</td><td>MNYLVMISLALLFMTGVESKKDGYIVDDKNTCTFFCGRNAYCNEDECKKKGAESGYCQWA SPYGNACYCYKLPDRVSTKKGGCNGR</td></tr><tr><td>P86408</td><td>Neurotoxin MeuNaTx-1</td><td>MVRDGYIADDKNCAYFCGRNAYCDEECKKKGAESGYCQWAGQYGNACWCYKLPDK VPIKVSGKCN</td></tr><tr><td>P60255</td><td>Toxin Boma6a</td><td>MVRDAYIAQNYNCVYDCARDAYCNDLCTKNGAKGYCEWFGPHGDACWCIDLPNNV PIKVEGKCHRK</td></tr><tr><td>P15225</td><td>Neurotoxin Os3</td><td>MGVRDGYIAQPHNCVYHCFPGSGGCDTLCKENGATOGGSSCFILGRGTACWCKDLPDR VGVIVDGEKCH</td></tr><tr><td>P45697</td><td>Alpha-like toxin BmK-M1; Chain (alpha-like toxin BmK-M1) [20–83] in alpha-like toxin BmK-M1</td><td>MNYLVMISFALLLLMTGVESVRDAYIAKPHNCVYECARNEYCNDLCTKNGAKSGYCQWV GKYGNGCWCIELPDNVPIRVPGKCHR</td></tr><tr><td>E4VP24</td><td>Chain [20–85] in sodium channel neurotoxin MeuNaTxalpha-1</td><td>MNSLVMISLALLVMTGVESVRDGYIADDKNCAYFCGRNAYCDEECKKKGAESGYCQW AGQYGNACWCYKLPDKVPIKVSGKCNGR</td></tr><tr><td>P55902</td><td>Alpha-insect toxin BotlT1</td><td>MVRDAYIAQNYNCVYFCMKDDYCNDLCTKNGASSGYCQWAGKYGNACWCYALPDNV PIRIPGKCHS</td></tr><tr><td>E7CAU3</td><td>Chain (neurotoxin BmK AGP-SYPU1) [2–65] in neurotoxin BmK AGP-SYPU1</td><td>MGRDAYIAQNYNCVYHCFRDDYCNGLCTENGADSGYCYLAGKYGHACW CINLPDDKPIRIPGKCHRR</td></tr><tr><td>Q1I178</td><td>Toxin Td9</td><td>MIGMVAECKDGYLVGDDGCKMHCFTRPGHYCASECSRVKGKDGYCYAW LACYCYNMPNWAPIWNSATNSCGGK</td></tr><tr><td>A0A146CJ90</td><td>Chain [20–87] in Venom toxin meuNa32</td><td>MNYLILISFALLVITGVESARDAYIAQNYNCVYFCLNPWSSYCDDDLCTKNGAK SGYCQIFGKYGNACWCIDLPDKVPIRIPGKCHFA</td></tr></tbody></table>
<table><thead><tr><th>Coverage (%)</th><th>Measured MW (Da)</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of unique peptides</th><th>No. of protein groups</th><th>No. of AAs</th><th>calc.pl</th></tr></thead><tbody><tr><td>98.46</td><td>6974.21</td><td>1</td><td>4</td><td>1</td><td>1</td><td>65</td><td>6.48</td></tr><tr><td>98.5</td><td>7012.14</td><td>1</td><td>1</td><td>1</td><td>1</td><td>67</td><td>6.71</td></tr><tr><td>98.52</td><td>7215.31</td><td>1</td><td>22</td><td>1</td><td>1</td><td>68</td><td>6.48</td></tr><tr><td>84</td><td>7062.13</td><td>1</td><td>1</td><td>1</td><td>1</td><td>75</td><td>7.58</td></tr><tr><td>77.64</td><td>7218.31</td><td>1</td><td>1</td><td>0</td><td>0</td><td>85</td><td>7.5</td></tr><tr><td>98.36</td><td>6564.78</td><td>1</td><td>1</td><td>1</td><td>1</td><td>61</td><td>4.84</td></tr><tr><td>88.4</td><td>6871.92</td><td>1</td><td>1</td><td>1</td><td>1</td><td>69</td><td>6.92</td></tr><tr><td>98.5</td><td>7307.23</td><td>1</td><td>4</td><td>1</td><td>1</td><td>67</td><td>7.2</td></tr><tr><td>80</td><td>7943.53</td><td>2</td><td>6</td><td>2</td><td>1</td><td>90</td><td>5.36</td></tr><tr><td>98.48</td><td>7791.58</td><td>1</td><td>6</td><td>1</td><td>1</td><td>66</td><td>8.46</td></tr><tr><td>100</td><td>6845.9</td><td>1</td><td>4</td><td>1</td><td>1</td><td>62</td><td>5.31</td></tr><tr><td>87.67</td><td>7289.18</td><td>1</td><td>5</td><td>1</td><td>1</td><td>73</td><td>7.53</td></tr><tr><td>75.29</td><td>7062.11</td><td>1</td><td>4</td><td>1</td><td>1</td><td>85</td><td>5.25</td></tr><tr><td>98.48</td><td>7289.18</td><td>1</td><td>6</td><td>1</td><td>1</td><td>66</td><td>5.31</td></tr><tr><td>98.48</td><td>7214.2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>66</td><td>8.12</td></tr><tr><td>75.29</td><td>7316.26</td><td>1</td><td>3</td><td>1</td><td>1</td><td>85</td><td>8.46</td></tr><tr><td>98.46</td><td>7218.31</td><td>1</td><td>6</td><td>1</td><td>1</td><td>65</td><td>7.85</td></tr><tr><td>98.5</td><td>7221.18</td><td>1</td><td>12</td><td>1</td><td>1</td><td>67</td><td>7.09</td></tr><tr><td>98.52</td><td>6957.15</td><td>2</td><td>6</td><td>2</td><td>1</td><td>68</td><td>6.71</td></tr><tr><td>76.19</td><td>7429.4</td><td>1</td><td>4</td><td>1</td><td>1</td><td>84</td><td>7.88</td></tr><tr><td>77.64</td><td>7336.32</td><td>1</td><td>1</td><td>1</td><td>1</td><td>85</td><td>7.85</td></tr><tr><td>98.48</td><td>7345.15</td><td>1</td><td>2</td><td>1</td><td>1</td><td>66</td><td>7.55</td></tr><tr><td>98.5</td><td>7488.32</td><td>2</td><td>8</td><td>2</td><td>1</td><td>67</td><td>7.61</td></tr><tr><td>86.48</td><td>7076.01</td><td>1</td><td>2</td><td>1</td><td>1</td><td>74</td><td>7.84</td></tr><tr><td>78.16</td><td>7690.37</td><td>1</td><td>1</td><td>1</td><td>1</td><td>87</td><td>7.53</td></tr></tbody></table>
<table><caption>**Table 2. Continued.**</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Identified Sequence</th></tr></thead><tbody><tr><td></td><td>P68410</td><td>Alpha-mammal toxin Ts2</td><td>MKEGYAMDHEGCKFSCFIRPAGFCDGYCKTHLKASSGYCAWPACYCYGV PDHIKVWDYATNKC</td></tr><tr><td></td><td>P68726</td><td>Chain (Insect toxin 2-53) [22-82] in Insect toxin 2-53</td><td>MKLLLLLIVSASMLIESLVNADGYIKRRDGCKVACLVGNEGCDKECKAYGGSY GYCWTWGLACWCEGLPDDTKWSETNTCGGK</td></tr><tr><td></td><td>Q1I163</td><td>Toxin Td8; chain (toxin Td8) [21-83] in toxin Td8</td><td>MTRFVLFLSCFFLIGMVVECKDGYLVGDDGCKMHCFTRPHGYCASESRVK GKDGYCYAWLACYCYNMPNWAPIWSATNRCRGK</td></tr><tr><td></td><td>P56569</td><td>Makatoxin-1</td><td>MGRDAYIADSENCTYTCALNPYCNDLCTKNGAKSGYCQWAGRYGNACWCID LDPKVPIRISGSCR</td></tr><tr><td></td><td>D8UWD3</td><td>Sodium channel neurotoxin MeuNaTxalpha-7</td><td>MARDGYIADDKNCAYFCGRNAYCDEECKKKGAESGYCQWAGQYGNACWC YKLPDKVPiPKVSGKNGR</td></tr><tr><td></td><td>P0DMH9</td><td>Chain (alpha-toxin BmalphaTx47) [20-83] in alpha-toxin BmalphaTx47</td><td>MNYLIVISFALLMTGVQSGRDAYIADSENCTYTCALNPYCNDLCTKNGAKSG YCQWAGRYGNACWCIDLDPKVPiRISGSCRGR</td></tr><tr><td></td><td>P01483</td><td>Neurotoxin Bot2</td><td>MGRDAYIAQPENCVYECAKNSYCNDLCTKNGAKSGYCQWLGRWGNACYC IDLDPKVPiRiEGKCHF</td></tr><tr><td></td><td>P17728</td><td>Chain (alpha-insect toxin LqhalT) [20-85] in alpha-insect toxin LqhalT</td><td>MNHLVIMISLALLLLGVESVRDAYIAKNYNCVYECFRDAYCNELCTKNGASS GYCQWAGKYGNACWCYALPDNVPIRVPGKCHRK</td></tr><tr><td></td><td>P01496</td><td>Chain (toxin-3) [15-76] in toxin-5</td><td>MLVVVCLLTAGTEGKKGDPYVEYNCAAYCWNYDNAYCDLKCKDKKADSGY CYVWHILCYCGLPDSEPTKTNGKCKSGKK</td></tr><tr><td></td><td>Q1EG64</td><td>Chain [20-85] in sodium toxin peptide BmKtb'</td><td>MNYLVMISFAFLLMTGVESARDAYIAQNYNCVYHCARDAYCNELCTKNGAKS GSCPYLGEHKFACYCKDLPDNVPIRVPGKCNGG</td></tr><tr><td></td><td>P01488</td><td>alpha-toxin Bot1</td><td>MGRDAYIAQPENCVYECAQNSYCNDLCTKNGATSGYCQWLGKYGNACWC KDLPDNVPIRIPGKCHF</td></tr><tr><td></td><td>P45698</td><td>Chain (neurotoxin BmK-M9) [15-78] in neurotoxin BmK-M9</td><td>MISFALLLMTGVESVRDAYIAKPENCVYHCATNEGNCKLCTDNGAESGYCQW GGRYGNACWCiCIKLPDVPIRVPGKCHRK</td></tr><tr><td></td><td>P83644</td><td>Toxin Lqh4</td><td>MGVRDAYIADDKNCVYTCGANSYCNTECTKNGAESGYCQWFGKYGNACWC IKLPDKVPiRIPGKCR</td></tr><tr><td></td><td>P01487</td><td>Alpha-insect toxin Lqq3</td><td>MVRDAYIAKNYNCVYECFRDSYCNDLCTKNGASSGYCQWAGKYGNACWC YALPDNVPIRVPGKCH</td></tr><tr><td></td><td>H1ZZI7</td><td>Toxin Tpa6</td><td>MSIFPIALALLLIGLEEGAARDGYPLSNNKNCKIYCPDTDVCKDTCKNARASAP DGKCDGWNSCYCFKVPDPHIPVWGDPKTKCMT</td></tr><tr><td></td><td>B8XGY6</td><td>Chain [20-85] in Putative alpha-toxin Tx17</td><td>MNYLILISLAVLLTSGVESVRDAYIAQNYNCVYTCFKDAYCNDLCTKNGATSGY COWWGKYGNGCWYALPDNVPIRVPGKCHSR</td></tr><tr><td></td><td>P81504</td><td>Insect toxin AaHIT5</td><td>MDGYIKHRDGCKVTCLINDNYCDTECKREGGYSGYCYSVGFACWCEGLPDD KAWKSETNTCD</td></tr><tr><td></td><td>P68722</td><td>Chain (beta-insect excitatory toxin LqhlT1b) [19-88] in beta-insect excitatory toxin LqhlT1b</td><td>MKFLLFLVVLPiMGVGLKKNGYAVDSKGKAPECSFLSNYCNNECTKVHYADK GYCCLLSCYCFGLNDDKKVLEISDTTKKYCDFTIIN</td></tr><tr><td></td><td>P60257</td><td>Toxin Boma6c</td><td>MVRDAYIAQNYNCVYITCFKDAHCNDLCTKNGASSGYCQWAGKYGNACWCY