ARTICLE

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Pseudechis australis Venomics: Adaptation for a Defense against Microbial Pathogens and Recruitment of Body Transferrin †,‡ †,§ € § § || Dessislava Georgieva,^ Jana Seifert, Michaela Ohler, Martin‡ von Bergen, Patrick Spencer, Raghuvir K. Arni, Nicolay Genov,# and Christian Betzel*, ‡ Institute of Biochemistry and Molecular Biology, University of Hamburg, Laboratory of Structural Biology of Infection and Inflammation, c/o DESY, Notkestrasse 85, Build. 22a, 22603 Hamburg, Germany § Department of Proteomics, Helmholtz Centre for Environmental Research-UFZ, Permoser Strasse 15, 04318 Leipzig, Germany Centro) de Biotecnologia, Instituto de Pesquisas Energeticas e Nucleares, Av. Lineeu Prestes 2242, 05508-000 S~ao Paulo, ^ Department of Physics, IBILCE/UNESP, Cristov~ao Colombo 2265, CEP 15054-000, S~ao Jose do Rio Preto, SP Brazil #Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

ABSTRACT: The venom composition of Pseudechis australis,a widely distributed in Australia , was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post- translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynth- esis of toxins destroying the hemostasis (PIII metallopro- teases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A2s, PLA2s). The three transferrin isoforms of the Australian P. australis ( ) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-50-nucleotidases (50-NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown . The results can be used for medical, scientific, and biotechnological purposes. KEYWORDS: snake venomics, Pseudechis australis, 2-D electrophoresis, electrospray mass spectrometry, venom transferrin, enzyme activity

’ INTRODUCTION application.2,4 15 During the past decade, investigations were focused on the molecular origin and evolution of the snake Human envenoming by poisonous is of public health 1623 significance and a serious medical problem. Snake venoms are venom proteome. Methods for the venom proteome anal- very rich and incompletely explored sources of pharmacologi- ysis with special attention to the structure, function and role of metalloproteinases in the viperid snakebite pathogenesis were cally important compounds. The best example for the develop- 2428 ment of pharmaceutical products based on investigations on developed. Proteomic and transcriptomic approaches were successfully combined in investigations on the venom composi- venom compounds is captopril, an inhibitor of the angiotensin 29,30 I-converting enzyme (ACE inhibitor), used for treatment of tions of South American snakes. hypertension.1 The knowledge of the venom composition is Australian elapid snakes are among the most venomous in the world.31 Their bites cause morbidity and in some cases necessary for the improvement of used for the 32 neutralization of snakebite consequences, for quality control of mortality. However, in comparison to viperid snakes, consider- venom preparations2 and for structure-based design of novel ably less information about the elapid venom proteome is available. Venom compositions of Naja naja atra,33 drugs, especially for the blood pressure regulation and the 34 fi 35 treatment of coagulopathy. components have a textilis, Micrurus surinamensis ( sh eating coral snake) and significant potential for clinical applications as diagnostic agents.3 The venomics of a large number of viperid snakes have been Received: December 16, 2010 investigated with respect to their pharmacological and medical Published: March 22, 2011

r 2011 American Chemical Society 2440 dx.doi.org/10.1021/pr101248e | J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE

Naja kaouthia36 were determined. Selected spots from 2-D Millipore) and reconstituted in 0.1% formic acid. Samples were PAGE of Australian snake venoms were analyzed and novel injected by an autosampler and concentrated on a trapping proteins identified.37 Transcriptomic approaches were applied column (nanoAcquity UPLC column, C18, 180 μm 2 cm, for analyzing venom gland genes of Oxyuranus scutellatus,38 5 μm, Waters) with water containing 0.1% formic acid at flow Micrurus corallinus39 and Bungarus flaviceps.40 rates of 15 μL/min. After 2 min the peptides were eluted onto a Snakes of the Pseudechis, also known as “Black Snakes” separation column (nanoAcquity UPLC column, C18, 75 μmx or “Bongani Sibanda”, are widespread in all Australian states 10 cm, 1.75 μm, Waters). Chromatography was performed with except for Tasmania. Nine were recognized: P. australis 0.1% formic acid in solvent A (100% water) and B (100% ACN). ( or mulga snake), P. butleri (Spotted mulga The peptides were eluted over 8 min with 20 80% solvent B snake), P. collettii (Collett’s snake), P. guttatus (Blue-bellied black gradient using a nano-HPLC system (nanoAcquity, Waters) snake), P. papuanus (), P. pailsi, P. porphyr- coupled to an LTQ-Orbitrap mass spectrometer (Thermo Fisher iacus (Red-bellied black snake), P. rossignolii (Papuan dwarf king Scientific). The capillary voltage in MS and MS/MS experiments brown) and P. weigeli (Pygmy mulga snake).41,42 P. australis is was set to 2000 V. The collision gas was helium at a pressure of one of the longest venomous snakes in the world (2.53min 0.1 MPa, and the collision energy was 40 V. For an unbiased length) and is encountered in most Australian states except for analysis, continuous scanning of eluted peptide ions was carried Victoria and Tasmania. out between 300 and 2000 m/z, automatically switching to In the present paper, we report the proteomic profile of the MS/MS CID mode on ions exceeding an intensity of 5000. Pseudechis australis venom. The venom components were ana- For MS/MS measurements, a dynamic precursor exclusion of lyzed by 2-D gel electrophoresis and electrospray mass spectro- 1 min with an isolation width of 4 m/z was used. metry, and classified into protein families. Raw data were applied to a database search using Thermo Proteome Discoverer software (v1.0 build 43) to carry out a ’ MATERIALS AND METHODS tandem ion search algorithm from the MASCOT house server (v2.2.1) by database comparison against all chordata entries in Collection of the Venom the National Center for Biotechnology Information (NCBInr database 2010) with 10 ppm tolerance for the precursor and Crude venom, pooled from several specimens of Pseudechis 2 australis, was a kind gift of Dr. P. Mirtschin (Venom Supplies Pyt. 0.8 Da for MS fragments. Further, trypsin with a maximum of fi Ltd., Australia). Snakes of both genders were milked and the two missed cleavages was selected and variable modi cations, lyophilized venom was stored at 4 C. such as methionine oxidation and carbamidomethylation of cysteine, were allowed. Peptides were considered to be identified 2-D Gel Electrophoresis and Electrospray Mass Spectrom- by Mascot when a probability <0.05 (probability based ion etry threshold scores >40) was achieved. Proteins were considered Two-dimensional electrophoresis was performed as described to be identified if at least two peptides were identified. In some previously.43 The venom was suspended in deionized water and cases there is a theoretical possibility that non-P. australis venom desalted using an Amicon-Ultra 0.5 mL filter (Millipore) with a peptides do not 100% mirror the corresponding fragments 10 kDa cutoff prior to the 2-D electrophoresis. 200 μg of the total because similar tryptic peptides of the same mass can possess protein were mixed with 135 μL DeStreak solution (GE nonidentical sequences due to different sequential order of pairs Healthcare) and 0.5% IPG (immobilized pH gradient), pH 3 of residues. 10, in nonlinear (NL) buffer (v/v) (GE Healthcare, Uppsala, Sweden). The sample was agitated for 15 min at room tempera- Enzymatic Activities ture and centrifuged for 30 min at 13 000 rpm to remove Proteolytic activity was determined by the method of Johnson precipitates. The equilibration was performed with the super- et al.47 The venom was assayed using 1.2% casein solution in natant loaded on 7 cm Immobiline DryStrip pH 3 10 NL (GE Tris-HCl buffer, pH 7.4, at 37 C. Undigested casein was fi Healthcare, Uppsala, Sweden). In the rst dimension, proteins precipitated with 0.5 M perchloric acid and centrifuged. Digested were separated by an IPGphore electrophoresis unit overnight casein in the supernatant was determined by measuring the (GE Healthcare, Uppsala, Sweden). After isoelectric focusing the absorbance at 280 nm. Unit definition: One CTA unit liberates strips were equilibrated for 15 min in equilibration buffer from cow casein 0.1 micro equivalents of tyrosine for 1 min at containing 0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v), 37.5 C. One CTA unit is equal to 0.096 proteolytic units as used 6 M urea, 4% sodium dodecyl sulfate and 2% dithioerythrithol. In a second equilibration step the strips were incubated with by SIGMA Chemical Corporation, St Louis, MO. 0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v), 6 M urea, Phospholipase A2 activity was determined using the Cayman Chemical Secretory PLA2 Assay kit (Ann Arbor, MI) containing 4% sodium dodecylsulfate and 2.5% iodoacetamide for 15 min. The strips were stored at 20 C until used in the second a bee venom PLA2 as a standard. 1,2 dithio analog of dimension, performed on a 10% Tris-tricine-polyacrylamide gel diheptanoyl phosphatidylcholine was used as a substrate. The 3 44 (100 100 1.0 mm ). release of free thiols upon the PLA2 catalyzed hydrolysis of the thioester bond at the sn-2 position was detected spectrophoto- After electrophoresis both gels were stained overnight with 0 coomassie brilliant blue and destained as described previously.45 metrically using 5,5 -dithiobis(2-nitrobenzoic acid). Gels were scanned and imported into the software Delta2D L-Amino acid oxidase activity was determined by the method 48 software package (Decodon, Greifswald, Germany). of Wellner et al. using L-phenylalanine as substrate. One unit of Protein spots of interest were cut from the polyacrylamide gels activity is the amount of enzyme required to give an absorbance and digested overnight using trypsin (Sigma, Munich, Germany) of 0.030 at 300 nm. according to the protocol from Shevchenko, modified in a Alkaline phosphatase activity was measured by the method of previous study.46 Peptides were desalted (ZipTip pipet tips, Sulkowski et al.49 using p-nitrophenylphosphate as a substrate.

2441 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE

Figure 1. 2-D gel pattern of the Pseudechis australis venom. Fractionation was performed under the conditions described in the Materials and Methods.