ALPDNVPIRIPGKCHRK</td></tr><tr><td></td><td>M1J7U4</td><td>Putative sodium channel alpha-toxin Acra5</td><td>MVRDGYIMIKDTNCKFSCNIFKKWEYCSPLCQSKGAETGYCYNFGCWCLDL PDDVPYGDRGVICRTR</td></tr><tr><td></td><td>Q9N682</td><td>Chain (neurotoxin BmK-M11) [20-83] in neurotoxin BmK-M11</td><td>MNYLVMISFALLLMTGVESVRDAYIAKPENCVYHCATNEGNCKLCTDNGAESG YCQWGGKYGNACWCiCIKLDDVPIRVPGKCHRK</td></tr><tr><td></td><td>P55903</td><td>beta-insect depressant toxin BotlT4</td><td>MDGYiRRRDGCKVSCLFGNEGCDKECKAYGGSYGYCWTWGLACWCEGLPDD KTWKSETNTCG</td></tr><tr><td></td><td>A0A0K0LBU9</td><td>Chain [20-83] in sodium channel blocker AbNaTx26</td><td>MRAALLLAFSSLILTGVLTKKGYPTQHDGCKiWCVFNHFCSNYCETYGGSGYCYT WKLACWCDNIHDWVPTWSYATTKCRAK</td></tr><tr><td></td><td>P0C910</td><td>Alpha-toxin Amm3</td><td>MGRDGYiVDTKNCVYHCYPPCDGLCKNQAKGSKGSCFLYPSGLACWCVALPENV PIKDPNDDCHK</td></tr><tr><td></td><td>P59360</td><td>Neurotoxin BmK-II</td><td>VRDAYIAKPHNCVYECARNEYCNDLCTKDGAKSGYCQWVGVKYGNGWCiELPDNV PIRIPGNCH</td></tr><tr><td></td><td>P81240</td><td>Insect toxin LqhlT5</td><td>MDGYiRGGDCKVSCVIDHVFCDNECKAAGGSYGYCWGWGLACWCEGLPADREWK YETNTCG</td></tr><tr><td></td><td>P01497</td><td>Chain (beta-insect excitatory toxin 1) [19-88] in beta-insect excitatory toxin 1</td><td>MKFLLFLVVLPiMGVFGKKNGYAVDSSGKAPECLLSNYCNNECTKVHYADKGYCCLL SCYCFGLNDDKKVLEISDTRKSYCDTTIIN</td></tr><tr><td></td><td>V9P3B8</td><td>Chain [23-82] in Chain [23-82] in Meutoxin-3</td><td>MKILTVFMIFIANFLSMTQVFLSKLDRFLLINGSYELCLYEE NLDEDCERLCKEQNASDG FCRQPHFCADMPDDYPTRPTTR</td></tr></tbody></table>
<table><thead><tr><th>Coverage (%)</th><th>Measured MW (Da)</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of unique peptides</th><th>No. of protein groups</th><th>No. of AAs</th><th>calc.pl</th></tr></thead><tbody><tr><td>98.41</td><td>6655.84</td><td>1</td><td>6</td><td>1</td><td>1</td><td>63</td><td>7.61</td></tr><tr><td>71.76</td><td>6739.87</td><td>1</td><td>1</td><td>1</td><td>1</td><td>85</td><td>7.5</td></tr><tr><td>73.25</td><td>6986.05</td><td>1</td><td>3</td><td>1</td><td>1</td><td>86</td><td>8.34</td></tr><tr><td>98.46</td><td>7240.24</td><td>1</td><td>2</td><td>0</td><td>0</td><td>65</td><td>7.5</td></tr><tr><td>98.5</td><td>7295.24</td><td>1</td><td>1</td><td>1</td><td>1</td><td>67</td><td>8.1</td></tr><tr><td>75.29</td><td>7240.24</td><td>1</td><td>1</td><td>0</td><td>0</td><td>85</td><td>7.87</td></tr><tr><td>98.48</td><td>7240.24</td><td>1</td><td>19</td><td>1</td><td>1</td><td>66</td><td>7.55</td></tr><tr><td>77.64</td><td>7173.2</td><td>1</td><td>12</td><td>1</td><td>1</td><td>85</td><td>8.12</td></tr><tr><td>76.54</td><td>7105.03</td><td>1</td><td>20</td><td>1</td><td>1</td><td>81</td><td>7.49</td></tr><tr><td>77.64</td><td>7321.09</td><td>1</td><td>2</td><td>1</td><td>1</td><td>85</td><td>7.58</td></tr><tr><td>98.48</td><td>7074.14</td><td>1</td><td>3</td><td>1</td><td>1</td><td>66</td><td>6.92</td></tr><tr><td>81.01</td><td>7015.19</td><td>1</td><td>1</td><td>1</td><td>1</td><td>79</td><td>7.88</td></tr><tr><td>98.48</td><td>7155.25</td><td>1</td><td>3</td><td>1</td><td>1</td><td>66</td><td>8.1</td></tr><tr><td>98.5</td><td>6980.01</td><td>2</td><td>12</td><td>2</td><td>1</td><td>65</td><td>7.87</td></tr><tr><td>74.41</td><td>7059.12</td><td>1</td><td>2</td><td>1</td><td>1</td><td>86</td><td>5.38</td></tr><tr><td>77.64</td><td>7313.2</td><td>1</td><td>2</td><td>1</td><td>1</td><td>85</td><td>7.87</td></tr><tr><td>98.38</td><td>6894.89</td><td>1</td><td>8</td><td>1</td><td>1</td><td>62</td><td>4.83</td></tr><tr><td>79.54</td><td>7924.56</td><td>1</td><td>1</td><td>1</td><td>1</td><td>88</td><td>7.87</td></tr><tr><td>98.5</td><td>7308.21</td><td>2</td><td>14</td><td>2</td><td>1</td><td>67</td><td>8.31</td></tr><tr><td>98.52</td><td>7741.51</td><td>1</td><td>1</td><td>1</td><td>1</td><td>68</td><td>7.5</td></tr><tr><td>77.38</td><td>7179.21</td><td>2</td><td>2</td><td>2</td><td>1</td><td>84</td><td>7.09</td></tr><tr><td>100</td><td>6837.96</td><td>1</td><td>4</td><td>1</td><td>1</td><td>62</td><td>5.31</td></tr><tr><td>77.1</td><td>7505.2</td><td>1</td><td>1</td><td>1</td><td>1</td><td>83</td><td>8.31</td></tr><tr><td>98.46</td><td>7011.14</td><td>1</td><td>1</td><td>1</td><td>1</td><td>65</td><td>7.09</td></tr><tr><td>100</td><td>7431.33</td><td>2</td><td>14</td><td>2</td><td>1</td><td>65</td><td>7.09</td></tr><tr><td>100</td><td>6611.8</td><td>1</td><td>3</td><td>1</td><td>1</td><td>62</td><td>4.72</td></tr><tr><td>79.54</td><td>7928.54</td><td>1</td><td>10</td><td>1</td><td>1</td><td>88</td><td>7.53</td></tr><tr><td>73.17</td><td>7074.13</td><td>1</td><td>1</td><td>1</td><td>1</td><td>82</td><td>4.75</td></tr></tbody></table>
<table><caption>**Table 2. Continued.**</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Identified Sequence</th></tr></thead><tbody><tr><td></td><td>Q8T3T0</td><td>Depressant insect toxin BmK1Ta1</td><td>MKLFLLLISASMLIDGLVNADGYIRGSNGCKVSCLWEGNECNKECGAYGASYGWCW TWGLACWCEGLPDDTKWSESNTCGKK</td></tr><tr><td></td><td>Q9GQW3</td><td>Chain (toxin BmKalT1) [20–83] in toxin BmKalT1</td><td>MNYLVMSIFAFFLMTGVESVRDAYIAQNYCNYHCARDAYCNELCTKNGAKSGSCPY LGEHKFACYCKDLPDNVPIRVPGKCHRR</td></tr><tr><td></td><td>Q95WX6</td><td>Beta-insect depressant toxin BmKITb</td><td>MKLFLLLVISASMLIDGLVNADGYIRGSNGCKVSCLWEGNECNKECKAFGAYYYGWCW TWGLACWCGLPPDDTKWSESNTCGKK</td></tr><tr><td></td><td>P0C5H1</td><td>Beta-toxin Isom1</td><td>MKKNGYAVDSSGKAPECLLSNYCNNECTKVVYADKGYCCLLSCYCFGLSDDKKVLEIS DTRKKYCDYTIIN</td></tr><tr><td></td><td>Q9GNG8</td><td>Toxin BmKaTX15</td><td>MNYLVFFSALLVMTGVESVRDGYIADDKNCAAYFCGRNAYCDDECKKNGAESGYCQW AGVYGNACWYCYKLPDKVPIRVPGKCNGG</td></tr><tr><td></td><td>M1JMR8</td><td>Sodium channel alpha-toxin Acra8</td><td>MVRDGYIVDDKNCTFFCGRNAYCNDECKKKGGESGYCQWASPYGNACWYCYKLPDRV PIKEKGRCNGR</td></tr><tr><td></td><td>A0A0U4RDS7</td><td>Chain [20–87] in sodium channel toxin NaTx4</td><td>MNHLVMSIFLAFLMTGVASVRDGYIAQPETCAYHCIPGSSGCTYLCKEKKGESGHCGWK SGHGSAWWCNDLPDKEGIIVDGKGCTRR</td></tr><tr><td></td><td>P82814</td><td>Insect toxin BsIT4</td><td>MDGYIKGNKGCKVSCVINVNFCSNMCCKSGGSYGCWSWGLACWCEGLPAKKWLY AATNTCG</td></tr><tr><td></td><td>B8XGX9</td><td>Chain [20–87] in Putative alpha-toxin Tx2</td><td>MNYLIMISLALLLLMTGVESGTGVRDAYIADDKNVCVYTCALNSYCNTECTKNGAESGYCQ WLGQYGNACWCIKLPDRVPIRIPGKCRG</td></tr><tr><td>Q17254</td><td>Alpha-insect toxin Bot14</td><td></td><td>MSSLIMISTAMKGKAPYRQVRDGYIAQPHNCAAYHCLKISSGCDTLCCKENGATSGHCGH KSGHGSACWCKDLPDKGIIVHGEKCHR</td></tr><tr><td>KScTx</td><td>A0A059UI30</td><td>Chain (potassium channel toxin Meg-beta-KTx1) [28–91] in potassium channel toxin Meg-beta-KTx1</td><td>MQRNLVVLLFLGMVALSSCGLREKHFOKLVKYAVPEGTLRTIIQTAVHKLGKTQFGCPA YQGYCDDHCQDIKKQEGFCHGFKCKCGIPMGF</td></tr><tr><td></td><td>Q9N661</td><td>Potassium channel toxin BmTXK-beta-2</td><td>MQRNLVVLLFLGMVALSSCGLREKHFOKLVKYAVPEGTLRTIIQTAVHKLGKTQFGCP AYQGYCDDHCQDIKKEEGFCHGFKCKCGIPMGF</td></tr><tr><td>AMP</td><td>A0A0A116E7</td><td>AMP AcrAP1</td><td>MEIKYLLTVFLVVLLIVSDHCUAFLSFLIPHAISGLISAFKGRKRDLDGDQIDRFRNFRKRD AELEELSKLPIY</td></tr><tr><td>Myotropic neuropeptide</td><td>F8THJ9</td><td>Putative orcokinin</td><td>MMFGWIWILCGTAFFCHVDAYLEYSNMAPGYNALVRRRSMKQPSEGRMFDNLGYNQE SLVKRNFDEIDNVGFNDFGPASRPGSGRSWFPKRNWELAYNLRRLVKRATOD ELMENKRQELDEIDKSGFGGFKHRNFEDEIDRGFNDFGKRSFDRFKLVRRAOFNN</td></tr><tr><td>Hypothetical secreted protein</td><td>F1CIZ9</td><td>Hypothetical secreted protein</td><td>MONIFWILIGVGCICITAQVCDESEMESSIRDIILTKRRYLYKARSVLLDDLNQOLDTLHKRSC VLNPLGMDCEYGDITGSKGDKDQDYWTSGRTPGKKRRSYCSLGIGNSEECLTKQLKDDM TDFNSWNDKFRPGKK</td></tr></tbody></table>

five of the entries were identified with 100% sequence coverage: neurotoxin BmK-II (P59360), beta-insect depressant toxin BotIT4 (P55903), beta-insect depressant toxin BaIT2 (P80962), insect toxin LqhIT5 (P81240), and insect toxin BsIT4 (P82814). These toxins were reported for the first time in this Moroccan venom, they corresponded to toxins already identified in other scorpion venom. The determined sequence of the neurotoxin BmK-II (P59360) showed 100% similarity with the database sequence, whereas the observed sequences of the other toxins showed methylation in the N-terminal part compared with sequences reported in Uniprot database (Fig. 4). Therefore, the other peptides corresponded approximately to toxins, previously identified in other scorpion species with a sequence identity ranging from 17% to 98% (Fig. S2).

Therefore, the detected peptides were divided into five categories on the basis of their molecular functions according to the UniProtKB database (https://www.uni prot.org); 63 neurotoxins acting on sodium channels

(NaScTxs), constitute 93% of the components and represent a MW from 6564.78 to 7943.53 Da; two neurotoxins acting on potassium channels (KScTxs) (2.94%, 2506.46–6889.3 Da); one antimicrobial peptide (AMP) (1.47%, 1959.13 Da); one myotropic neuropeptide (1.47%, 3112.45 Da); and one hypothetical secreted protein (1.47%, 3939.79 Da) (Fig. 5A).

Additionally, we have observed, that between these 68 peptides, 27 of them (40%) were detected as chains or fragments, for example, venom toxin meuNa32 (A0A146CJ90); potassium channel toxin Meg-beta-KTx1 (A0A059UI30); putative alpha-toxin Tx2 (B8XGX9); sodium channel toxin NaTx4 (A0A0U4RDS7); toxin BmKaIT1(Q9GQW3); sodium channel blocker AbNaTx26 (A0A0K0LBU9); neurotoxin BmK-M11 (Q9N682); beta-insect excitatory toxin LqhIT1b (P68722); toxin-5 (P01496); toxin Td8 (Q1I163); alpha-like toxin BmK-M1 (P45697); toxin AahP1005 (Q9BLM4); makatoxin-2 (Q86BW9); and alpha-mammal toxin Bot3 (P01485) (Table 2).
<table><thead><tr><th>Coverage (%)</th><th>Measured MW (Da)</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of unique peptides</th><th>No. of protein groups</th><th>No. of AAs</th><th>calc.pl</th></tr></thead><tbody><tr><td>71.76</td><td>6632.71</td><td>1</td><td>18</td><td>1</td><td>1</td><td>85</td><td>6.38</td></tr><tr><td>75.29</td><td>7012.23</td><td>1</td><td>3</td><td>1</td><td>1</td><td>85</td><td>8.12</td></tr><tr><td>71.76</td><td>6775.93</td><td>1</td><td>4</td><td>1</td><td>1</td><td>85</td><td>7.85</td></tr><tr><td>98.59</td><td>7895.47</td><td>1</td><td>35</td><td>1</td><td>1</td><td>71</td><td>7.53</td></tr><tr><td>77.64</td><td>7211.14</td><td>1</td><td>1</td><td>1</td><td>1</td><td>85</td><td>6.4</td></tr><tr><td>98.5</td><td>7218.3</td><td>1</td><td>1</td><td>1</td><td>1</td><td>67</td><td>8.29</td></tr><tr><td>78.16</td><td>7243.29</td><td>2</td><td>4</td><td>2</td><td>1</td><td>87</td><td>7.66</td></tr><tr><td>100</td><td>6954.15</td><td>1</td><td>1</td><td>1</td><td>1</td><td>63</td><td>8.31</td></tr><tr><td>78.16</td><td>7394.28</td><td>1</td><td>3</td><td>1</td><td>1</td><td>87</td><td>7.5</td></tr><tr><td>78.82</td><td>7184.3</td><td>1</td><td>5</td><td>1</td><td>1</td><td>85</td><td>8.5</td></tr><tr><td>70.32</td><td>6889.3</td><td>1</td><td>9</td><td>1</td><td>1</td><td>91</td><td>8.76</td></tr><tr><td>25.27</td><td>2506.46</td><td>1</td><td>1</td><td>1</td><td>1</td><td>91</td><td>8.57</td></tr><tr><td>24.32</td><td>1959.13</td><td>1</td><td>1</td><td>1</td><td>1</td><td>74</td><td>9.31</td></tr><tr><td>16.96</td><td>3112.45</td><td>1</td><td>1</td><td>1</td><td>1</td><td>165</td><td>9.29</td></tr><tr><td>25.75</td><td>3939.79</td><td>1</td><td>1</td><td>1</td><td>1</td><td>132</td><td>7.99</td></tr></tbody></table>

## Bottom-up proteomics

For the bottom-up workflow, two digest methods were performed: (a) in-solution digestion, the flow-through containing toxin < 30 kDa was directly reduced with DTT, alkylated with IAA, and digested with trypsin; and (b) in-gel digestion, the gel spot corresponding to peptides under 30 kDa (Fig. S1) was excised to small cubes, which after series of washings, were reduced, alkylated, and digested.

The results generated by the bottom-up approach using the in-gel digestion yielded the identification of 36 peptides, whereas 37 was the total of the identified peptide by in-solution digestion. The detected peptides showed similarity of sequences with peptides from other scorpion species, and with their sequence coverage ranging from 10.23% (P68721) to 86.15% (P01489) and from 8.75% (P0C294) to 92.86% (P80669) for the in-gel and in-solution digestions, respectively.

The identified categories of peptides using the in-gel digestion were as follows: 27 NaScTxs; seven KscTxs; and two ClTxs (Table 3). While, through the in-solution digestion, we identified in addition to 24 NaScTxs, eight KScTxs and three ClScTxs, one entry that shares 60% of similarity with neurotoxin Tx-2 (P83406) purified from *Hottentotta judaicus*, could correspond to a calcium channel activator ‘CaScTx’ scorpion. Besides neurotoxins, one amphipathic peptide was detected by this digestion method (Table 4).

According to the results, 23 of the entries were detected by both digestion methods (Tables 3 and 4). Thus, 14 peptides were identified only by the in-solution digestion method, for example, alpha-toxin Amm5 (P01482), alpha-mammal toxin Bot3 (P01485), potassium channel toxin alpha-KTx 9.3 (P80669), neurotoxin Tx-2 (P83406), neurotoxin P2 (P01498), and amphipathic peptide Tx348 (B8XH50). Otherwise, regarding the in-gel digestion results, 13 peptides were identified only by this method of digestion, for example,
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /><figcaption>**Fig. 4.** The detected amino acid sequences of the five toxins identified with 100% coverage by the top-down LC-MS/MS; neurotoxin BmK-II (P59360); beta-insect depressant toxin BaIT2 (P80962); insect toxin BsIT4 (P82814); insect toxin LqhIT5 (P81240); and beta-insect depressant toxin BotIT4 (P55903).</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Fig. 5.** (A) Relative abundance of the different peptide categories identified in reduced/alkylated *B. occitanus* venom filtrate by the top-down LC-MS/MS analysis. Peptides were divided on the basis of their molecular functions into: neurotoxins active on sodium channels (NaScTxs), neurotoxins active on potassium channels (KScTxs), myotropic neuropeptide, AMP, and hypothetical secreted protein. (B) Relative abundance of the different peptide categories identified in reduced/alkylated and digested *B. occitanus* venom by bottom-up LC-MS/MS analysis. The peptides were divided on the basis of their molecular functions into: neurotoxins active on sodium channels (NaScTxs), neurotoxins active on potassium channels (KScTxs), neurotoxins active on chloride channels (ClScTxs), neurotoxins active on calcium channels (CaScTx), toxin Acra, and amphipathic peptide.</figcaption></figure>

potassium channel toxin alpha-KTx 9.11 (B3EWX9); sodium channel alpha-toxin Acra4 (M1JBC0); sodium channel alpha-toxin Acra8 (M1JMR8), alpha-toxin Ac3 (fragment) (D5HR52); and beta-insect depressant toxin BotIT5 (P55904).

Since the aim of using two methods of digestions was to identify the maximum of peptide, the data generated by bottom-up approaches using in-gel and in-solution digestions were then summarized in Table 5; the repeated molecules were deleted and thus allowed the detection of a total of 50 peptides, which were divided into different categories according to their molecular functions. The generated data from the bottom-up process confirmed that the family with the most diverse members in this venom is neurotoxins, with 31 NaScTxs (62%, 4.3–10.2 kDa), 13 KScTxs (26%, 2.9–10.4 kDa), three ClScTxs (6%, 3.6–4 kDa), one CaScTx (2%, 2.9 kDa), and one toxin Acra (2%, 8.8 kDa).

In addition to these neurotoxins, we identified one amphipathic peptide (2%, 7.8 kDa) (Fig. 5B). Also, some peptides were detected as fragments (10% of

total): alpha-toxin Ac1 (D5HR50) and Ac3 (D5HR52); alpha-mammal toxin Bot3 (P01485); and neurotoxin 8 (P04098).

As we mentioned above, we aimed to gain a deeper understanding of the *B. occitanus* peptidome (under 30 kDa), so the molecular diversity of its toxins. In this context, we combined data from the top-down and bottom-up analyses and then analyzed the generated data to infer a global and comprehensive characterization of this venom.

According to this study, a total of 118 peptides were identified from *B. occitanus* venom; among them, 16 were identified by both approaches, for example, potassium channel toxin BmTXK-beta-2 (Q9N661); toxin AaHIT4 (P21150); and alpha-mammal toxin Bot3 (Fragment) (P01485).