One unit of activity is defined as the amount of enzyme which of spots with identical or very similar molecular masses but liberates 1 μmole of p-nitrophenol per min. different isoelectric points. Acid phosphatase activity was determined by the method of Tu and Chua.50 o-Carboxyphenylphosphate (0.0036 M) was PIII metalloproteases used as a substrate and the initial rate of hydrolysis of the PIII SVMPs are the most widely represented family of toxins substrate at 25 C was determined from the increase of the in the P. australis venom accounting for 53% of the identified absorbance at 300 nm due to the liberation of salicylic acid. toxins (Figure 2). A group of high molecular mass enzymes was Venom concentration was adjusted so that the increase of the identified from spots of multiple horizontal trains in the upper absorbance was linear for at least 5 min. One unit of acid left part of the 2-D gel (Figure 1, Table 1). Spots 15 contain phosphatase activity is equivalent to 1 μmole of the substrate PIII metalloproteases with molecular masses of 100105 kDa hydrolyzed per min. and pI values between 4.7 and 5.2. A second group of multiple isoforms of PIII SVMPs is shown in the upper right panel of the gel (Figure 1). Again, horizontal trains of spots with identical ’ RESULTS molecular masses, but differing in the pI values were observed. Metalloproteases (6085 kDa) were identified in spots 726, 2-D Gel Electrophoresis of the Pseudechis australis Venom 29, 3035, 42, 4447, 5974, 87, and 88. The peptide analyses The proteomic composition of the P. australis venom was showed a high degree of sequence similarity between the PIII investigated by 2-D electrophoresis. The separated protein bands SVMPs from the P. australis venom and their counterparts from were subjected to tryptic digestion and the venom components the other elapid Australian snakes: Austrelaps superbus (the fi were identi ed by MS/MS and MASCOT search program Lowland copperhead), Pseudechis porphyriacus (Red-bellied (Figure 1, Tables 1 and 2). The oxidized methionine residues black snake), Oxyuranus scutellatus (the Coastal taipan) and are indicated by Mox. The oxidation of methionine is due Notechis scutatus (Tables 1 and 2). probably to the procedure of harvesting and sample handling. Proteins with molecular masses in the region of 3343 kDa The samples were dried in the presence of ambient air which is and pI values between 4.0 and 7.2 were isolated from spots known to cause oxidation of methionine. The gel ensured 3641, 7577 and 80. They form another group of processed detailed information about the components with molecular PIII SVMPs (Figure 1). The molecular masses are character- masses 9 110 kDa and pI values between 3 and 10. A total of istic for the medium size class II proteases, but sequence fi 110 spots were detected and identi ed on the 2-D gel. The similarities with the PIII group suggest that these proteins isolated proteins were assigned to the following protein families: belong to a group of processed PIII enzymes. metalloproteases, phospholipases A2, L-amino acid oxidases, 0 transferrin-like proteins, ecto-5 -nucleotidases, nerve growth Antimicrobial Proteins: L-Amino Acid Oxidases and Trans- factors and serine protease inhibitors. The major group is that ferrin-Like Proteins of the hemostasis-related SVMPs including 53% of the identified Multiple isoforms of L-amino acid oxidases were found in the proteins. The isoforms of LAAOs comprise the second largest processed spots of the 2-DE gel (20% of the identified proteins; protein family (20% of the identified toxins). In the third position Figure 1, Tables 1 and 2). Spots 47, 49, 51, 53, 55, 57, and 61 form fi are PLA2s representing 18.5% of the identi ed venom compo- a horizontal train in the pI range from 6.7 to 7.8. The proteins nents. The representatives of the other five protein families possess similar molecular masses from 58 to 62 kDa. A second comprise 8.5% of the analyzed toxins. A characteristic feature train in the same pI interval is formed by spots 48, 50, 52, 54, 56, of the 2-D gel is the presence of several multiple horizontal trains 58 and 60, containing proteins with molecular masses of

2442 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III III III III III III III III III III III III III MS/MS derived sequence protein family Venom to Protein Families by MS/MS and MASCOT zz / ion m Pseudechis australis 861.7 3 AAKDDCDLPESCTGQSAECPTDR 593.8 2 NDNAQLLTGIK 593.8 2 NDNAQLLTGIK 861.7 3 AAKDDCDLPESCTGQSAECPTDR 861.7 2 AAKDDCDLPESCTGQSAECPTDR 955.9 2 NGHPCQNNQGYCYNGK 593.8 2 NDNAQLLTGIK 593.8 2 NDNAQLLTGIK 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1292 2 AAKDDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 3 DDCDLPESCTGQSAECPTDR peptide a matched peptides score Mascot MW I 5.95 71183 94 2 593.8 2 NDNAQLLTGIK P 5.76 70518 154 3 955.9 2 NGHPCQNNQGYCYNGK P 5.95 71183 149.6 2 861.7 3 AAKDDCDLPESCTGQSAECPTDR P 5.95 71183 234 3 861.7 3 AAKDDCDLPESCTGQSAECPTDR P 5.95 71183 149.6 2 861.7 3 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 130 3 955.9 2 NGHPCQNNQGYCYNGK P 5.95 71183 122.9 2 593.8 2 NDNAQLLTGIK P 5.95 71183 318 4 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.95 71183 133.7 3 861.7 3 AAKDDCDLPESCTGQSAECPTDR P 5.95 71183 314 3 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.95 71183 211 2 671.9 2 RNDNAQLLTGIK P 5.95 71183 161 3 671.9 2 RNDNAQLLTGIK P 5.76 70518 151 2 671.9 2 RNDNAQLLTGIK P 5.95 71183 211 2 861.7 3 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 151 2 955.9 2 NGHPCQNNQGYCYNGK P 5.95 71183 318 3 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 180 3 955.9 2 NGHPCQNNQGYCYNGK P 5.95 71183 267 3 861.7 3 AAKDDCDLPESCTGQSAECPTDR P with a homology protein from p Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus Austrelaps superbus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus Austrelaps superbus Austrelaps superbus Austrelaps superbus Austrelaps superbus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus code accession porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 no. protein 1 asrin 111572527 2 asrin 111572527 11 asrin 111572527 3 asrin 111572527 4 asrin 111572527 12 asrin 111572527 5 asrin 111572527 13 asrin 111572527 7 asrin 111572527 14 asrin 111572527 8 asrin 111572527 9 asrin 111572527 10 asrin 111572527 spot Table 1. Assignment of the Proteins Isolated from the Spots of the 2-D-Gel Electrophoresis of the

2443 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III III III III III III III III III III III III MS/MS derived sequence protein family zz / ion m 861.7 2 AAKDDCDLPESCTGQSAECPTDR 690.3 2 CPLMTNQCLAR P 690.3 2 CPLMTNQCLAR 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 861.7 2 AAKDDCDLPESCTGQSAECPTDR 690.3 2 CPLMTNQCLAR 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 861.7 2 AAKDDCDLPESCTGQSAECPTDR 690.3 2 CPLMTNQCLAR 690.3 2 CPLMTNQCLAR 349.7 2 TVLLPR 690.3 2 CPLMTNQCLAR 593.8 2 NDNAQLLTGIK 490.3 3 KRNDNAQLLTGIK P 1156.9 2 DDCDLPESCTGQSAECPTDR P 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR 1156.9 2 DDCDLPESCTGQSAECPTDR peptide a matched peptides score Mascot MW I 5.95 71183 155 3 671.9 2 RNDNAQLLTGIK P 5.95 71183 313 3 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 139 2 593.8 2 NDNAQLLTGIK 5.76 70518 152 2 593.8 2 NDNAQLLTGIK P 5.95 71183 190 2 1156.9 2 DDCDLPESCTGQSAECPTDR 5.95 71183 1945.76 70518 112 2 1156.9 2 2 DDCDLPESCTGQSAECPTDR 955.9 2 NGHPCQNNQGYCYNGK P P 5.95 71183 266 3 1156.9 2 DDCDLPESCTGQSAECPTDR 5.95 71183 2275.76 70518 118 2 1156.9 2 2 DDCDLPESCTGQSAECPTDR 955.9 2 NGHPCQNNQGYCYNGK P P 5.76 70518 235 3 955.9 2 NGHPCQNNQGYCYNGK 5.95 71183 316 3 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.55 68267 90 2 413.7 2 KTVLLPR 5.76 70518 139 2 593.8 2 NDNAQLLTGIK P 5.95 71183 213 3 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.95 71183 271 4 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 205 3 593.8 2 NDNAQLLTGIK P 5.95 71183 308 2 861.7 2 AAKDDCDLPESCTGQSAECPTDR with a homology protein from p Austrelaps superbus Austrelaps superbus Pseudechis porphyriacus Pseudechis porphyriacus Austrelaps superbus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus Austrelaps superbus Pseudechis porphyriacus Pseudechis porphyriacus Austrelaps superbus Demansia vestigata Pseudechis porphyriacus Austrelaps superbus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus code accession porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 no. protein 15 asrin 111572527 16 asrin 111572527 22 asrin 111572527 17 asrin 111572527 23 asrin 111572527 18 asrin 111572527 19 asrin 111572527 24 metalloproteinase precursor 118151738 25 asrin 111572527 20 asrin 111572527 21 asrin 111572527 spot Table 1. Continued