Among the 102 identified peptides, the most representative category is neurotoxins, mainly NaScTxs (77%), followed by KScTxs (14%), ClScTxs (3%), CaScTx (1%), and toxin Acra (1%). We also characterized other peptides with low percentage such as
<table><caption>**Table 3.** Bottom-up data generated from in-gel digestion of *B. occitanus* venom filtrate using nano-LC-MS/MS. Data sets generated from the mass spectrometer were analyzed by the PROTEOME DISCOVER 2.2 software, against UniProtKB/Swiss-Prot database.</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Score</th><th>Coverage</th><th>No. of proteins</th><th>No. of unique peptides</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of AAs</th><th>MW [kDa]</th><th>calc. pI</th></tr></thead><tbody><tr><td rowspan="10">NaScTx</td><td><u>Q86SE0</u></td><td>Toxin Aam2 OS = <i>Androctonus amoreuxi</i><br/>PE = 1 SV = 1 - [SCX2_ANDAM]</td><td>198.74</td><td>24.42%</td><td>9</td><td>2</td><td>3</td><td>6</td><td>86</td><td>9.3</td><td>7.87</td></tr><tr><td><u>P21150</u></td><td>Toxin AaHIT4 OS = <i>Androctonus australis</i><br/>PE = 1 SV = 1 - [SIX4_ANDAU]</td><td>85.81</td><td>29.23%</td><td>2</td><td>1</td><td>2</td><td>5</td><td>65</td><td>7.8</td><td>8.46</td></tr><tr><td><u>P13488</u></td><td>Alpha-like toxin Bom3 OS = <i>Buthus occitanus mardochei</i> PE = 1 SV = 1 - [SCX3_BUTOM]</td><td>169.75</td><td>56.06%</td><td>2</td><td>3</td><td>3</td><td>15</td><td>66</td><td>6.9</td><td>6.71</td></tr><tr><td><u>P68721</u></td><td>Beta-insect excitatory toxin LqhIT1a<br/>OS = <i>Leiurus quinquestriatus hebraeus</i><br/>PE = 3 SV = 1 - [SIX1A_LEIQH]</td><td>54.81</td><td>10.23%</td><td>2</td><td>1</td><td>2</td><td>3</td><td>88</td><td>9.9</td><td>8.09</td></tr><tr><td><u>P0DJH8</u></td><td>Alpha-toxin Bu1 OS = <i>Buthacus macrocentrus</i> PE = 1 SV = 1 - [SCX1_BUTMA]</td><td>346.32</td><td>71.64%</td><td>1</td><td>3</td><td>5</td><td>7</td><td>67</td><td>7.5</td><td>8.48</td></tr><tr><td><u>P86406</u></td><td>Neurotoxin MeuNaTx-6 OS = <i>Mesobuthus eupeus</i> PE = 1 SV = 1 - [SCXN6_MESEU]</td><td>134.56</td><td>15.15%</td><td>3</td><td>1</td><td>1</td><td>4</td><td>66</td><td>7.8</td><td>7.87</td></tr><tr><td><u>P83644</u></td><td>Toxin Lqh4 OS = <i>Leiurus quinquestriatus hebraeus</i> PE = 1 SV = 1 - [SCX4_LEIQH]</td><td>305.53</td><td>46.15%</td><td>8</td><td>1</td><td>3</td><td>7</td><td>65</td><td>7.2</td><td>8.1</td></tr><tr><td><u>P01489</u></td><td>Alpha-toxin Lqq4 OS = <i>Leiurus quinquestriatus quinquestriatus</i> PE = 1 SV = 1 - [SCX4_LEIQU]</td><td>531.95</td><td>86.15%</td><td>9</td><td>2</td><td>5</td><td>11</td><td>65</td><td>7.2</td><td>8.1</td></tr><tr><td><u>P01486</u></td><td>Alpha-toxin Bot11 OS = <i>Buthus occitanus tunetanus</i> PE = 1 SV = 1 - [SCXB_BUTOC]</td><td>106.19</td><td>35.38%</td><td>7</td><td>1</td><td>3</td><td>7</td><td>65</td><td>7.5</td><td>7.87</td></tr><tr><td><u>P60255</u></td><td>Toxin Boma6a OS = <i>Buthus occitanus mardochei</i> PE = 3 SV = 1 - [SCXA_BUTOM]</td><td>65.84</td><td>15.15%</td><td>2</td><td>1</td><td>1</td><td>2</td><td>66</td><td>7.5</td><td>7.09</td></tr><tr><td><u>P17728</u></td><td>Alpha-insect toxin LqhIT OS = <i>Leiurus quinquestriatus hebraeus</i> PE = 1 SV = 2 - [SCXA_LEIQH]</td><td>174.28</td><td>31.76%</td><td>4</td><td>1</td><td>2</td><td>3</td><td>85</td><td>9.6</td><td>8.12</td></tr><tr><td><u>P04098</u></td><td>Neurotoxin 8 (Fragment) OS = <i>Buthus occitanus tunetanus</i> PE = 1 SV = 1 - [SCX8_BUTOC]</td><td>202.83</td><td>72.22%</td><td>2</td><td>2</td><td>2</td><td>4</td><td>36</td><td>4.1</td><td>6.24</td></tr><tr><td><u>P55902</u></td><td>Alpha-insect toxin Bot1T OS = <i>Buthus occitanus tunetanus</i> PE = 1 SV = 1 - [SIX1_BUTOC]</td><td>211.59</td><td>41.54%</td><td>2</td><td>1</td><td>2</td><td>4</td><td>65</td><td>7.3</td><td>7.55</td></tr><tr><td><u>P01488</u></td><td>Alpha-toxin Bot1 OS = <i>Buthus occitanus tunetanus</i> PE = 1 SV = 2 - [SCX1_BUTOC]</td><td>136.52</td><td>20.00%</td><td>1</td><td>1</td><td>1</td><td>2</td><td>65</td><td>7.3</td><td>6.92</td></tr></tbody></table>
<table><caption>**Table 3.** (Continued).</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Score</th><th>Coverage</th><th>No. of proteins</th><th>No. of unique peptides</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of AAs</th><th>MW [kDa]</th><th>calc. pI</th></tr></thead><tbody><tr><td></td><td>P81504</td><td>Insect toxin AaHIT5 OS = <i>Androctonus australis</i> PE = 1 SV = 1 - [SIX5_ANDAU]</td><td>51.44</td><td>24.59%</td><td>1</td><td>1</td><td>1</td><td>2</td><td>61</td><td>6.9</td><td>4.83</td></tr><tr><td></td><td>P59863</td><td>Beta-toxin BotIT2 OS = <i>Buthus occitanus</i> tunetanus PE = 1 SV = 1 - [SIX2_BUTOC]</td><td>109.66</td><td>43.33%</td><td>1</td><td>2</td><td>2</td><td>3</td><td>60</td><td>6.9</td><td>4.84</td></tr><tr><td></td><td>Q17254</td><td>Alpha-insect toxin Bot14 OS = <i>Buthus occitanus tunetanus</i> PE = 2 SV = 1 - [SCXE_BUTOC]</td><td>44.99</td><td>18.82%</td><td>1</td><td>1</td><td>1</td><td>3</td><td>85</td><td>9.2</td><td>8.5</td></tr><tr><td></td><td>D5HR52</td><td>Alpha-toxin Ac3 (Fragment) OS = <i>Androctonus crassicauda</i> PE = 3 SV = 1 - [SCX3A_ANDCR]</td><td>139.86</td><td>63.77%</td><td>10</td><td>2</td><td>4</td><td>10</td><td>69</td><td>7.8</td><td>7.87</td></tr><tr><td></td><td>P55904</td><td>Beta-insect depressant toxin BotIT5 OS = <i>Buthus occitanus tunetanus</i> PE = 1 SV = 1 - [SIX5_BUTOC]</td><td>64.67</td><td>27.87%</td><td>21</td><td>2</td><td>2</td><td>9</td><td>61</td><td>6.8</td><td>5.31</td></tr><tr><td></td><td>O77091</td><td>Beta-insect excitatory toxin BmK IT-AP OS = <i>Mesobuthus martensii</i> GN = IT-AP PE = 1 SV = 1 - [SIXP_MESMA]</td><td>126.26</td><td>17.78%</td><td>8</td><td>2</td><td>2</td><td>4</td><td>90</td><td>10.2</td><td>5.36</td></tr><tr><td></td><td>P59864</td><td>Beta-insect depressant toxin BotIT6 OS = <i>Buthus occitanus tunetanus</i> PE = 1 SV = 1 - [SIX6_BUTOC]</td><td>32.37</td><td>11.29%</td><td>1</td><td>1</td><td>1</td><td>1</td><td>62</td><td>7.3</td><td>8.1</td></tr><tr><td></td><td>P68723</td><td>Beta-insect excitatory toxin LqhIT1c OS = <i>Leiurus quinquestriatus hebraeus</i> PE = 1 SV = 1 - [SIX1C_LEIQH]</td><td>182.31</td><td>11.36%</td><td>1</td><td>2</td><td>3</td><td>8</td><td>88</td><td>9.9</td><td>8.1</td></tr><tr><td></td><td>P59360</td><td>Neurotoxin BmK-II OS = <i>Mesobuthus martensii</i> PE = 1 SV = 1 - [SCX2_MESMA]</td><td>48.26</td><td>15.63%</td><td>3</td><td>1</td><td>1</td><td>1</td><td>64</td><td>7.2</td><td>7.09</td></tr><tr><td></td><td>P15224</td><td>Toxin Os1 OS = <i>Orthochirus scrobiculosus</i> PE = 1 SV = 1 - [SCX1_ORTSC]</td><td>39.14</td><td>19.70%</td><td>1</td><td>1</td><td>1</td><td>1</td><td>66</td><td>7.6</td><td>7.88</td></tr><tr><td></td><td>D5HR50</td><td>Alpha-toxin Ac1 (Fragment) OS = <i>Androctonus crassicauda</i> PE = 2 SV = 1 - [SCX1A_ANDCR]</td><td>37.66</td><td>11.11%</td><td>2</td><td>1</td><td>1</td><td>2</td><td>81</td><td>8.7</td><td>7.55</td></tr><tr><td></td><td>M1JMR8</td><td>Sodium channel alpha-toxin Acra8 OS = <i>Androctonus crassicauda</i> PE = 3 SV = 1 - [SCX8_ANDCR]</td><td>66.82</td><td>40.91%</td><td>3</td><td>2</td><td>3</td><td>5</td><td>66</td><td>7.5</td><td>8.29</td></tr><tr><td></td><td>M1JBC0</td><td>Sodium channel alpha-toxin Acra4 OS = <i>Androctonus crassicauda</i> PE = 1 SV = 1 - [SCX4_ANDCR]</td><td>37.39</td><td>29.23%</td><td>1</td><td>1</td><td>2</td><td>4</td><td>65</td><td>7.1</td><td>8.31</td></tr></tbody></table>
<table><caption>**Table 3. (Continued).**</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Score</th><th>Coverage</th><th>No. of proteins</th><th>No. of unique peptides</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of AAs</th><th>MW [kDa]</th><th>calc. pI</th></tr></thead><tbody><tr><td rowspan="5">KScTx</td><td>P0C161</td><td>Potassium channel toxin alpha-KTx 2.8<br>OS = <i>Centruroides elegans</i> PE = 1<br>SV = 1 - [KAX28_CENEL]</td><td>45.57</td><td>17.95%</td><td>2</td><td>1</td><td>1</td><td>1</td><td>39</td><td>4.3</td><td>8.94</td></tr><tr><td><u>O9NJC6</u></td><td>Potassium channel toxin BmTXK-beta<br>OS = <i>Mesobuthus martensii</i> PE = 2<br>SV = 1 - [KBX2_MESMA]</td><td>264.78</td><td>23.33%</td><td>2</td><td>1</td><td>2</td><td>6</td><td>90</td><td>10.4</td><td>8.82</td></tr><tr><td>P59869</td><td>Potassium channel toxin alpha-KTx 5.4<br>OS = <i>Mesobuthus tamulus</i> PE = 1<br>SV = 1 - [KAX54_MESTA]</td><td>40.7</td><td>22.58%</td><td>2</td><td>1</td><td>2</td><td>2</td><td>31</td><td>3.5</td><td>8.02</td></tr><tr><td>B8XH40</td><td>Potassium channel toxin ButTXK-beta<br>OS = <i>Buthus occitanus israelis</i> PE = 2<br>SV = 1 - [KBX1_BUTOS]</td><td>298.65</td><td>42.86%</td><td>2</td><td>2</td><td>5</td><td>18</td><td>91</td><td>10.2</td><td>8.57</td></tr><tr><td><u>O9N661</u></td><td>Potassium channel toxin BmTXK-beta-2<br>OS = <i>Mesobuthus martensii</i> PE = 2<br>SV = 1 - [KBX1_MESMA]</td><td>230.62</td><td>42.86%</td><td>2</td><td>1</td><td>4</td><td>13</td><td>91</td><td>10.2</td><td>8.57</td></tr><tr><td rowspan="5">CIScTx</td><td>B3EWX9</td><td>Potassium channel toxin alpha-KTx 9.11<br>OS = <i>Mesobuthus gibbosus</i> PE = 1<br>SV = 1 - [KAX9B_MESGB]</td><td>85.33</td><td>40.74%</td><td>4</td><td>1</td><td>1</td><td>2</td><td>27</td><td>2.9</td><td>5.01</td></tr><tr><td>B8XH42</td><td>Potassium channel toxin alpha-KTx 16.6<br>OS = <i>Buthus occitanus israelis</i> PE = 2<br>SV = 1 - [KA166_BUTOS]</td><td>23.25</td><td>12.07%</td><td>1</td><td>1</td><td>1</td><td>1</td><td>58</td><td>6.5</td><td>8.12</td></tr><tr><td><u>P45639</u></td><td>Chlorotoxin OS = <i>Leiorus quinquestriatus</i><br><i>quinquestriatus</i> PE = 1 SV = 1 - [CTXL_LEIQU]</td><td>41.56</td><td>38.89%</td><td>1</td><td>1</td><td>1</td><td>3</td><td>36</td><td>4</td><td>8.13</td></tr><tr><td><u>P86436</u></td><td>Chlorotoxin-like peptide OS = <i>Androctonus australis</i> PE = 1 SV = 1 - [CTXL_ANDAU]</td><td>290.9</td><td>44.12%</td><td>1</td><td>1</td><td>1</td><td>7</td><td>34</td><td>3.6</td><td>8.34</td></tr></tbody></table>