2444 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III III III III III III III III III MS/MS derived sequence protein family zz / ion m 553.8 2 RPLEECFR 959939.9 GGPGVNLSPDICFTINQK LQHEAQCDSGECCER 981 2 IQQNAEDVRVTYQTPAK 630.3 2 SDDLFSYEKR 959 2 GGPGVNLSPDICFTINQK 939.9 2 LQHEAQCDSGECCER 509.3 2 VTLLEASER 728.8 2 EADYEEFLEIAK 593.8 2 NDNAQLLTGIK 593.8 2 NDNAQLLTGIK 690.3 CPLMTNQCLAR 735.4 2 KRNDNAQLLTGIK 690.3 2 CPLMTNQCLAR 735.9 2 KRNDNAQLLTGIK 636.9 3 NGHPCQNNQGYCYNGK 593.8 2 NDNAQLLTGIK 690.3 2 CPLMTNQCLAR 1156.9 2 DDCDLPESCTGQSAECPTDR peptide a matched peptides score Mascot MW I 6.26 59049 6635.45 71238 597 65.76 70518 436 549.6 96.26 59049 3 RFDEIVGGFDQLPR 1375 981.7 68.99 59032 17 AAKDDCDLPESCTGQSAECPTDSFQR 595 981.75.45 71238 746.9 AAKDDCDLPESCTGQSAECPTDSFQR 422 7 25.76 FDEIVGGFDQLPR 70518 P 323 608.8 68.44 51406 2 P FWEADGIHGGK 281 981.7 LAAO 66.26 59049 3 AAKDDCDLPESCTGQSAECPTDSFQR 953 981.7 45.95 71183 3 AAKDDCDLPESCTGQSAECPTDSFQR 15 289 608.8 P 2 FWEADGIHGGK 746.9 LAAO 4 2 P FDEIVGGFDQLPR 861 LAAO 3 AAKDDCDLPESCTGQSAECPTDR P LAAO LAAO 5.76 70518 162 2 690.3 2 CPLMTNQCLAR P 5.76 70518 92 2 593.8 NDNAQLLTGIK P 5.95 71183 174 3 861.7 2 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 6135.45 71238 272 95.95 71183 256 981.7 64.99 68020 3 AAKDDCDLPESCTGQSAECPTDSFQR 100 981.7 4 3 AAKDDCDLPESCTGQSAECPTDSFQR 861 2 P 3 AAKDDCDLPESCTGQSAECPTDR 732.8 P 2 CPIMTNQCIALK P P 5.95 71183 1605.76 70518 135 2 861 2 3 AAKDDCDLPESCTGQSAECPTDR 593.8 2 NDNAQLLTGIK P P with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Oxyuranusscutellatus Pseudechis australis scutellatus Pseudechis porphyriacus Naja atra Pseudechis australis Austrelaps superbus Pseudechis porphyriacus Pseudechis porphyriacus Austrelaps superbus Pseudechis porphyriacus Pseudechis australis Austrelaps superbus Notechis scutatus Austrelaps superbus Pseudechis porphyriacus code accession -amino-acid oxidase 123916679 -amino-acid oxidase-amino-acid oxidase precursor 12391680 123916679 -amino-acid oxidase-amino-acid oxidase 126035677 123916679 australease-1porphyriacase-1 145982758 145982756 australease-1porphyriacase-1 145982758 145982756 asrin 111572527 L L L L L porphyriacase-1 145982756 porphyriacase-1 145982756 australease-1asrinscutatease-1 145982758 111572527 145982766 porphyriacase-1 145982756 no. protein 32 33 26 asrin 111572527 29 porphyriacase-1 145982756 30 asrin 111572527 31 spot Table 1. Continued

2445 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III III III III III III III III III III III MS/MS derived sequence protein family zz / ion m 728.8 2 EADYEEFLEIAK 690.3 2 CPLMTNQCLAR 728.8 2 EADYEEFLEIAK 671.9 2 RNDNAQLLTGIK 607.8 2 DVNLASQKPSR 891.7 3 DDCDLPESCTGQSAECPTDSFQR 735.9 2 KRNDNAQLLTGIK 955.9 2 NGHPCQNNQGYCYNGK 690.3 2 CPLMTNQCLAR 955.9 2 NGHPCQNNQGYCYNGK 707.3 2 DDPDYGMVEAGTK 959 2 GGPGVNLSPDICFTINQK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 959 2 GGPGVNLSPDICFTINQK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 959 2 GGPGVNLSPDICFTINQK 891.7 3 DDCDLPESCTGQSAECPTDSFQR 891.7 3 DDCDLPESCTGQSAECPTDSFQR 1156.9 2 DDCDLPESCTGQSAECPTDR peptide a matched peptides score Mascot MW I 6.26 59049 9885.76 70518 13 252 746.9 2 2 FDEIVGGFDQLPR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P LAAO 6.26 59049 514.85.76 70518 4 299.56.26 59049 552.2 5 583.4 2 SDDIFSYEK 690.3 7 2 CPLMTNQCLAR 728.8 2 EADYEEFLEIAK LAAO P LAAO 5.76 70518 355.7 6 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.95 71183 414.3 5 861 3 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 128 6 735.9 2 KRNDNAQLLTGIK P 5.95 71183 141 4 861 3 AAKDDCDLPESCTGQSAECPTDR P 5.76 70518 290.7 5 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 356.3 5 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 305.4 6 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 572.55.76 70518 8 281.4 981.7 5 3 AAKDDCDLPESCTGQSAECPTDSFQR 690.3 2 CPLMTNQCLAR P P 5.45 71238 5645.76 70518 307.4 8 5 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 690.3 2 CPLMTNQCLAR P P 5.45 71238 6065.76 70518 10 334.7 981.7 6 3 AAKDDCDLPESCTGQSAECPTDSFQR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P P 5.45 71238 516.4 8 981.3 3 AAKDDCDLPESCTGQSAECPTDSFQR P with a homology protein from p Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Austrelaps superbus Pseudechis porphyriacus Austrelaps superbus Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis code accession -amino-acid oxidase 123916679 -amino-acid oxidase-amino-acid oxidase 123916679 123916679 porphyriacase-1 145982756 porphyriacase-1 145982756 L L L porphyriacase-1 145982756 porphyriacase-1 145982756 australease-1 145982758 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 no. protein 36 asrin 111572527 43 34 asrin 111572527 42 35 37 australease-1 145982758 38 australease-1 145982758 39 australease-1 145982758 40 australease-1 145982758 41 australease-1 145982758 spot Table 1. Continued

2446 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III MS/MS derived sequence protein family zz / ion m 824.9 2 RFDEIVGGFDQLPR 684.4 2489.2 CGLVPILTEIPR 2483.2 GSGGEGGLSEK 2574.8 LFGSQGTQK 2 DFPELICVR 626.2 3 LQHEAQCDSGECCER 690.3 2 CPLMTNQCIAR 607.8 2 DVNLASQKPSR 707.3 2 DDPDYGMVEAGTK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 755.9 2489.2 LKQECFSQQQSK 2574.8 GSGGEGGLSEK 2 DFPELICVR 607.8 2 DVNLASQKPSR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 684.4 2635.3 CGLVPILTEIPR 2489.2 QECFSQQQSK 2483.2 GSGGEGGLSEK 2 LFGSQGTQK 728.8 2 EADYEEFLEIAK 626.2 3 LQHEAQCDSGECCER 553.8 2 RPLEECFR 728.8 2 EADYEEFLEIAK peptide a matched peptides score Mascot MW I 6.26 59049 996.26.4 78321 13 291.7 746.9 5 2 FDEIVGGFDQLPR5.76 755.9 70518 2 220.9 LKQECFSQQQSK5.45 71238 4 516.46.26 59049 690.3 8 680.95.45 2 71238 CPLMTNQCLAR 707.3 5 372.65.76 2 LAAO 70518 DDPDYGMVEAGTK 824.9 5 311.86.4 2 RFDEIVGGFDQLPR 78321 transferrin 981.7 5 268 3 AAKDDCDLPESCTGQSAECPTDSFQR 690.36.26 4 2 59049 CPLMTNQCIAR 658.1 684.45.76 P P 70518 2 P 7 CGLVPILTEIPR 316.66.4 78321 824.9 LAAO 6 175.9 2 RFDEIVGGFDQLPR 690.3 5 2 CPLMTNQCIAR6.26 755.9 59049 P 2 814.3 LKQECFSQQQSK5.76 70518 9 241.76.26 transferrin 59049 824.9 5 737.76.26 2 LAAO 59049 RFDEIVGGFDQLPR 690.3 8 667.7 2 CPLMTNQCIAR 824.9 P 8 2 RFDEIVGGFDQLPR transferrin 746.9 2 FDEIVGGFDQLPR LAAO P LAAO LAAO with a homology protein from p Pseudechis australis Lamprophis fuliginosus Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis porphyriacus Lamprophis fuliginosus Pseudechis australis Pseudechis porphyriacus Lamprophis fuliginosus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis australis code accession -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase-amino-acid oxidase 123916679 -amino-acid oxidase 123916679 123916679 transferrinporphyriacase-1 108792441 australease-1 145982756 australease-1porphyriacase-1 145982758 transferrin 145982758 145982756 porphyriacase-1 108792441 transferrin 145982756 108792441 porphyriacase-1 145982756 L L L L L L no. protein 45 46 47 48 49 44 spot Table 1. Continued

2447 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III MS/MS derived sequence protein family zz / ion m 536.8 2 IQQNAEDVR 668.8 2 EQIQALCYPSK 746.9 2 FDEIVGGFDQLPR 536.8 2 IQQNAEDVR 728.8 2 EADYEEFLEIAK 607.8 2 DVNLASQKPSR 939.9 2 LQHEAQCDSGECCER 668.8 2 EQIQALCYPSK 608.8 2 FWEADGIHGGK 454.2 2 VTYQTPAK 728.8 2 EADYEEFLEIAK 728.8 2 EADYEEFLEIAK 854.9 2 NEKEGWYVNLGPMR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 636.9 3 NGHPCQNNQGYCYNGK 1338.1 2 DDCDLPESCTGQSAECPTDSFQR 1338 2 DDCDLPESCTGQSAECPTDSFQR 1338 2 DDCDLPESCTGQSAECPTDSFQR peptide a matched peptides score Mascot MW I 6.26 59049 667.76.26 59049 6 835.16.26 59049 11 607.8 687.56.26 2 59049 DVNLASQKPSR 662.3 5 686 26.26 DGWYVNLGPMR 59049 607.8 820.7 66.26 2 59049 DVNLASQKPSR 10 835.1 607.85.76 70518 2 746.9 DVNLASQKPSR 7 135.3 26.26 FDEIVGGFDQLPR 59049 824.9 2 9186.26 2 LAAO 59049 RFDEIVGGFDQLPR 682.8 12 944 LAAO 5.76 2 70518 FSSCSVQEHQR 746.9 136.26 2 LAAO 59049 FDEIVGGFDQLPR 728.8 510.46.26 2 59049 LAAO EADYEEFLEIAK LAAO 3 965.65.76 70518 982.5 13 607.8 LAAO 478.2 3 2 AAKDDCDLPESCTGQSAECPTDSFQR DVNLASQKPSR 746.9 9 2 FDEIVGGFDQLPR P 981.7 LAAO P 6.26 3 59049 AAKDDCDLPESCTGQSAECPTDSFQR 1284.88.99 LAAO 59032 17 648.25.76 P 70518 746.9 8 284.3 2 FDEIVGGFDQLPR LAAO 669.3 LAAO 5 2 EGWYVNLGPMR 690.3 2 CPLMTNQCLAR LAAO LAAO P 5.45 71238 422.3 7 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 4.98 68626 115.5 2 578.2 2 CGDGMVCSNR P with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Oxyuranus scutellatus Pseudechis porphyriacus Pseudechis australis Pseudonaja textilis code accession -amino-acid oxidase-amino-acid oxidase-amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 123916679 -amino-acid oxidase-amino-acid oxidase 123916679 123916680 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 L L L L L L L L L L L L australease-1 145982758 textilease-1 145982770 no. protein 51 52 53 54 55 56 57 59 58 50 60 spot Table 1. Continued

2448 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP III III III III III III III III III III III III III III III -nucleotidase 0 MS/MS derived sequence protein family zz / ion m 728.8 2 EADYEEFLEIAK 959 2 GGPGVNLSPDICFTINQK 636.9 3 NGHPCQNNQGYCYNGK 959 2 GGPGVNLSPDICFTINQK 620.8 2 AYVGTLCSLEK 726476.3 3 GDSSNHSSGNLDISIVGDYIK 2 VGIIGYTTK 959 22 GGPGVNLSPDICFTINQK 981.7 AAKDDCDLPESCTGQSAECPTDSFQR 959 2 GGPGVNLSPDICFTINQK 959 2 GGPGVNLSPDICFTINQK 620.8 2 AYVGTLCSLEK 959 2 GGPGVNLSPDICFTINQK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 959 2 GGPGVNLSPDICFTINQK 959 2 GGPGVNLSPDICFTINQK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 1338 2 DDCDLPESCTGQSAECPTDSFQR peptide a matched peptides score Mascot MW I 6.26 59049 8575.76 70518 10 565.1 746.9 9 2 FDEIVGGFDQLPR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P LAAO 8.65 64441 153.4 3 653.9 2 QVPVVQAYAFGK 5 4.89 68542 143.7 2 586.2 2 CGDGMVCSNR P 5.45 71238 494.5 9 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 152 2 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 169.65.76 70518 3 147 981.7 3 3 AAKDDCDLPESCTGQSAECPTDSFQR 690.3 3 CPLMTNQCLAR P P 5.45 71238 321.4 3 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 181.75.76 70518 3 149.4 981.7 3 3 AAKDDCDLPESCTGQSAECPTDSFQR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P P 5.45 71238 725.15.76 70518 6 305.2 981.7 5 3 AAKDDCDLPESCTGQSAECPTDSFQR 690.3 2 CPLMTNQCLAR P P 5.45 71238 159.5 2 690.3 2 CPLMTNQCLAR P 5.45 71238 598.85.76 70518 6 254.6 981.7 5 3 AAKDDCDLPESCTGQSAECPTDSFQR 690.3 2 CPLMTNQCLAR P P ffi with a homology protein from p brevicaudus Pseudechis australis Pseudechis porphyriacus Gloydius blomho Oxyuranus scutellatus Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis porphyriacus code accession -nucleotidase 211926754 0 -amino-acid oxidase 123916679 porphyriacase-1 145982756 L scutellatease-1 145982762 australease-1 145982758 porphyriacase-1 145982756 5.76 70518 134.2 2 515.7 2 CGMLYCVK P porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1 145982756 ecto-5 porphyriacase-1 145982756 no. protein 61 62 australease-1 145982758 63 australease-1 145982758 66 australease-1 145982758 64 australease-1 145982758 67 australease-1 145982758 65 australease-1 145982758 68 australease-1 145982758 spot Table 1. Continued

2449 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP fragment SVMP fragment III III III III III III III III III III III III III III III III -nucleotidase -nucleotidase 0 0 MS/MS derived sequence protein family zz / ion m 959 2 GGPGVNLSPDICFTINQK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 726 3 GDSSNHSSGNLDISIVGDYIK 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR 959 2 GGPGVNLSPDICFTINQK 959 2 GGPGVNLSPDICFTINQK 939.9 2 LQHEAQCDSGECCER 959 2 GGPGVNLSPDICFTINQK 939.9 2 LQHEAQCDSGECCER 741.9 2 KYIELYVVVDNK 959 2 GGPGVNLSPDICFTINQK 939.9 2 LQHEAQCDSGECCER 741.9 KYIELYVVVDNK 959 2 GGPGVNLSPDICFTINQK 939.9 2 LQHEAQCDSGECCER 959 2 GGPGVNLSPDICFTINQK P 939.9 2 LQHEAQCDSGECCER 1211.1 2 FHECNLGNLICDAVIYNNVR peptide a matched peptides score Mascot MW I 8.65 64441 125.4 2 653.28.65 2 64441 QVPVVQAYAFGK 183 5 778 3 GDSSNHSSGNLDISIVGDYIKR 5 5 5.76 70518 240.7 5 690.3 2 CPLMTNQCLAR P 5.45 71238 601.6 7 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.76 70518 192.1 4 690.3 2 CPLMTNQCLAR P 5.45 71238 486 8 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.45 71238 592.35.76 70518 9 208.3 981.7 6 3 AAKDDCDLPESCTGQSAECPTDSFQR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P P 5.45 71238 734.75.76 70518 10 257.14.89 68542 981.7 7 189.9 3 AAKDDCDLPESCTGQSAECPTDSFQR 981.7 3 3 AAKDDCDLPESCTGQSAECPTDSFQR 677.9 P 2 YIELYVVVDNK P P 5.45 71238 7385.76 70518 12 277.54.89 68542 981.7 7 201.6 3 AAKDDCDLPESCTGQSAECPTDSFQR 981.7 3 32 AAKDDCDLPESCTGQSAECPTDSFQR 677.9 2 P YIELYVVVDNK P P 5.45 71238 5815.76 70518 10 215.3 981.7 6 3 AAKDDCDLPESCTGQSAECPTDSFQR 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P P 5.45 71238 471.8 7 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR P 5.76 70518 267.1 5 981.7 3 AAKDDCDLPESCTGQSAECPTDSFQR ffi ffi with a homology protein from p brevicaudus brevicaudus Gloydius blomho Gloydius blomho Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Oxyuranus scutellatus Pseudechis australis Pseudechis porphyriacus Oxyuranus scutellatus Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus code accession -nucleotidase 211926754 -nucleotidase 211926754 0 0 porphyriacase-1ecto-5 145982756 ecto-5 porphyriacase-1 145982756 porphyriacase-1 145982756 porphyriacase-1scutellatease-1 145982756 145982762 porphyriacase-1scutellatease-1 145982756 145982762 porphyriacase-1 145982756 porphyriacase-1 145982756 no. protein 69 australease-1 145982758 70 australease-1 145982758 71 australease-1 145982758 72 australease-1 145982758 73 australease-1 145982758 74 australease-1 145982758 75 australease-1 145982758 spot Table 1. Continued

2450 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP fragment SVMP fragment SVMP fragment 2 2 2 2 2 2 2 III III III IQCANK PLA MS/MS derived sequence protein family ox DYGCYCGWGGSGTPVDELDRDYGCYCGWGGSGTPVDELDR PLA PLA DYGCYCGWGGSGTPVDELDR PLA ox ox ox CPLMTNQCLAR NEKDGWYVNLGPMR SDDLFSYEKR zz / ion m 626.2 3 552.2 2 VTLLEASER 454.2 2 728.8 2 EADYEEFLEIAK 707.3 2 DDPDYGMVEAGTK 847.9 2 GGPGVNLSPDICFTINQK 478.5 3 VHDDCYDQAGKK 959 2 874.9 2 LWNSYCTTTQTFVK PLA 590.9 3 CCQTHDNCYEQAGK 938.9 2 TECKDFTCACDAEAAK 501.3 2 GTPVDELDR 1037.5 3 VVVVGAGMAGLSAAYVLAGAGHQVTLLEASER 1037.5 3 VVVVGAGMAGLSAAYVLAGAGHQVTLLEASER 1056 2 LTLYSWDCTGNVPICSPK 1136 2 ITWYSWDCTENVPTCNPK 1142 2 ITWYSWDCTENVPTCNPK peptide a matched peptides score Mascot MW I 6.26 59049 750.8 11 746.96.26 59049 2 FDEIVGGFDQLPR 879.8 13 1037.56.26 3 59049 VVVVGAGMAGLSAAYVLAGAGHQVTLLEASER 828 LAAO fragment 8.99 13 59032 374.88.44 LAAO 746.9 fragment 51406 2 6 197 FDEIVGGFDQLPR 630.3 3 2 SDDIFSYEK 509.36.26 59049 2 VTYQTPAK 351.25.45 71238 5 303 LAAO fragment 746.9 6 2 FDEIVGGFDQLPR 959 2 LAAO GGPGVNLSPDICFTINQK fragment LAAO fragment LAAO fragment P 5.45 71238 235.6 4 959 2 LQHEAQCDSGECCER P 8.84 13798 190.1 2 833.9 2 NLIQFGNM 8.52 13018 85.4 2 848.96.84 2 13941 NLIQFSNMIQCANK 286.27.76 15760 5 244.1 904.4 4 3 HYM 904.47.53 3 13914 HYM 160.38.52 14002 3 95.8 1069.5 2 2 LTLYSWDCTGNVPICNPK 1086.5 2 LTWYSWDCTGDAPTCNPK PLA PLA 5.75 27138 326.3 4 1004 2 GNTVTVEVDVNLNNEVYK nerve growth factor 5.45 71238 343.9 6 690.3 2 GGPGVNLSPDICFTINQK P 6.77 16768 181.8 3 904.4 3 HYM with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Oxyuranusscutellatus Naja atra scutellatus Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis code accession 123916680 129471 129397 129447 295841609 129474 isozyme isozyme isozyme isozyme isozyme PA-5 129452 isozyme 2 2 2 2 2 2 PA-12C precursor PA-10A PA-3 HI-2009 PA-13 -amino-acid oxidase 123916679 -amino-acid oxidase 123916679 -amino-acid oxidase-amino-acid oxidase 123916679 -amino-acid oxidase 126035677 -amino-acid oxidase 123916679 australease-1 145982758 australease-1 145982758 L L L L L phospholipase A L australease-1 145982758 PA-17 precursorphospholipase A 71066780 phospholipase A phospholipase A phospholipase A no. protein 78 79 venom nerve growth factor 1 83288314 82 phospholipase A 80 76 77 spot Table 1. Continued