Underlined peptide entries were identified by in-gel and in-solution digestion methods.
<table><caption>**Table 4.** Bottom-up data generated from in-solution digestion of *B. occitanus* venom filtrate using nano-LC-MS/MS. Data sets generated from the mass spectrometer were analyzed by the PROTEOME DISCOVER 2.2 software, against UniProtKB/Swiss-Prot database.</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Score</th><th>Coverage</th><th>No. of proteins</th><th>No. of unique peptides</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of AAs</th><th>MW (kDa)</th><th>calc. pI</th></tr></thead><tbody><tr><td rowspan="14">NaScTxs</td><td><u>O86SE0</u></td><td>Toxin Aam2 OS = Androctonus<br>amoreuxi PE = 1 SV = 1 -<br>[SCX2_ANDAM]</td><td>250.68</td><td>24.42%</td><td>8</td><td>2</td><td>4</td><td>15</td><td>86</td><td>9.3</td><td>7.87</td></tr><tr><td><u>P21150</u></td><td>Toxin AaHIT4 OS = Androctonus<br>australis PE = 1 SV = 1 -<br>[SIX4_ANDAU]</td><td>192.23</td><td>30.77%</td><td>2</td><td>2</td><td>3</td><td>12</td><td>65</td><td>7.8</td><td>8.46</td></tr><tr><td>P01482</td><td>Alpha-toxin Amm5<br>OS = Androctonus mauretanicus<br>mauretanicus PE = 1 SV = 1 -<br>[SCX5_ANDMA]</td><td>96.57</td><td>28.13%</td><td>1</td><td>1</td><td>1</td><td>2</td><td>64</td><td>7.3</td><td>7.5</td></tr><tr><td>P01481</td><td>Alpha-mammal toxin Lq5<br>OS = Leiurus quinquestratus<br>quinquestratus PE = 1 SV = 1 -<br>[SCX5_LEIQU]</td><td>77.71</td><td>25.00%</td><td>2</td><td>1</td><td>2</td><td>4</td><td>64</td><td>7.3</td><td>8.1</td></tr><tr><td><u>P13488</u></td><td>Alpha-like toxin Bom3 OS = Buthus<br>occitanus mardochei PE = 1<br>SV = 1 - [SCX3_BUTOM]</td><td>155.6</td><td>59.09%</td><td>2</td><td>2</td><td>4</td><td>18</td><td>66</td><td>6.9</td><td>6.71</td></tr><tr><td>P45698</td><td>Neurotoxin Bmk-M9<br>OS = Mesobuthus martensii<br>PE = 1 SV = 1 - [SCX9_MESMA]</td><td>124.54</td><td>26.58%</td><td>11</td><td>1</td><td>3</td><td>15</td><td>79</td><td>8.8</td><td>7.88</td></tr><tr><td><u>P68721</u></td><td>Beta-insect excitatory toxin<br>LqhTTla OS = Leiurus<br>quinquestratus hebraeus PE = 3<br>SV = 1 - [SIX1A_LEIQH]</td><td>55.48</td><td>10.23%</td><td>2</td><td>1</td><td>2</td><td>4</td><td>88</td><td>9.9</td><td>8.09</td></tr><tr><td><u>P0DJH8</u></td><td>Alpha-toxin Bu1 OS = Buthacus<br>macrocentrus PE = 1 SV = 1 -<br>[SCX1_BUTMA]</td><td>272.84</td><td>71.64%</td><td>1</td><td>2</td><td>4</td><td>9</td><td>67</td><td>7.5</td><td>8.48</td></tr><tr><td>P83644</td><td>Toxin Lqh4 OS = Leiurus<br>quinquestratus hebraeus PE = 1<br>SV = 1 - [SCX4_LEIQH]</td><td>293.2</td><td>46.15%</td><td>7</td><td>1</td><td>3</td><td>10</td><td>65</td><td>7.2</td><td>8.1</td></tr><tr><td><u>P01489</u></td><td>Alpha-toxin Lq4 OS = Leiurus<br>quinquestratus quinquestratus<br>PE = 1 SV = 1 - [SCX4_LEIQU]</td><td>569.9</td><td>90.77%</td><td>8</td><td>2</td><td>6</td><td>18</td><td>65</td><td>7.2</td><td>8.1</td></tr><tr><td><u>P01486</u></td><td>Alpha-toxin Bot11 OS = Buthus<br>occitanus tunetanus PE = 1<br>SV = 1 - [SCXB_BUTOC]</td><td>76.63</td><td>35.38%</td><td>2</td><td>1</td><td>3</td><td>7</td><td>65</td><td>7.5</td><td>7.87</td></tr><tr><td><u>P60255</u></td><td>Toxin Boma6a OS = Buthus<br>occitanus mardochei PE = 3<br>SV = 1 - [SCXA_BUTOM]</td><td>46.01</td><td>15.15%</td><td>2</td><td>1</td><td>1</td><td>2</td><td>66</td><td>7.5</td><td>7.09</td></tr></tbody></table>
<table><caption>**Table 4.** (Continued).</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Score</th><th>Coverage</th><th>No. of proteins</th><th>No. of unique peptides</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of AAs</th><th>MW (kDa)</th><th>calc. pI</th></tr></thead><tbody><tr><td></td><td>P17728</td><td>Alpha-insect toxin Lqhait OS = Leiurus quinquestriatus hebraeus PE = 1 SV = 2 - [SCXA_LEIQH]</td><td>369.61</td><td>51.76%</td><td>4</td><td>2</td><td>5</td><td>13</td><td>85</td><td>9.6</td><td>8.12</td></tr><tr><td></td><td>P04098</td><td>Neurotoxin 8 (Fragment) OS = Buthus occitanus tunetanus PE = 1 SV = 1 - [SCX8_BUTOC]</td><td>536.34</td><td>77.78%</td><td>2</td><td>3</td><td>3</td><td>13</td><td>36</td><td>4.1</td><td>6.24</td></tr><tr><td></td><td>P55902</td><td>Alpha-insect toxin BotIT1 OS = Buthus occitanus tunetanus PE = 1 SV = 1 - [SIX1_BUTOC]</td><td>296.35</td><td>61.54%</td><td>1</td><td>1</td><td>3</td><td>9</td><td>65</td><td>7.3</td><td>7.55</td></tr><tr><td></td><td>P01488</td><td>Alpha-toxin Bot1 OS = Buthus occitanus tunetanus PE = 1 SV = 2 - [SCX1_BUTOC]</td><td>185.35</td><td>20.00%</td><td>1</td><td>1</td><td>1</td><td>3</td><td>65</td><td>7.3</td><td>6.92</td></tr><tr><td></td><td>P81504</td><td>Insect toxin AaHIT5 OS = Androctonus australis PE = 1 SV = 1 - [SIX5_ANDAU]</td><td>49.42</td><td>24.59%</td><td>1</td><td>1</td><td>1</td><td>2</td><td>61</td><td>6.9</td><td>4.83</td></tr><tr><td></td><td>P01485</td><td>Alpha-mammal toxin Bot3 (Fragment) OS = Buthus occitanus tunetanus PE = 1 SV = 2 - [SCX3_BUTOC]</td><td>436.17</td><td>61.11%</td><td>3</td><td>2</td><td>5</td><td>61</td><td>72</td><td>8.1</td><td>7.53</td></tr><tr><td></td><td>P59863</td><td>Beta-toxin BotIT2 OS = Buthus occitanus tunetanus PE = 1 SV = 1 - [SIX2_BUTOC]</td><td>164.41</td><td>41.67%</td><td>1</td><td>2</td><td>2</td><td>4</td><td>60</td><td>6.9</td><td>4.84</td></tr><tr><td></td><td>Q17254</td><td>Alpha-insect toxin Bot14 OS = Buthus occitanus tunetanus PE = 2 SV = 1 - [SCXE_BUTOC]</td><td>91.78</td><td>18.82%</td><td>1</td><td>1</td><td>1</td><td>9</td><td>85</td><td>9.2</td><td>8.5</td></tr><tr><td></td><td>O77091</td><td>Beta-insect excitatory toxin BmK IT-AP OS = Mesobuthus martensii GN = IT-AP PE = 1 SV = 1 - [SIXP_MESMA]</td><td>50.93</td><td>17.78%</td><td>8</td><td>1</td><td>2</td><td>5</td><td>90</td><td>10.2</td><td>5.36</td></tr><tr><td></td><td>P59864</td><td>Beta-insect depressant toxin BotIT6 OS = Buthus occitanus tunetanus PE = 1 SV = 1 - [SIX6_BUTOC]</td><td>78.83</td><td>53.23%</td><td>1</td><td>2</td><td>3</td><td>7</td><td>62</td><td>7.3</td><td>8.1</td></tr><tr><td></td><td>P0C294</td><td>Toxin AcrA I-3 OS = Androctonus crassicauda PE = 2 SV = 1 - [TX13_ANDCR]</td><td>43.58</td><td>8.75%</td><td>1</td><td>1</td><td>1</td><td>1</td><td>80</td><td>8.8</td><td>8.25</td></tr><tr><td></td><td>P59360</td><td>Neurotoxin BmK-II OS = Mesobuthus martensii PE = 1 SV = 1 - [SCX2_MESMA]</td><td>62.88</td><td>15.63%</td><td>3</td><td>1</td><td>1</td><td>2</td><td>64</td><td>7.2</td><td>7.09</td></tr></tbody></table>
<table><caption>**Table 4.** (Continued).</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>Score</th><th>Coverage</th><th>No. of proteins</th><th>No. of unique peptides</th><th>No. of peptides</th><th>No. of PSMs</th><th>No. of AAs</th><th>MW (kDa)</th><th>calc. pI</th></tr></thead><tbody><tr><td rowspan="10">KScTxs</td><td rowspan="2">P0CC12</td><td>Potassium channel toxin alpha-KTx</td><td>86.25</td><td>48.28%</td><td>2</td><td>1</td><td>1</td><td>2</td><td>29</td><td>3.2</td><td>5.1</td></tr><tr><td>8.5 OS = Odontobuthus doriae<br>PE = 1 SV = 1 - [KAX85_OODO]</td><td>82.22</td><td>32.26%</td><td>1</td><td>1</td><td>1</td><td>5</td><td>31</td><td>3.3</td><td>8.73</td></tr><tr><td rowspan="2">P83407</td><td>Potassium channel toxin alpha-KTx</td><td>19.1 OS = Mesobuthus martensii</td><td>79.79</td><td>16.36%</td><td>1</td><td>1</td><td>1</td><td>5</td><td>55</td><td>6.2</td><td>8</td></tr><tr><td>PE = 1 SV = 1 - [KA191_MESMA]</td><td>17.1 OS = Mesobuthus martensii</td><td>211.78</td><td>92.86%</td><td>3</td><td>2</td><td>2</td><td>9</td><td>28</td><td>3</td><td>6.98</td></tr><tr><td rowspan="2">Q95NJ8</td><td>Potassium channel toxin alpha-KTx</td><td>9.3 OS = Leiurus quinquestriatus</td><td>135.05</td><td>27.78%</td><td>2</td><td>2</td><td>2</td><td>3</td><td>90</td><td>10.4</td><td>8.82</td></tr><tr><td>hebraeus PE = 1 SV = 1 - [KAX93_LEIQH]</td><td>96.9</td><td>42.86%</td><td>3</td><td>2</td><td>4</td><td>7</td><td>91</td><td>10.2</td><td>8.57</td></tr><tr><td rowspan="2">Q9NJC6</td><td>Potassium channel toxin BmTXK-<br>beta OS = Mesobuthus martensii</td><td>86.02</td><td>48.28%</td><td>2</td><td>1</td><td>1</td><td>2</td><td>29</td><td>3.2</td><td>5.1</td></tr><tr><td>PE = 2 SV = 1 - [KBX2_MESMA]</td><td>Neurotoxin lamda-Meutx</td><td>262</td><td>25.00%</td><td>2</td><td>1</td><td>1</td><td>8</td><td>64</td><td>7.2</td><td>7.12</td></tr><tr><td rowspan="2">Q9N661</td><td>Potassium channel toxin BmTXK-<br>beta-2 OS = Mesobuthus<br>martensii PE = 2 SV = 1 - [KBX1_MESMA]</td><td>287.63</td><td>60.71%</td><td>1</td><td>2</td><td>2</td><td>10</td><td>28</td><td>2.9</td><td>4.89</td></tr><tr><td>Neurotoxin lamda-Meutx</td><td>OS = Mesobuthus eupeus PE = 1<br>SV = 2 - [TXL_MESEU]</td><td>993.18</td><td>67.65%</td><td>1</td><td>3</td><td>3</td><td>65</td><td>34</td><td>3.6</td><td>8.34</td></tr><tr><td rowspan="2">P80670</td><td>Toxin GaTx2 OS = Leiurus<br>quinquestriatus hebraeus PE = 1</td><td>Neurotoxin Tx-2 OS = Buthotus</td><td>288.38</td><td>38.89%</td><td>1</td><td>2</td><td>2</td><td>38</td><td>36</td><td>4</td><td>8.13</td></tr><tr><td>SV = 1 - [KAX83_LEIQH]</td><td>Neurotoxin Tx-2 OS = Buthotus<br>judaicus PE = 1 SV = 1 - [SCBT2_BUTJU]</td><td>188.48</td><td>71.43%</td><td>1</td><td>2</td><td>2</td><td>5</td><td>35</td><td>3.7</td><td>7.88</td></tr><tr><td rowspan="2">P83406</td><td>Neurotoxin P2 OS = Androctonus</td><td>Chlorotoxin-like peptide</td><td>87.27</td><td>19.40%</td><td>4</td><td>1</td><td>1</td><td>1</td><td>67</td><td>7.8</td><td>9.19</td></tr><tr><td>mauretanicus mauretanicus</td><td>OS = Androctonus australis</td><td>Chlorotoxin OS = Leiurus</td><td>88.38</td><td>38.89%</td><td>1</td><td>2</td><td>2</td><td>38</td><td>36</td><td>4</td><td>8.13</td></tr><tr><td rowspan="2">CIScTxs</td><td rowspan="2">P86436</td><td>Neurotoxin P2 OS = Androctonus</td><td>Neurotoxin P2 OS = Androctonus</td><td>88.38</td><td>38.89%</td><td>1</td><td>2</td><td>2</td><td>38</td><td>36</td><td>4</td><td>8.13</td></tr><tr><td>mauretanicus mauretanicus</td><td>PE = 1 SV = 1 - [SCXL_LEIQU]</td><td>Neurotoxin P2 OS = Androctonus</td><td>OS = Mesobuthus martensii</td><td>188.48</td><td>71.43%</td><td>1</td><td>2</td><td>2</td><td>5</td><td>35</td><td>3.7</td><td>7.88</td></tr><tr><td rowspan="2">B8XH50</td><td rowspan="2">B8XH50</td><td>Amphipathic peptide Tx348</td><td>OS = Buthus occitanus israelis</td><td>87.27</td><td>19.40%</td><td>4</td><td>1</td><td>1</td><td>1</td><td>67</td><td>7.8</td><td>9.19</td></tr><tr><td>PE = 2 SV = 1 - [NDB5R_BUTOS]</td><td>OS = Buthus occitanus israelis</td><td>Neurotoxin P2 OS = Androctonus</td><td>OS = Mesobuthus martensii</td><td>88.38</td><td>38.89%</td><td>1</td><td>2</td><td>2</td><td>38</td><td>36</td><td>4</td><td>8.13</td></tr></tbody></table>

Underlined peptide entries were identified by in-gel and in-solution digestion methods.
<table><caption>**Table 5.** List of the 50 peptides detected by the bottom-up analysis of the reduced/alkylated *B. occitanus* venom filtrate. Data sets generated from the mass spectrometer were analyzed by the PROTEOME DISCOVER 2.2 software, against UniProtKB/Swiss-Prot database.</caption><thead><tr><th>Category</th><th>Accession</th><th>Description</th><th>MW (kDa)</th><th>Species</th><th>Digestion method</th></tr></thead><tbody><tr><td rowspan="20">NaScTx</td><td>P86406</td><td>Neurotoxin MeuNaTx-6</td><td>7.8</td><td>Mesobuthus eupeus</td><td>In-gel digestion</td></tr><tr><td><b>P59863</b></td><td>Beta-toxin BotIT2</td><td>6.9</td><td>Buthus occitanus tunetanus</td><td>Both</td></tr><tr><td>D5HR52</td><td>Alpha-toxin Ac3 (Fragment)</td><td>7.8</td><td>Androctonus crassicauda</td><td>In-gel digestion</td></tr><tr><td>P55904</td><td>Beta-insect depressant toxin BotIT5</td><td>6.8</td><td>Buthus occitanus tunetanus</td><td>In-gel digestion</td></tr><tr><td><b>O77091</b></td><td>Beta-insect excitatory toxin BmK IT-AP</td><td>10.2</td><td>Mesobuthus martensii</td><td>Both</td></tr><tr><td>P68723</td><td>Beta-insect excitatory toxin LqhIT1c</td><td>9.9</td><td>Leiurus quinquestriatus hebraeus</td><td>In-gel digestion</td></tr><tr><td><b>P59360</b></td><td>Neurotoxin BmK-II</td><td>7.2</td><td>Mesobuthus martensii</td><td>Both</td></tr><tr><td>P15224</td><td>Toxin Os1</td><td>7.6</td><td>Orthochirus scrobiculosus</td><td>In-gel digestion</td></tr><tr><td>D5HR50</td><td>Alpha-toxin Ac1 (Fragment)</td><td>8.7</td><td>Androctonus crassicauda</td><td>In-gel digestion</td></tr><tr><td><b>M1JMR8</b></td><td>Sodium channel alpha-toxin Acra8</td><td>7.5</td><td>Androctonus crassicauda</td><td>Both</td></tr><tr><td>M1JBC0</td><td>Sodium channel alpha-toxin Acra4</td><td>7.1</td><td>Androctonus crassicauda</td><td>In-gel digestion</td></tr><tr><td>Q86SE0</td><td>Toxin Aam2</td><td>9.3</td><td>Androctonus amoreuxi</td><td>Both</td></tr><tr><td><b>P21150</b></td><td>Toxin AaHIT4</td><td>7.8</td><td>Androctonus australis</td><td>Both</td></tr><tr><td>P01482</td><td>Alpha-toxin Amm5</td><td>7.3</td><td>Androctonus mauretanicus<br>mauretanicus</td><td>In-solution digestion</td></tr><tr><td>P01481</td><td>Alpha-mammal toxin Lqq5</td><td>7.3</td><td>Leiurus quinquestriatus<br>quinquestriatus</td><td>In-solution digestion</td></tr><tr><td><b>P13488</b></td><td>Alpha-like toxin Bom3</td><td>6.9</td><td>Buthus occitanus mardochei</td><td>Both</td></tr><tr><td><b>P45698</b></td><td>Neurotoxin BmK-M9</td><td>8.8</td><td>Mesobuthus martensii</td><td>In-solution digestion</td></tr><tr><td>P68721</td><td>Beta-insect excitatory toxin LqhIT1a</td><td>9.9</td><td>Leiurus quinquestriatus hebraeus</td><td>Both</td></tr><tr><td>P0DJH8</td><td>Alpha-toxin Bu1</td><td>7.5</td><td>Buthacus macrocentrus</td><td>Both</td></tr><tr><td><b>P83644</b></td><td>Toxin Lqh4</td><td>7.2</td><td>Leiurus quinquestriatus hebraeus</td><td>Both</td></tr><tr><td>P01489</td><td>Alpha-toxin Lqq4</td><td>7.2</td><td>Leiurus quinquestriatus<br>quinquestriatus</td><td>Both</td></tr><tr><td rowspan="13">KScTx</td><td>P01486</td><td>Alpha-toxin Bot11</td><td>7.5</td><td>Buthus occitanus tunetanus</td><td>In-solution digestion</td></tr><tr><td><b>P60255</b></td><td>Toxin Boma6a</td><td>7.5</td><td>Buthus occitanus mardochei</td><td>Both</td></tr><tr><td><b>P17728</b></td><td>Alpha-insect toxin LqhIT</td><td>9.6</td><td>Leiurus quinquestriatus hebraeus</td><td>Both</td></tr><tr><td>P04098</td><td>Neurotoxin 8 (Fragment)</td><td>4.1</td><td>Buthus occitanus tunetanus</td><td>Both</td></tr><tr><td><b>P55902</b></td><td>Alpha-insect toxin BotIT1</td><td>7.3</td><td>Buthus occitanus tunetanus</td><td>Both</td></tr><tr><td><b>P01488</b></td><td>Alpha-toxin Bot1</td><td>7.3</td><td>Buthus occitanus tunetanus</td><td>Both</td></tr><tr><td><b>P81504</b></td><td>Insect toxin AaHIT5</td><td>6.9</td><td>Androctonus australis</td><td>Both</td></tr><tr><td><b>P01485</b></td><td>Alpha-mammal toxin Bot3 (Fragment)</td><td>8.1</td><td>Buthus occitanus tunetanus</td><td>In-solution digestion</td></tr><tr><td>P83406</td><td>Neurotoxin Tx-2</td><td>2.9</td><td>Buthotus judaicus</td><td>In-solution digestion</td></tr><tr><td><b>Q17254</b></td><td>Alpha-insect toxin Bot14</td><td>9.2</td><td>Buthus occitanus tunetanus</td><td>Both</td></tr><tr><td>P59864</td><td>Beta-insect depressant toxin BotIT6</td><td>7.3</td><td>Buthus occitanus tunetanus</td><td>In-solution digestion</td></tr><tr><td>P0C294</td><td>Toxin Acra I-3</td><td>8.8</td><td>Androctonus crassicauda</td><td>In-solution digestion</td></tr><tr><td>B3EWX9</td><td>Potassium channel toxin alpha-KTx 9.11</td><td>2.9</td><td>Mesobuthus gibbosus</td><td>In-gel digestion</td></tr><tr><td>P0C161</td><td>Potassium channel toxin alpha-KTx 2.8</td><td>4.3</td><td>Centruroides elegans</td><td>In-gel digestion</td></tr><tr><td>B8XH42</td><td>Potassium channel toxin alpha-KTx 16.6</td><td>6.5</td><td>Buthus occitanus israelis</td><td>Both</td></tr><tr><td>P0CC12</td><td>Potassium channel toxin alpha-KTx 8.5</td><td>3.2</td><td>Odontobuthus doriae</td><td>In-solution digestion</td></tr><tr><td>P59869</td><td>Potassium channel toxin alpha-KTx 5.4</td><td>3.5</td><td>Mesobuthus tamulus</td><td>In-gel digestion</td></tr><tr><td>B8XH40</td><td>Potassium channel toxin BuTXK-beta</td><td>10.2</td><td>Buthus occitanus israelis</td><td>In-gel digestion</td></tr><tr><td>Q95NJ8</td><td>Potassium channel toxin alpha-KTx 17.1</td><td>6.2</td><td>Odontobuthus doriae</td><td>In-solution digestion</td></tr><tr><td>P83407</td><td>Potassium channel toxin alpha-KTx 19.1</td><td>3.3</td><td>Mesobuthus martensii</td><td>in-solution digestion</td></tr><tr><td>P80669</td><td>Potassium channel toxin alpha-KTx 9.3</td><td>3</td><td>Leiurus quinquestriatus hebraeus</td><td>In-solution digestion</td></tr><tr><td>P86399</td><td>Neurotoxin lamda-MeuTx</td><td>7.2</td><td>Mesobuthus eupeus</td><td>In-solution digestion</td></tr><tr><td>Q9NJC6</td><td>Potassium channel toxin BmTXK-beta</td><td>10.4</td><td>Mesobuthus martensii</td><td>Both</td></tr><tr><td><b>Q9N661</b></td><td>Potassium channel toxin BmTXK-beta-2</td><td>10.2</td><td>Mesobuthus martensii</td><td>Both</td></tr><tr><td>P01498</td><td>Neurotoxin P2</td><td>3.7</td><td>Androctonus mauretanicus<br>mauretanicus</td><td>In-solution digestion</td></tr><tr><td rowspan="2">CIscTx</td><td>P86436</td><td>Chlorotoxin-like peptide</td><td>3.6</td><td>Androctonus australis</td><td>Both</td></tr><tr><td>P45639</td><td>Chlorotoxin</td><td>4</td><td>Leiurus quinquestriatus<br>quinquestriatus</td><td>Both</td></tr><tr><td rowspan="2">Amphipathic<br>peptide</td><td>P80670</td><td>Toxin GaTx2</td><td>3.2</td><td>Leiurus quinquestriatus hebraeus</td><td>In-solution digestion</td></tr><tr><td>B8XH50</td><td>Amphipathic peptide Tx348</td><td>7.8</td><td>Buthus occitanus israelis</td><td>In-solution digestion</td></tr></tbody></table>

Peptide entries in bold were identified by both top-down and bottom-up approaches.
AMPs (1%), amphipathic peptides (1%), hypothetical secreted proteins (1%), and myotropic neuropeptides (1%) (Fig. 6).

The majority of described peptides were identified for the first time in this Moroccan *B. occitanus*

scorpion venom. The identified peptides showed sequence similarities with toxins previously detected from several genera of scorpions (Fig. 7), principally Mesobuthus sp (30%), Buthus Sp (20%), and Androctonus sp (18%).

<figure><img src="image_17.png" /><figcaption>**Fig. 6.** Summary of the total peptides identified by top-down and bottom-up approaches. The 102 peptides were divided into neurotoxins, including NaScTxs, KScTxs, CIScTxs, CaScTx and toxin Acra, amphipathic peptide, myotropic neuropeptide, AMPs, and hypothetical secreted protein.</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 7.** Percentage of *B. occitanus* peptides, which showed similarity of sequences with others from several scorpion genera.</figcaption></figure>
Discussion

Envenomation following scorpion stings constitutes one of the most encountered emergencies in large parts of the world, especially in North Africa, where the data show the highest incidence and lethality [1]. Morocco is a country known for a high risk of envenomation owing to its huge and diversified scorpion fauna. Among the different scorpion species living in this country, the yellow scorpion *B. occitanus* is one of the most dangerous species with venom responsible for severe cases of envenomation.