2451 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 IQCANK PLA IQCANK PLA MS/MS derived sequence protein family ox OX DYGCYCGWGGSGTPVDELDR DYGCYCGWGGSGTPVDELDR PLA DYGCYCGWGGSGTPVDELDR PLA DYGCYCGWGGSGTPVDELDRDYGCYCGWGGSGTPVDELDR PLA PLA DYGCYCGWGGSGTPVDELDR PLA ox ox ox ox ox ox zz / ion m 904.4 3 HYM 761.3 2 APYNDANWNIDTK 894.9 2 TECKDFACACDAAAAK 667.6 3 AAWHYLDYGCYCGPGGR 761.3 2 APYNDANWNIDTK 885.8 2 CCQTHDNCYEQAGK 903.7 3 HYMDYGCYCGWGGSGTPVDELDR 825.9 2 NLIQFGNMIQCANK 844.9 2 CCQVHDNCYEQAGK 793.8 2 CKDFVCACDAAAAK 793.8 2 CKDFVCACDAAAAK 903.7 3 HYMDYGCYCGWGGSGTPVDELDR 894.9 2 TECKDFACACDAAAAK 1070.1 3 GSRPSLDYADYGCYCGWGGSGTPVDELDR 1086.5 2 LTWYSWDCTGDAPTCNPK 1069.5 2 LTLYSWDCTGNVPICNPK 1056 21070.1 LTLYSWDCTGNVPICSPK 3 GSRPSLDYADYGCYCGWGGSGTPVDELDR 1136 2 ITWYSWDCTENVPTCNPK 1142 2 ITWYSWDCTENVPTCNPK peptide a matched peptides score Mascot MW I 8.48 15937 256.1 3 904.47.76 3 15937 HYM 256.1 3 904.4 3 HYM 8.48 15937 412 6 904.4 3 HYM 7.76 15937 280 4 938.9 2 TECKDFTCACDAEAAK PLA 6.84 13941 321.17.53 13914 4 267 833.9 5 2 NLIQFGNM 1069.58.52 14002 2 LTLYSWDCTGNVPICNPK 95.8 28.84 14798 1086.5 197.7 2 LTWYSWDCTGDAPTCNPK 35.61 PLA 13815 1070.8 147.68.52 3 14002 GSRPSLDYADYGCYCGWGGSGTPVDELDR 2 479.58.52 13816 PLA 763.3 6 PLA 439.48.74 2 13755 ATYNDANWNIDTK 793.8 4 435.1 2 CKDFVCACDAAAAK 840.9 56.84 2 13941 NLIQFSNM 1070.8 351.17.53 3 13914 GSRPSLDYADYGCYCGWGGSGTPVDELDR 5 349.97.94 14087 PLA 904.4 4 296 PLA 3 HYM 1069.5 PLA 3 2 LTLYSWDCTGNVPICNPK 653.8 2 VHDECYGEAVK PLA PLA 7.87 16718 244.7 4 1063.5 2 LTLYSWDCTGNVPTCNPK8.6 16900 160.6 3 PLA 884.9 2 CCQVHDNCYEQAGK PLA 8.6 16900 282.9 4 884.96.77 16768 2 CCQVHDNCYEQAGK 246.3 4 904.4 3 HYM PLA 7.87 16718 248.2 4 1063.5 2 LTLYSWDCTGNVPTCNPK PLA with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis code accession 295841605 295841609 295841605 295841609 isozyme PA-3 129447 isozyme PA-5 129452 isozyme PA-13 129474 isozyme PA-12C 129471 isozyme PA-1G 129477 isozyme PA-13 129474 isozyme PA-10A 129397 isozyme PA-11 129415 isozyme PA-3 129447 isozyme PA-5 129452 isozyme PA-9C 129454 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PA-18 precursorphospholipase A phospholipase A 71066782 phospholipase A PA-19 precursor 71066784 phospholipase A phospholipase A PA-19 precursorphospholipase A 71066784 PA-17 precursor 71066780 phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A PA-18 precursor 71066782 no. protein 83 phospholipase A 84 phospholipase A 86 phospholipase A spot Table 1. Continued

2452 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE SVMP SVMP 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 III III MS/MS derived sequence protein family zz / ion m 611.7 2 AHDDCYGEAGK 619.3 2 CTGNVPTCNSK 619.3 2 CTGNVPTCNSK 611.3 2 IVCDCDAAVAK 679.8 2 DFTCACDAEAAK 725.3 2 GTPVDELDRCCK 590.9 2 CCQTHDNCYEQAGK 679.8 2 DFTCACDAEAAK 590.9 3 CCQVHDNCYEQAGK 679.8 2 DFTCACDAEAAK 679.8 2 DFTCACDAEAAK 633.6 3 CCQTHDNCYEQAGKK 590.9 3 CCQVHDNCYEQAGK 938.9 2 TECKDFTCACDAEAAK 679.8 2 DFTCACDAEAAK 632.9 3 CCQTHDNCYEQAGKK 601.8 2 GGSGTPVDELDR 692.3 2 TAAICFAGAPYNK 707.3 2 DDPDYGMVEAGTK 626.2 3 LQHEAQCDSGECCER peptide a matched peptides score Mascot MW 16498 144.8 2 528.2 2 AFICNCDR PLA I 8.78 15724 224.8 4 611.38.78 2 15724 IVCDCDAAVAK 119.57.76 15937 2 186.1 3 450.9 3 AHDDCYGEAGKK 938.9 2 TECKDFTCACDAEAAK PLA 7.76 15937 1927.76 15937 192 3 PLA PLA 626.3 3 3 TECKDFTCACDAEAAK 626.37.76 15937 3 TECKDFTCACDAEAAK 345.3 58.48 15937 761.4 258.35.05 2 14252 APYNDANWNIDTK PLA 3 190.3 PLA 761.3 3 2 APYNDANWNIDTK 761.3 2 APYNDANWNIDTK PLA PLA PLA 8.84 13758 153.58.74 13755 3 150.6 590.2 3 3 CCQVHDNCYEQAGK 590.2 3 CCQVHDNCYEQAGK7.53 13914 120.8 PLA 2 PLA 939.9 2 TECKDFTCACDAEAAK PLA 5 16932 98.4 2 528.2 2 AFICNCDR PLA 5.43 16929 100.5 2 528.26.77 2 16768 AFICNCDR 205.45.23 16878 3 68.9 679.8 26.77 2 16768 DFTCACDAEAAK 268.8 528.2 2 AFICNCDR 4 633.6 PLA 3 CCQTHDNCYEQAGKK PLA PLA PLA 5.45 71238 299.4 6 690.3 2 CPLMTNQCLAR P 6.77 16768 122.1 2 679.8 2 DFTCACDAEAAK PLA 5.45 71238 306.1 6 707.3 2 DDPDYGMVEAGTK P with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Notechis scutatus scutatus Pseudechis australis Pseudechis australis Pseudechis australis Lapemis hardwickii Pseudechis australis Pseudechis australis Oxyuranus microlepidotus Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis code accession 295841613 295841613 295841609 295841595 295841609 295841609 295841609 295841605 24638107 isozyme PA-12A 129458 isozyme PA-11 129415 isozyme PA-5 129452 57 24638081 2 2 2 2 2 2 2 2 2 2 2 2 2 phospholipase A phospholipase A phospholipase A phospholipase A PLA-1 precursor 71066734 PA-17 precursorPLA-3 precursor 71066780 PA-17 precursor 71066722 phospholipase A phospholipase A 71066780 phospholipase A no. protein 87 australease-1 145982758 92 phospholipase A 94 phospholipase A 95 PA-17 precursor96 71066780 phospholipase A 97 phospholipase A 98 phospholipase A 88 australease-1 145982758 90 phospholipase A spot Table 1. Continued