Due to the limited knowledge about the composition and toxin arsenal of *B. occitanus* venom, we aimed in this study to elaborate the first exhaustive view of this scorpion venom peptidome and its molecular diversity, using mass spectrometry-based top-down and bottom-up approaches.

Top-down data sets showed that the venom of *B. occitanus* is very complex, counting around 200 MWs ranging from 1868 to 16 720 Da. A similar number of components have been revealed by previous studies [32–34], others showed fewer components, as well as *Leiurus abdullahbayrami* (45 masses) and *Opisthacanthus elatus* (106 masses) [35, 36], whereas some other scorpion venoms were more complex, such as the *Pandinus cavimanus* (390 masses) and *Centruroides limpidus* (395 masses) [37, 38]. Additionally, the repartition of MWs showed that < 1% were components with molecular masses < 2000 Da, 14% were those from 2000 to 5000 Da, 74% were those between 5000 and 8000 Da, and 10% were those over than 8000 Da, while the repartition of MW from the French *B. occitanus* scorpion venom showed an abundance of molecules ranging from 2000 to 3000 Da and those less than 2000 Da [39]. Most importantly, the whole sequences of five toxins were identified with 100% sequence coverage using the top-down approach. These neurotoxins were detected for the first time in this venom; they all belong to the NaScTxs category and shared high similarities of sequence with toxins identified from other scorpion species: neurotoxin BmK-II (P59360), beta-insect depressant toxin BotIT4 (P55903), beta-insect depressant toxin BaIT2 (P80962), insect toxin LqhIT5 (P81240), and insect toxin BsIT4 (P82814). It is important to stress that the observed sequence of the P59360 entry with a MW of 7431.33 Da showed 100% similarity with the sequence of neurotoxin BmK-II isolated from the Chinese scorpion *Mesobuthus martensii*, this neurotoxin is active in mammal and insect Nav channel [40]. In contrast, the detected sequence of the P81240 entry (6611.8 Da)

showed the presence of methionine in the N-terminal compared with the database sequence of the Insect toxin LqhIT5, an excitatory insect beta-toxin from the *Leiurus hebraeus* scorpion [41]. Similar to the P82814 entry (6954.15 Da), in which the observed sequence corresponds 100% to the insect toxin BsIT4, a depressant insect beta-toxins was isolated from *Hottentotta tamulus sindicus* [42]. Also, the observed sequence of the peptide corresponding to the depressant toxin BotIT4 (6837. 96 Da) presents methionine in N-terminal compared with the database sequence. This toxin, identified for the first time from the Tunisian *Buthus tunetanus* [43], showed also 100% sequence identity with the P80962 entry (6845.9 Da), referred to the beta-insect depressant toxin BaIT2 isolated from the *Buthacus arenicola* scorpion [44]. The high similarity of the amino acid sequence, in both detected depressant toxins and in the other peptides is commonly observed in scorpion toxins.

Interestingly, the combined top-down and bottom-up data sets of *B. occitanus* venom provide the identification of 102 different peptides, whereas 147 proteins were characterized from the yellow Brazilian scorpion *Tityus serrulatus*, 60 of which were detected by the top-down approach [45]. The major representative category of components identified in our venom was neurotoxins, mainly NaScTxs (77%), these neurotoxins are abundant in species from the Buthidae family [38,46,47] and less representative in scorpions from the non-Buthidae family [33,48,49]. Those toxins are the ones responsible for envenomation symptoms [39]; their high content in the *B. occitanus* venom could explain the involvement of this scorpion in lethal cases of envenoming in the country.

Between the entries corresponding to NaScTxs, there are alpha-like toxins, this type of toxins had been already identified in several Buthus sp; yet, the alpha-toxin Bot1 (P01488) has never been found in other Moroccan Buthus subspecies except from *Buthus mar-doehei* [39,50–53], but identified herein with a high sequence coverage (98.48% on top-down data set). We should mention also that we identified for the first time, in this scorpion venom, peptides corresponding to atypical NaScTxs, as well as makatoxin-1, fragment from makatoxin-2, toxin Cg2, chain [20-87] in venom toxin meuNa32, and AaHIT4 toxin (which could bind on receptor site 3 or 4 of sodium channel) [33].

Besides NaScTxs and KScTxs (14%), ClScTxs (3%) were identified, these categories of peptides showed activities against autoimmune disease and cancers, respectively [54–58]; also, we identified one entry that shared 60% of similarity with neurotoxin Tx-2 (P83406), a calcium channel activator identified for the
first time from the *Buthotus judaicus*, this category of toxins was identified in few scorpion species, for example, *Parabuthus transvaalicus* (Kurtoxin) and *Parabuthus granulatus* (Kurtoxin-like I) but never been detected in a Moroccan scorpion venom [59, 60]. And last but not least, peptides referring to toxin Acra category have also been screened in *B. occitanus* venom, these toxins probably acting on ion channels.

Some peptides with antibacterial activities were also found, for example, amphipathic peptide (B8XH50) and AMP AcrAP1 (A0A059UI30); this category was commonly present in scorpion venom due to its role in the protection of venom glands and its involvement in the neurotoxic effects [61–65]. Additionally, other components were identified with a low percentage, such as orcokinin, a myotropic neuropeptide identified from crustaceans, insects, and arachnids [17, 66], and hypothetical secreted proteins, which are proteins with unknown activities. Finally, we notice that some of the detected toxins were identified as fragments and chains, which may be due to the proteolysis of toxins. This process seems to be a usual PTM in scorpion and snake venoms, whereas its biological pertinence remains obscure [17, 45].

This study decrypted the peptidome arsenal of the Moroccan *B. occitanus* scorpion venom through proteomic view without the *de novo* sequence annotation. These findings constitute a step forward to a 'deeper' understanding of this scorpion venom; nevertheless, complete identification of this complex matrix is still a challenging task, especially with the lack of a specific database and/or a complete sequenced genome of this venom.

## Conclusion

Herein; we reported the first proteomic study of the Moroccan *B. occitanus* scorpion peptidome, using mass spectrometry-based top-down and bottom-up venomic approaches. The combination of these approaches allowed the identification of 102 components classified, with approximation, on different categories, mainly neurotoxins (96%), including NaScTxs (77%), KScTxs (14%), ClScTxs (3%), CaScTx (1%), and toxin Acra (1%). We also identified AMPs (1%), amphipathic peptides (1%), hypothetical secreted proteins (1%), and myotropic neuropeptides (1%). This study constitutes for sure a step forward to a deeper understanding of the *B. occitanus* venom; nevertheless, complete identification of this complex matrix is still a challenging task, especially with the lack of a specific database and a complete sequenced genome.

## Acknowledgements

We declare that this study received financial support from the French government through the grant No 870634D.

## Conflict of interest

The authors declare no conflict of interest.

## Data Accessibility

All generated data during this study are included in this article.

## Author contributions

NO and JCR conceived the research. KD and CM performed experiments. KD and CM analyzed the data. KD interpreted data and wrote the manuscript. AL, BD, and SC participated in writing. JMS and RC reviewed the manuscript. NO designed the project, supervised the study, and reviewed the manuscript. All authors read and approved the final version for publication.

## References

1 Chippaux JP (2012) Emerging options for the management of scorpion stings. *Drug Des Devel Ther* 6, 165-173.

2 Sunagar K, Undheim EA, Chan AH, Koludarov I, Muñoz-Gómez SA, Antunes A and Fry BG (2013) Evolution stings: the origin and diversification of scorpion toxin peptide scaffolds. *Toxins* 12, 2456-2487.

3 Possani LD, Becerril B, Delepierre M and Tytgat J (1999) Scorpion toxins specific for Na⁺ channels. *Eur J Biochem* 2, 287-300.

4 Rodríguez de la Vega RC and Possani LD (2005) Overview of scorpion toxins specific for Na⁺ channels and related peptides: biodiversity, structure-function relationships and evolution. *Toxicon* 8, 831-844.

5 Almaaytah A, Zhou M, Wang L, Chen T, Walker B and Shaw C (2012) Antimicrobial/cytolytic peptides from the venom of the North African scorpion, *Androctonus amoreuxi*: biochemical and functional characterization of natural peptides and a single site-substituted analog. *Peptides* 2, 291-299.

6 Cao L, Dai C, Li Z, Fan Z, Song Y, Wu Y, Cao Z and Li W (2012) Antibacterial activity and mechanism of a scorpion venom peptide derivative *in vitro* and *in vivo*. *PLoS One* 7, e40135.

7 El Oufir R (2019) Piqûres et Envenimations Scorpioniques (PES) Centre Anti Poison du Maroc
(CAPM). Rapports général et spécifiques. Revue toxicologie Maroc **43**:11.

8 Touloun O, Slimani T and Boumezzough A (2001) Epidemiological survey of scorpion envenomation in Southwestern Morocco. *J Venom Anim Toxins Incl Trop Dis* **7**, 99–218.

9 Ghalim N, Ghalim N, Sebti F, El-Hafny B, Lazar N, Moustanir R and Heikel J (2000) Scorpion envenomation and serotherapy in Morocco. *Am J Trop Med Hyg* **2**, 277–283.

10 Aboumaâd B, Lahssaini M, Tiger A and Benhassain SM (2014) Clinical comparison of scorpion envenomation by *Androctonus mauritanicus* and *Buthus occitanus* in children. *Toxicon* **90**, 337–343.

11 Oukkache N, El Jaoudi R, Ghalim N, Chgoury F, Bouhaouala B, Mdaghri NE and Sabatier JM (2014) Evaluation of the lethal potency of scorpion and snake venoms and comparison between intraperitoneal and intravenous injection routes. *Toxins* **6**, 1873–1881.

12 Daoudi K, Chgoury F, Rezzak M, Bourouah O, Boussadda L, Soukri A, Sabatier JM and Oukkache N (2017) Consequences of *Androctonus mauretanicus* and *Buthus occitanus* scorpion venoms on electrolyte levels in rabbits. *Heliyon* **1**, e00221.

13 Emerich BL, De Lima ME, Martin-Eauclaire M and Bougis PE (2018) Comparative analyses and implications for antivenom serotherapy of four Moroccan scorpion *Buthus occitanus* venoms: subspecies tunetanus, paris, malhommei, and mardochei. *Toxicon* **149**, 26–36.

14 Pimenta AM, Stöcklin R, Favreau P, Bougis PE and Martin-Eauclaire MF (2001) Moving pieces in a proteomic puzzle: mass fingerprinting of toxic fractions from the venom of *Tityus serrulatus* (Scorpiones, Buthidae). *Rapid Commun Mass Spectrom* **17**, 1562–1572.

15 Batista CV, D'Suze G, Gómez-Lagunas F, Zamudio FZ, Encarnación S, Sevcik C and Possani LD (2006) Proteomic analysis of *Tityus discrepans* scorpion venom and amino acid sequence of novel toxins. *Proteomics* **12**, 3718–3727.

16 Favreau P, Menin L, Michalet S, Perret F, Cheneval O, Stöcklin M, Bulet P and Stöcklin R (2006) Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen. *Toxicon* **6**, 676–687.

17 Rates B, Ferraz KK, Borges MH, Richardson M, De Lima ME and Pimenta AM (2008) *Tityus serrulatus* venom peptidomics: assessing venom peptide diversity. *Toxicon* **5**, 611–618.

18 Verano-Braga T, Rocha-Resende C, Silva DM, Ianzer D, Martin-Eauclaire MF, Bougis PE, de Lima ME, Santos RA and Pimenta AM (2008) *Tityus serrulatus* Hypotensins: a new family of peptides from scorpion venom. *Biochem Biophys Res Commun* **3**, 515–520.

19 Hempel BF, Damm M, Mrinalini, Göçmen B, Karış M, Nalbantsoy A, Kini RM and Süssmuth RD (2020) Extended snake venomics by top-down in-source decay: investigating the newly discovered Anatolian meadow viper subspecies, *Vipera anatolica senliki*. *J Proteome Res* **4**, 1731–1749.

20 Compton PD and Kelleher NL (2012) Spinning up mass spectrometry for whole protein complexes. *Nat Methods* **11**, 1065–1066.

21 Smith LM and Kelleher NL (2013) Consortium for top down proteomics. Proteoform: a single term describing protein complexity. *Nat Methods* **3**, 186–187.

22 Mayne J, Ning Z, Zhang X, Starr AE, Chen R, Deeke S, Chiang CK, Xu B, Wen M, Cheng K *et al*. (2016) Bottom-up proteomics (2013–2015): keeping up in the era of systems biology. *Anal Chem* **1**, 95–121.

23 Fornelli L, Toby TK, Schachner LF, Doubleday PF, Srzentić K, DeHart CJ and Kelleher NL (2018) Top-down proteomics: where we are, where we are going? *J Proteomics* **175**, 3–4.

24 Melani RD, Skinner OS, Fornelli L, Domont GB, Compton PD and Kelleher NL (2016) Mapping proteoforms and protein complexes from king cobra venom using both denaturing and native top-down proteomics. *Mol Cell Proteomics* **7**, 2423–2434.

25 Petras D, Heiss P, Harrison RA, Süssmuth RD and Calvete JJ (2016) Top-down venomics of the East African green mamba, *Dendroaspis angusticeps*, and the black mamba, *Dendroaspis polylepis*, highlight the complexity of their toxin arsenals. *J Proteomics* **146**, 148–164.

26 Melani RD, Nogueira FCS and Domont GB (2017) It is time for top-down venomics. *J Venom Anim Toxins Incl Trop Dis* **23**, 44.

27 Trevisan-Silva D, Bednaski AV, Fischer JSG, Veiga SS, Bandeira N, Guthals A, Marchini FK, Leprevost FV, Barbosa VC, Senff-Ribeiro A *et al*. (2017) A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the *Loxosceles intermedia* venom. *Sci Data* **4**, 170090.

28 Estrada-Gomez S, Cardoso FC, Vargas-Muñoz LJ, Quintana-Castillo JC, Arenas Gómez CM, Pineda SS and Saldarriaga-Cordoba MM (2019) Venomic, transcriptomic, and bioactivity analyses of pamphobeteus verdolaga venom reveal complex disulfide-rich peptides that modulate calcium channels. *Toxins* **9**, 496.

29 Ghezellou P, Garikapati V, Kazemi SM, Strupat K, Ghassempour A and Spengler B (2019) A perspective view of top-down proteomics in snake venom research. *Rapid Commun Mass Spectrom* **1**, 20–27.

30 Oukkache N, Chgoury F, Lalaoui M, Cano AA and Ghalim N (2013) Comparison between two methods of scorpion venom milking in Morocco. *J Venom Anim Toxins Incl Trop Dis* **1**, 5.
31 Shevchenko A, Wilm M, Vorm O and Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. *Anal Chem* **5**, 850–858.

32 Miyashita M, Otsuki J, Hanai Y, Nakagawa Y and Miyagawa H (2007) Characterization of peptide components in the venom of the scorpion *Liocheles australasiae* (Hemiscorpiidae). *Toxicon* **3**, 428–437.

33 Luna-Ramírez K, Quintero-Hernández V, Vargas-Jaimes L, Batista CV, Winkel KD and Possani LD (2013) Characterization of the venom from the Australian scorpion *Urodacus yaschenkoi*: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity. *Toxicon* **63**, 44–54.

34 Cid-Uribe JI, Santibáñez-López CE, Meneses EP, Batista CVF, Jiménez-Vargas JM, Ortiz E and Possani LD (2018) The diversity of venom components of the scorpion species *Paravaejovis schwenkmeyerii* (Scorpiones: Vaejovidae) revealed by transcriptome and proteome analyses. *Toxicon* **151**, 47–62.

35 Erdeş E, Doğan TS, Coşar I, Danışman T, Kunt KB, Seker T, Yücel M and Ozen C (2014) Characterization of *Leiurus abdullahbayrami* (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity. *J Venom Anim Toxins Incl Trop Dis* **1**, 48.

36 Estrada-Gómez S, Vargas Muñoz LJ, Saldarriaga-Córdoba M and Quintana Castillo JC (2016) Venom from *Opisthacanthus elatus* scorpion of Colombia, could be more hemolytic and less neurotoxic than thought. *Acta Trop* **153**, 70–78.

37 Diego-García E, Peigneur S, Clynen E, Marien T, Czech L, Schoofs L and Tytgat J (2012) Molecular diversity of the telson and venom components from *Pandinus cavimanus* (Scorpionidae: Latreille 1802): transcriptome, venomics and function. *Proteomics* **2**, 313–328.

38 Cid-Uribe JI, Meneses EP, Batista CVF, Ortiz E and Possani LD (2019) Dissecting toxicity: the venom gland transcriptome and the venom proteome of the highly venomous scorpion *Centruroides limpidus* (Karsch, 1879). *Toxins* **5**, 247.

39 Martin-Eauclaire MF, Bosmans F, Céard B, Diochot S and Bougis PE (2014) A first exploration of the venom of the *Buthus occitanus* scorpion found in southern France. *Toxicon* **79**, 55–63.

40 Ji YH, Mansuelle P, Terakawa S, Kopeyan C, Yanaihara N, Hsu K and Rochat H (1996) Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity. *Toxicon* **9**, 987–1001.

41 Moskowitz H, Herrmann R, Jones AD and Hammock BD (1998) A depressant insect-selective toxin analog from the venom of the scorpion *Leiurus quinquestriatus* hebraeus-purification and structure/function characterization. *Eur J Biochem* **1**, 44–49.

42 Ali SA, Stoeva S, Grossmann JG, Abbasi A and Voelter W (2001) Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (*Buthus sindicus*) venom. *Arch Biochem Biophys* **2**, 197–206.

43 Borchani L, Stankiewicz M, Kopeyan C, Mansuelle P, Kharrat R, Cestèle S, Karoui H, Rochat H, Pelhate M and el Ayeb M (1997) Purification, structure and activity of three insect toxins from *Buthus occitanus tunetanus* venom. *Toxicon* **3**, 365–382.

44 Cestèle S, Kopeyan C, Oughideni R, Mansuelle P, Granier C and Rochat H (1997) Biochemical and pharmacological characterization of a depressant insect toxin from the venom of the scorpion *Buthacus arenicola*. *Eur J Biochem* **1–2**, 93–99.

45 Verano-Braga T, Dutra AA, León IR, Melo-Braga MN, Roepstorff P, Pimenta AM and Kjeldsen F (2013) Moving pieces in a venomic puzzle: unveiling post-translationally modified toxins from *Tityus serrulatus*. *J Proteome Res* **7**, 3460–3470.

46 DeBin JA, Maggio JE and Strichartz GR (1993) Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. *Am J Physiol* **264** (2 Pt 1), C361–C369.

47 Zeng XC, Peng F, Luo F, Zhu SY, Liu H and Li WX (2001) Molecular cloning and characterization of four scorpion K(+) toxin-like peptides: a new subfamily of venom peptides (alpha–KTx14) and genomic analysis of a member. *Biochimie* **9**, 883–889.

48 Abdel-Rahman MA, Quintero-Hernandez V and Possani LD (2013) Venom proteomic and venomous glands transcriptomic analysis of the Egyptian scorpion *Scorpio maurus palmatus* (Arachnida: Scorpionidae). *Toxicon* **74**, 193–207.

49 Quintero-Hernández V, Ramírez-Carreto S, Romero-Gutiérrez MT, Valdez-Velázquez LL, Becerril B, Possani LD and Ortiz E (2015) Transcriptome analysis of scorpion species belonging to the Vaejovis genus. *PLoS One* **2**, e0117188.

50 Volkova TM, Garsia AF, Telezhinskaia I N, Potapenko NA and Grishin EV (1985) Neurotoxins from the venom of the Central Asian scorpion Buthus eupeus. *Bioorg Khim* **11**, 1445–1456.

51 Vargas O, Martin MF and Rochat H (1987) Characterization of six toxins from the venom of the Moroccan scorpion *Buthus occitanus mardochei*. *Eur J Biochem* **3**, 589–599.

52 Koo GC, Blake JT, Talento A, Nguyen M, Lin S, Sirotina A, Shah K, Mulvany K, Hora D Jr, Cunningham P *et al*. (1997) Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses *in vivo*. *J Immunol* **11**, 5120–5128.

53 Liu ZR, Ye P and Ji YH (2011) Exploring the obscure profiles of pharmacological binding sites on voltage-
gated sodium channels by BmK neurotoxins. *Protein Cell* **6**, 437–444.

54 Lyons SA, O'Neal J and Sontheimer H (2002) Chlorotoxin, a scorpion-derived peptide, specifically binds to gliomas and tumors of neuroectodermal origin. *Glia* **2**, 162–173.

55 Sidach SS and Mintz IM (2002) Kurtoxin, a gating modifier of neuronal high- and low-threshold ca channels. *J Neurosci* **6**, 2023–2034.

56 Varga Z, Gurrola-Briones G, Papp F, Rodríguez de la Vega RC, Pedraza-Alva G, Tajhya RB, Gaspar R, Cardenas L, Rosenberg Y, Beeton C et al. (2012) Vm24, a natural immunosuppressive peptide, potently and selectively blocks Kv1.3 potassium channels of human T cells. *Mol Pharmacol* **3**, 372–382.

57 Yue PJ, He L, Qiu SW, Li Y, Liao YJ, Li XP, Xie D and Peng Y (2014) OX26/CTX-conjugated PEGylated liposome as a dual-targeting gene delivery system for brain glioma. *Mol Cancer* **13**, 191.

58 Misra SK, Ye M, Kim S and Pan D (2015) Defined nanoscale chemistry influences delivery of peptido-toxins for cancer therapy. *PLoS One* **6**, e0125908.

59 Olamendi-Portugal T, García BI, López-González I, Van Der Walt J, Dyason K, Ulens C, Tytgat J, Felix R, Darszon A and Possani LD (2002) Two new scorpion toxins that target voltage-gated Ca2+ and Na+ channels. *Biochem Biophys Res Commun* **4**, 562–568.

60 Carballar-Lejarazú R, Rodríguez MH, de la Cruz Hernández-Hernández F, Ramos-Castañeda J, Possani LD, Zurita-Ortega M, Reynaud-Garza E, Hernández-Rivas R, Loukeri T, Lycett G et al. (2008) Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens. *Cell Mol Life Sci* **19**, 3081–3092.

61 Pascual N, Castresana J, Valero ML, Andreu D and Bellés X (2004) Orcokinins in insects and other invertebrates. *Insect Biochem Mol Biol* **11**, 1141–1146.

62 Hernández-Aponte CA, Silva-Sanchez J, Quintero-Hernández V, Rodríguez-Romero A, Balderas C, Possani LD and Gurrola GB (2011) Vejovine, a new

antibiotic from the scorpion venom of *Vaejovis mexicanus*. *Toxicon* **1**, 84–92.

63 Parente AMS, Daniele-Silva A, Furtado AA, Melo MA, Lacerda AF, Queiroz M, Moreno C, Santos E, Rocha HAO, Barbosa EG et al. (2018) Analogs of the scorpion venom peptide stigmurin: structural assessment, toxicity, and increased antimicrobial activity. *Toxins* **4**, 161.

64 Das Neves RC, Mortari MR, Schwartz EF, Kipnis A and Junqueira-Kipnis AP (2019) Antimicrobial and antibiofilm effects of peptides from venom of social wasp and scorpion on multidrug-resistant *Acinetobacter baumannii*. *Toxins* **4**, 216.

65 Amorim-Carmo B, Daniele-Silva A, Parente AMS, Furtado AA, Carvalho E, Oliveira JWF, Santos ECG, Silva MS, Silva SRB, Silva-Júnior AA et al. (2019) Potent and broad-spectrum antimicrobial activity of analogs from the scorpion peptide stigmurin. *Int J Mol Sci* **3**, 623.

66 Hofer S, Dircksen H, Tollbäck P and Homberg U (2005) Novel insect orcokinins: characterization and neuronal distribution in the brains of selected dicondylian insects. *J Comp Neurol* **1**, 57–71.

## Supporting information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**Fig. S1.** SDS/PAGE profile of the < 30 kDa filtrate of *Buthus occitanus* venom. Molecular weight markers (MM) are indicated in kDa. Proteins/Peptides were stained with Coomassie Brilliant Blue R (InstantBlue, Expedeon, CA, USA). Stained bands corresponding to proteins/peptides with massed < 30 kDa were manually excised into equal small cubes of 1 mm³ and subjected to a nanoLC-MS/MS analysis.

**Fig. S2.** Detected amino acid sequences of the 68 peptides identified by Top-down approach.