2453 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 MS/MS derived sequence protein family DYGCYCGWGGSGTPVDELDR PLA DYGCYCGWGGSGTPVDELDR OX OX zz / ion m 822.8 2 CKDFVCACDAEAAK 679.8 2 DFTCACDAEAAK 922.9 2 AECKDFVCACDAEAAK 679.8 2 DFTCACDAEAAK 501.2 2 GTPVDELDR 590.9 3 CCQVHDDCYGEAEK 679.8 2 DFTCACDAEAAK 950.4 2 CCQTHDNCYGEAEKK 938.9 2 TECKDFTCACDAEAAK 938.9 2 TECKDFTCACDAEAAK 950.4 2 CCQTHDNCYGEAEKK 885.8 2 CCQTHDNCYEQAGK 501.2 2 GTPVDELDR 682.3 2 ALTMEGNQASWR PLA 1056 2 LTLYSWDCTGNVPICSPK 1056 2 LTLYSWDCTGNVPICSPK 1136 2 ITWYSWDCTENVPTCNPK peptide a matched peptides score Mascot MW 15919 260.6 3 904.4 3 HYM I 7.76 15937 276.2 5 938.9 2 TECKDFTCACDAEAAK7.76 15937 257.2 5 PLA 938.9 27.76 TECKDFTCACDAEAAK 15937 419.4 6 1136 2 ITWYSWDCTENVPTCNPK PLA 7.76 15937 500 6 PLA 1136 2 ITWYSWDCTENVPTCNPK PLA 5.61 13815 256.4 3 763.3 2 ATYNDANWNIDTK6.84 13941 545.1 7 PLA 904.4 3 HYM 6.84 13941 636.6 9 922.9 2 AECKDFVCACDAEAAK8.84 13798 272.18.52 14002 3 114.7 825.9 2 PLA 2 NLIQFGNMIQCANK 649.8 2 DFVCACDAAAAK PLA PLA 6.77 16768 212.98.48 15825 4 1705.43 16929 633.6 100.5 3 3 CCQTHDNCYEQAGKK 3 678.8 2 DFVCACDAEAAK 528.2 2 AFICNCDR PLA 5.75 27138 263 PLA 2 874.9 2 LWNSYCTTTQTFVK PLA 6.77 16768 423.6 5 1356 2 HYMDYGCYCGWGGSGTPVDELDR Nerve growth factor PLA 6.77 16650 463.3 6 1056 2 LTLYSWDCTGNVPICSPK PLA 6.77 16650 375 5 1056 2 LTLYSWDCTGNVPICSPK PLA with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Oxyuranus microlepidotus Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis code accession 295841609 295841609 295841609 295841609 isozyme PA-1G 129477 isozyme PA-3 129477 isozyme PA-3 129477 isozyme PA-12C 129471 isozyme PA-13 129474 2 2 2 2 2 2 2 2 2 PLA-2 precursor 71066792 phospholipase A PA-17 precursorPLA-3 precursorPLA-1 precursor 71066780 71066794 71066734 phospholipase A venom nerve growth factor 83288314 PA-17 precursorphospholipase A 71066780 phospholipase A PA-16 precursor 71066778 phospholipase A PA-16 precursor 71066778 no. protein 99 phospholipase A 102 phospholipase A 100 phospholipase A 101 phospholipase A spot Table 1. Continued

2454 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 IQCANK PLA IQCANK PLA MS/MS derived sequence protein family OX OX DYGCYCGWGGSGTPVDELDR PLA DYGCYCGWGGSGTPVDELDR PLA OX ox zz / ion m 938.9 2 TECKDFTCACDAEAAK 938.9 2 TECKDFTCACDAEAAK 922.9 2 AECKDFVCACDAEAAK 793.8 2 CKDFVCACDAAAAK 939.9 2 TECKDFTCACDAEAAK 885.8 2 CCQTHDNCYEQAGK 894.9 2 TECKDFACACDAAAAK 793.8 2 CKDFVCACDAAAAK 649.8 2 DFVCACDAAAAK 894.9 2 TECKDFACACDAAAAK 763.4 2 ATYNDANWNIDTK 793.8 2 CKDFVCACDAAAAK 793.8 2 CKDFVCACDAAAAK 679.8 2 DFTCACDAEAAK 601.8 2 GGSGTPVDELDR 692.3 2 TAAICFAGAPYNK 1136 21069.5 ITWYSWDCTENVPTCNPK 2 LTLYSWDCTGNVPICNPK 1069.5 2 LTLYSWDCTGNVPICNPK peptide a matched peptides score Mascot MW I 7.76 15937 641.5 88.48 15937 1136 481.3 2 ITWYSWDCTENVPTCNPK 6 904.4 3 HYM PLA 7.87 16718 366.4 56.84 13941 1063.5 310.6 2 LTLYSWDCTGNVPTCNPK 48.48 15937 833.9 354.9 2 NLIQFGNM 3 761.3 PLA 2 APYNDANWNIDTK PLA 6.84 13941 542.5 7 904.48.52 3 13816 HYM 430.77.53 13914 6 359.78.84 13798 840.9 6 291.7 2 NLIQFSNM 1069.5 38.52 2 14002 LTLYSWDCTGNVPICNPK 825.9 251.27.94 2 14087 NLIQFGNMIQCANK 4 235.57.53 13914 649.8 3 520 2 DFVCACDAAAAK 793.8 PLA 7 2 CKDFVCACDAAAAK 840.97.76 15937 2 NLIQFSNMIQCANK 322.6 PLA 5 761.3 2 PLA APYNDANWNIDTK PLA 8.6 16900 244.9 PLA 3 625.6 3 TECKDFTCACDAEAAK PLA PLA 8.52 13816 485.1 6 840.9 2 NLIQFSNMIQCANK PLA 5.05 14252 185.25 16932 3 113.2 761.3 2 2 APYNDANWNIDTK 528.2 2 AFICNCDR PLA PLA 6.77 16768 476.3 5 1356 2 HYMDYGCYCGWGGSGTPVDELDR7.87 16718 276.4 PLA 5 1063.5 2 LTLYSWDCTGNVPTCNPK PLA 7.94 14087 304.6 4 653.8 2 VHDECYGEAVK PLA with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Notechis scutatus scutatus Lapemis hardwickii Pseudechis australis Pseudechis australis Pseudechis australis code accession 295841609 295841605 71066782 129477 295841605 24638107 24638081 isozyme PA-3 129477 isozyme PA-10 A 129397 isozyme PA-5 129452 isozyme PA-12C 129471 isozyme PA-13 129474 isozyme PA-9C 129454 isozyme PA-5isozyme PA-10 A 129452 isozyme PA-3isozyme PA-9C 295841609 57 71066784 1069.5 2 LTLYSWDCTGNVPICNPK 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PA-18 precursor 129397 phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A PA-17 precursor 71066780 phospholipase A PA-18 precursorphospholipase A phospholipase A 71066782 phospholipase A PA-19 precursorphospholipase A phospholipase A 129454 phospholipase A phospholipase A phospholipase A no. protein 103 phospholipase A 104 phospholipase A spot Table 1. Continued

2455 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 IQCANK PLA IQCANK PLA IQCANK IQCANK PLA MS/MS derived sequence protein family OX OX OX OX zz / ion m 894.9 2 TECKDFACACDAAAAK 939.9 2 TECKDFTCACDAEAAK 938.9 2 TECKDFTCACDAEAAK 793.8 2 CKDFVCACDAAAAK 592.3 2 FVCACDAAAAK 793.8 2 CKDFVCACDAAAAK 793.8 2 CKDFVCACDAAAAK 484.2 2 VHDDCYGEAEKK 601.8 2 GGSGTPVDELDR 675.8 2 CTENVPICDSR 718.3 2 VHDDCYDQAGKK 904.4 3 HYMDYGCYCGWGGSGTPVDELDR 793.8 2 CKDFVCACDAAAAK 793.8 2 CKDFVCACDAAAAK 654.3 2 VHDDCYDQAGK 833.9 2 NLIQFGNM 635.8 2 SFVCACDAAAAK peptide a matched peptides score Mascot MW I 7.76 15937 285 4 761.3 2 APYNDANWNIDTK8.48 15937 190.2 35.05 14252 761.3 143.6 2 APYNDANWNIDTK PLA 2 761.3 2 APYNDANWNIDTK8.48 PLA 15937 241.77.76 15760 3 215.6 PLA 904.4 3 3 HYMDYGCYCGWGGSGTPVDELDR 904.4 3 HYMDYGCYCGWGGSGTPVDELDR PLA PLA 7.53 13914 295.6 48.52 13816 840.9 279.6 28.84 NLIQFSNMIQCANK 13798 5 221.27.94 14087 840.9 3 194.5 2 NLIQFSNMIQCANK 825.9 4 26.84 NLIQFGNMIQCANK 13941 653.8 188.1 2 PLA VHDECYGEAVK 37.94 14087 833.9 340.2 2 PLA 8.52 NLIQFGNM 13816 5 308.8 PLA 6.84 13941 793.8 4 291.5 28.52 CKDFVCACDAAAAK 14002 793.8 4 PLA 281 2 CKDFVCACDAAAAK 833.9 3 2 NLIQFGNM 649.88.84 2 13758 DFVCACDAAAAK 209.5 PLA 8.74 13755 3 205.1 PLA 635.8 3 2 SFVCACDAAAAK 833.9 2 NLIQFGNM PLA PLA 7.87 16718 298.2 5 1063.5 2 LTLYSWDCTGNVPTCNPK PLA with a homology protein from p Pseudechis australis Pseudechis australis Notechis scutatus scutatus Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis code accession 295841609 295841605 24638107 295841605 295841609 isozyme PA-5 129452 isozyme PA-10 A 129397 isozyme PA-12C 129471 isozyme PA-9C 129454 isozyme PA-3 129477 isozyme PA-9C 129454 isozyme PA-10 A 129397 isozyme PA-3 129477 isozyme PA-13 129474 isozyme PA-12 A 129458 isozyme PA-11 129415 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A no. protein 105 PA-18 precursor 71066782 106 phospholipase A spot Table 1. Continued

2456 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 IQCANKIQCANK PLA IQCANK PLA PLA IQCANKIQCANK PLA PLA IQCANK MS/MS derived sequence protein family OX OX OX OX OX OX zz / ion m 619.3 2 CTGNVPTCNSK 844.9 2 CCQVHDNCYEQAGK 884.9 2 CCQVHDNCYEQAGK 793.8 2 CKDFVCACDAAAAK 718.3 2 VHDDCYDQAGKK 793.8 2 CKDFVCACDAAAAK 763.3 2 ATYNDANWNIDTK 654.3 2 VHDDCYDQAGK 680.3 3 IHDDCYIEAGKDGCYPK 844.9 2 CCQVHDNCYEQAGK 884.8 2 CCQVHDNCYEQAGK 826.3 2 CTGNAPTCNSKPGCK 793.8 2 CKDFVCACDAAAAK 625.6 3 TECKDFTCACDAEAAK 793.8 2 CKDFVCACDAAAAK 938.9 2 TECKDFTCACDAEAAK 833.9 2 NLIQFGNM 633.3 2 IDTACVCVISK PLA 1086.5 2 LTWYSWDCTGDAPTCNPK 1003.5 2 GCYPVLTLYSWECTEK 1067.5 2 KGCYPVLTLYSWECTEK peptide a matched peptides score Mascot MW I 8.78 15724 449.2 6 1067.5 2 KGCYPVLTLYSWECTEK8.48 15937 278.3 47.76 15760 PLA 197.4 761.3 2 APYNDANWNIDTK 3 761.3 2 APYNDANWNIDTK PLA 8.48 15937 PLA 325.8 4 761.37.76 15760 2 APYNDANWNIDTK 188.6 2 761.3 2 APYNDANWNIDTK PLA PLA 8.52 14002 485.4 78.74 13755 793.8 364.38.84 13758 2 CKDFVCACDAAAAK 5 355.77.94 14087 5 325.2 833.98.52 13816 2 NLIQFGNM 3 298.8 833.9 2 NLIQFGNM 5 653.86.84 13941 2 VHDECYGEAVK 793.8 246.7 PLA 2 CKDFVCACDAAAAK 4 833.9 2 NLIQFGNM 8.52 14002 428 PLA 8.74 PLA 13755 411.88.84 5 13758 4 406.8 1086.58.84 13798 2 LTWYSWDCTGDAPTCNPK 5 406.3 833.9 2 NLIQFGNM 4 833.9 2 NLIQFGNM 885.87.94 14087 2 CCQTHDNCYEQAGK 221.2 PLA 35.61 13815 157 778.4 2 APYNKDNYNIDTK 2 PLA 763.4 2 ATYNDANWNIDTK PLA PLA 8.33 15667 130.8 2 501.2 2 GTPVDELDR 8.59 16783 538.5 7 1003.5 2 GCYPVLTLYSWECTEK PLA 6.94 26736 163.9 2 682.3 2 ALTMEGNQASWR8.6 16900 Nerve growth factor 224.5 3 884.9 2 CCQVHDNCYEQAGK PLA with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis porphyriacus Pseudechis australis Pseudechis porphyriacus Pseudechis australis code accession 295841613 295841605 295841609 295841605 295841609 isozyme PA-13 129474 isozyme PA-11 129415 isozyme PA-12 A 129458 isozyme PA-9 C 129454 isozyme PA-10 A 129397 isozyme PA-3 129477 isozyme PA-13 129474 isozyme PA-11 129415 isozyme PA-12 A 129458 isozyme PA-12 C 129471 isozyme PA-9 C 129454 isozyme PA-1 G 129477 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 PLA-4 precursor 71066796 phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A phospholipase A venom nerve growth factor 83288328 phospholipase A phospholipase A phospholipase A phospholipase A PA-19 precursorphospholipase A phospholipase A 71066784 phospholipase A phospholipase A phospholipase A phospholipase A no. protein 108 PA-20 precursor 71066788 107 phospholipase A spot Table 1. Continued

2457 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE 2 2 2 2 2 2 2 2 2 2 inhibitor IQCANKIQCANK PLA MS/MS derived sequence protein family OX OX zz / ion m 826.3 2 CTGNAPTCNSKPGCK 793.8 2 CKDFVCACDAAAAK 718.3 2 VHDDCYDQAGKK 763.3 2 ATYNDANWNIDTK 833.9 2 NLIQFGNM 679.8 2 DFTCACDAEAAK 793.8 2 CKDFVCACDAAAAK 661.8 2 VHDDCYGEAEK 698.8 2 FCELPPDSGSCK 1136 2 ITWYSWDCTENVPTCNPK 1136 2 ITWYSWDCTENVPTCNPK peptide a matched peptides score Mascot MW I 7.76 15937 394.1 68.48 15937 938.9 318 2 TECKDFTCACDAEAAK 5 761.3 2 APYNDANWNIDTK PLA PLA 8.84 13798 351 58.52 13816 316.5 885.8 2 CCQTHDNCYEQAGK 56.84 13941 793.8 252.88.74 2 13755 CKDFVCACDAAAAK 4 252.7 833.9 38.52 2 14002 NLIQFGNM PLA 844.9 196.7 2 CCQVHDNCYEQAGK 4 PLA 649.8 2 DFVCACDAAAAK PLA PLA 6.77 16768 265.3 4 679.8 2 DFTCACDAEAAK7.87 16718 136.1 3 894.9 PLA 2 TECKDFACACDAAAAK PLA 6.04 9549 212.6 3 1069.4 2 TCLEFIYGGCEGNDNNFK Serine proteinase 8.6 16900 231.9 3 884.9 2 CCQVHDNCYEQAGK PLA with a homology protein from p Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis Pseudechis australis code accession 295841609 295841605 isozyme PA-12 C 129471 isozyme PA-10 A 129397 isozyme PA-3 129447 isozyme PA-11 129415 isozyme PA-13 129474 2 2 2 2 2 2 2 phospholipase A phospholipase A PA-17 precursorphospholipase A 71066780 phospholipase A PA-18 precursor 71066782 phospholipase A phospholipase A PA-19 precursor 71066784 no. protein 109 mulgin-2 82201571 110 phospholipase A spot Here the real number of the matched peptides is shown. Only representative peptide sequences are shown in the next column. Table 1. Continued a

2458 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE

Table 2. Summary of the Protein Families in the Pseudechis australis Venom Identified after 2-DE

spot no. homologous protein homology with protein from protein family

15; 726; 29, 3035; asrin Austrelaps superbus PIII metalloprotease 3641; 42, 4447; 5974; porphyricase-1 Pseudechis porphyriacus PIII metalloprotease 7577, 80, 87, 88 australease-1 Pseudechis australis PIII metalloprotease scutatease 1 Notechis scutatus PIII metalloprotease scutellatease Oxyuranus scutellatus PIII metalloprotease 32, 33, 35, 4758; 60, 61 L-amino acid oxidase Pseudechis australis L-amino acid oxidase Oxyuranus scutellatus L-amino acid oxidase Naja atra L-amino acid oxidase Austrelaps superbus L-amino acid oxidase 90, 94 108; 110 Pa-1G, Pa3, Pa-5, Pa-9C, Pa-10A, Pa-11, Pa-12A, Pa-12C, Pseudechis australis phospholipase A2 Pa-13, Pa-16, Pa-17, Pa-18, Pa-19

PLA2 isoforms Oxyuranus microlepidotus phospholipase A2

Notechis scutatus scutatus phospholipase A2

Lapemis hardwickii phospholipase A2

Pseudechis porphyriacus phospholipase A2 4446 transferrin Lamprophis fuliginosus transferrin 65, 69, 70 ecto-50-nucleotidase Gloydius b. brevicaudus 50-nucleotidase 79, 101, 107 nerve growth factor Pseudechis australis nerve growth factor nerve growth factor Pseudechis porphyriacus nerve growth factor 109 mulgin Pseudechis australis serine protease inhibitor

the respective segments in the body transferrin of the African house snake Lamprophis fuliginosus.53 The exception is a se- quence that has partial identity to the respective segment of the body protein. The snake plasma protein and the three venom TFLPs possess identical molecular weights. This result suggests recruitment of body transferrin into the snake venom. Compar- ison of the five P. australis peptides with the sequence of human plasma transferrin54 showed identity of the sequences CGLVPXL and LFGSXXT.

Phospholipases A2 The proteomic analysis showed a large diversity of PLA2sin the venomics of P. australis (Figure.1, Table 1). These enzymes represent 18.5% of all identified toxins (Figure 2). Acidic and Figure 2. Percent of toxin sequences found in the Pseudechis australis basic monomeric PLA2s were isolated from spots 90, 94 108 venom proteome. and 110. These spots form a long of proteins with molecular weights of approximately 16 kDa in the pI interval from 5 to 9, fi 5358 kDa. LAAOs with pI values in the acidic region, from 3.7 again due probably to post-translational modi cations. The fi to 4.3 and molecular masses of 5665 kDa were identified in identi ed enzymes likely belong to Group I since elapid snake spots 32, 33, and 35 (Figure 1, Table 1). Peptides from proteins venom PLA2s are members of this group. Most probably, 13 of with molecular weight of 80 kDa (spots 4246) showed the proteins correspond to those isolated from the P. australis sequence similarity to other snake venom LAAOs. However, venom PLA2s labeled as Pa-1G, Pa-3, Pa-5, Pa-9c, Pa-10A, Pa-11, 5558 the molecular weight of these proteins is not characteristic of the Pa-12A, Pa-12c, Pa-13, Pa-16, Pa-17, Pa-18, and Pa-19. The monomeric oxidases, since these proteins oligomerize.51 All others are isoforms of these enzymes, which have not been detected isoforms have a sequence similarity to both LAAOs characterized before, with different pI values and/or molecular 52 isolated previously from the P. australis venom. Peptides from masses. It should be mentioned that PLA2s isolated from spots the proteins in spots 32 and 60 have a sequence similarity to the 90100 are acidic proteins with pI values between 3.6 and 6.6 LAAOs from the Oxyuranus scutellatus scutellatus and Naja atra (Figure 1). Usually, the acidic PLA2s are neither catalytically venoms. active (or possess very low enzymatic activity) nor neurotoxic.59 Isoforms of transferrin-like proteins were identified from spots However, Pa-1G is an exception to this rule and it is the first μ 44, 45, and 46. The three proteins are in a horizontal in the pI acidic phospholipase A2 with high neurotoxicity (0.13 gs/g range of 7.57.8 (Figure 1). They have the same molecular body wt).55 One peculiarity of the P. australis venomics is the ff masses of 80 kDa and di erent isoelectric points, which suggests high content of acidic PLA2s, while the single chain phospholi- possible post-translational modifications. All but one sequences polytic enzymes from other Australian elapids are almost exclu- 31 of the isolated tryptic peptides (Table 1) are 100% identical to sively basic. Basic PLA2s are highly homologous in their amino

2459 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE

Table 3. Enzymatic Activities of the Pseudechis australis Venom and Comparison of Elapidae and Snake Venom activities

species PLA2 U/mg proteinase U/mg LAAO U/g alkaline phosphatase U/g acidic phosphatase U/g

Pseudechis australis 11.44 0.96 100 360 0 Bothropsa alternatus 3.20 1.12 70 180 0 Crotalusa durissus terrificus 5.90 0.13 0 460 0 Viperaa ammodytes ammodytes 4.30 0.33 50 400 0 Viperaa ammodytes meridionalis 6.75 0.27 100 400 0 Daboiaa russelli siamensis 13.42 0.06 40 240 0 a Data from ref 43 and references therein. acid sequences and, at the same time, differ considerably in their ’ DISCUSSION catalytic properties and toxicity. Thus, Pa-11 is enzymatically 30 0 PIII Metalloproteases, Phospholipases A and 5 -Nucleo- times more active than Pa-13 and considerably more toxic than 2 57 tidases in Relation to the Pharmacological Activities of the the second protein. Pa-13 showed no lethal activity at a dose P. australis Venom μ 56 60 of 7.4 g/g mouse and Pa10A is another lethal PLA2. Australian elapid snakes are among the most toxic in the Isoforms with higher molecular masses were isolated from 31 ff world. The major pathological e ects of the P. australis spots 82 84, 86 (basic proteins with molecular masses of envenomation are severe disruption of hemostasis,31,61 muscle 3234 kDa) and 92 (also basic protein with molecular mass 62 fi damage and necrosis. Mulga is a member of the nonprocoa- of 45 46 kDa). The enzymes from the rst group are dimers gulant group of elapid snakes.31 The coagulopathy should be because their molecular masses correspond to that of two-chain attributed to the PIII SVMPs which predominate in the complexes. The protein in spot 92 could be an isoform of a venomics of P. australis (53% of all identified toxins). The class multichain PLA2. Australian snakes contain such proteins; for III metalloproteases are composed of metalloprotease, disinte- example, the 45.6 kDa taipoxin, the principle toxin of the O. s. grin-like and cysteine-rich domains.28 The metalloprotease do- 31 fi scutellatus venom. The identi ed PLA2 isoforms showed main is responsible for the degradation of matrix proteins while sequence homology to phospholipolytic enzymes from the the nonprotease domains exert anticoagulant effects.63 The venoms of Oxyuranus microlepidotus, Notechis scutatus scutatus, cysteine-rich domain inhibits the collagen-stimulated platelet Lapemis hardwickii,andPseudechis porphyriacus. aggregation.63 The PIII-enzymes induce also muscle damage and myonecrosis.64 In this way the metalloproteases contribute Other Proteins significantly to the pathogenesis of the P. australis induced A serine protease inhibitor was isolated from spot 109 of the envenomation. 2-D gel (Figure 1, Tables 1 and 2). This is a basic polypeptide of The other feature of the investigated venom composition is 9 kDa molecular mass and pI value of 7.4. It is homologous to the absence of serine proteases including enzymes with throm- mulgin-2, a 9.2 kDa protein with serine- endopeptidase bin-like activity. The severe disruption of hemostasis caused by inhibitor activity (GenBank: AAT4540.1). Three isoforms of the P. australis bites proceeds without fibrinolysis,31 which is in ecto-50-nucleotidase with pI values between 8.5 and 8.7 were line with the lack of serine proteases/fibrinogenases in the venom identified in the spots 65, 69, and 70. Spots 79, 101, and 107 proteome. contain isoforms of venom nerve growth factor (Figure 1, The severe coagulopathic effect, caused by the PIII metallo- Tables 1 and 2). proteases, is strengthened by the high quantities of PLA2s, the third largest group of toxins in the venomics of P. australis. Enzymatic Activities Anticoagulant phospholipases A2 can bind and block factors of the coagulation cascade.31 A hemotoxic PLA , potent inhibitor of We determined phospholipase A2, proteinase, L-amino acid 2 the platelet aggregation, was isolated from the venom of another oxidase, alkaline phosphatase and acidic phosphatase activities of 65 the Pseudechis australis venom. The results are presented in Australian elapid, Austrelaps superbus. It is homologous to an Table 3. The enzymatic activities of the Elapidae snake P. australis enzyme from the P. australis venom (Table 1). venom are compared with the respective activities of Viperidae The king brown snake venom caused rhabdomyolysis fol- lowed by myoglobinuria and nephropathy.62 Neurotoxicity can snakes. The data are comparable because the activities were be supposed due to the presence of PLA s in the venom. determined using the same methods and equipment. The venom 2 However, myotoxicity is the major pharmacological effect fol- PLA2 activity of the Elapidae snake is considerably higher than lowing the P. australis bites.66 This can be explained by a strong that of alternatus, Crotalus d. terrificus, Vipera a. ammo- and direct myotoxic action of a large quantity of PLA2s on the dytes, and Vipera a. meridionalis, but similar (even less) than the muscles. Myotoxicity is independent of the enzymatic activity.67 phospholipolytic activity of the Daboia russelli siamensis venom Analysis of the structurefunction relationships and crystal- (Table 3). The venom proteinase activity is similar to that of the lographic investigations on snake venom PLA2s demonstrated B. alternatus venom, but considerably higher than that of the that the C-terminal part of the polypeptide chain, an exposed other Viperidae snakes. Both P. australis and Vipera a. ammodytes hydrophobic surface and interfacial surface charge are important venoms show the highest LAAO activity. The alkaline phospha- structural determinants of the myotoxicity.68,69 Investigations of fi tase activity of the mulga venom is similar to the activities of the the action of ve P. australis venom PLA2s on nerves and muscles other snakes. No acidic phosphatase activity was detected. demonstrated that the predominant pharmacological effect is

2460 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE myotoxicity.70 However, neuromuscular effects of some isolated bacterial survival, that is, transferrin has a bactericidal effect. king brown snake venom phospholipases A2 have been Transferrins play a major role in iron transport and defense observed.60,71 These toxins produce muscle paralysis by reducing against microbial pathogens.53 One of the reasons for the acetylcholine release60 or act postsynaptically to depress the incorporation of TFLPs into the snake venom could be strength- 71 ff muscle contractility. The high content of PLA2s is in accor- ening of the antimicrobial e ect of the venom. dance with the myotoxic effects of the P. australis .66 Enzymatic Activities PIII metalloproteases also contribute significantly to the The snake venom enzymatic activities contribute considerably myotoxicity. 0 to the total toxic effect. For this reason they are an important 5 -Nucleotidases inhibit the platelet aggregation via increased 72 characteristic of the venom. The relatively high PLA activity of adenosine signaling acting as anticoagulants. In this way the 2 the king brown snake venom is in agreement with its destructive three isoforms described in Table 1 strengthen additionally the effects on the body tissues. PLA s hydrolyze membrane phos- coagulopathic effects of the P. australis venom. 2 pholipids and liberate lysophospholipids and fatty acids, includ- ing arachidonate. In this way they exert pathological effects on Adaptation of the P. australis venom for defense against the prey. The damage of biological membranes leads to changes microbial pathogens. Recruitment of body transferrin into 77 the snake venom in the permeability to ions and drugs. On the other hand lysophospholipids are involved in cell lysis77 and arachidonate is The venomics of mulga snake reveal a high content of a precursor of mediators of inflammation such as thromboxanes, antibacterial proteins, LAAOs and transferrin-like proteins prostaglandins and leukotriens.78 The catalytic activity of PLA (22.5% of the identified proteins). Potent antibacterial activity 2 leads to a serious disturbance of important physiological pro- of the P. australis venom was demonstrated against Gram- cesses in the prey. Taking into consideration the relatively high positive and Gram-negative bacteria.52 This snake feeds upon content of these enzymes in P. australis, it can be concluded that frogs containing the Aeromonas hydrophila,73 a heterotrophic, phospholipolytic enzymes play an important role in the life Gram-negative bacterium widespread among amphibians and threatening effects caused by the mulga snakebite. fish. The pathogen is toxic to many organisms and can survive in The high LAAO activity corresponds to the large quantity of aerobic and anaerobic environments. The P. australis venom these enzymes in the investigated venom. The catalytic activity of showed the highest antibacterial activity toward A. hydrophila these enzymes results in the formation of the highly cytotoxic among 21 tested Elapidae snake venoms,52 which correlates with hydrogen peroxide, which accounts for the strong antimicrobial features of the snake diet. The presence of a large diversity of effect of the P. australis venom. L-Amino acid oxidases induce LAAO isoforms in the venomics of the king brown snake (20% of necrotic and apoptotic cell death.75 Probably, this effect is used the identified proteins) can account for the bactericidal effects of by the snake as a defense against pathogens from the prey. the venom because these enzymes are active against various 52,74,75 Of course, LAAOs contribute to the total toxicity of the venom bacteria. L-Amino acid oxidases exert their antibacterial aimed at the killing of the small used as food. effect through the hydrogen peroxide liberated after the oxidative The proteolytic activity of the P. australis venom is higher than deamination of amino acids. Two L-amino acid oxidases, LAO1 that of a number of Viperidae snake venoms but, in principle, it is and LAO2, were isolated from the venom of P. australis.52 Both not as high as it can be expected from the large quantity of enzymes possess subunit molecular masses of 56 kDa and form metalloproteases. Most probably, the low level of this activity is aggregates of 142 kDa. The correlation of the subunit molecular due to the high specificity of the PIII metalloproteases in the masses with those of the proteins from spots 32 and 35 suggests a venom, hydrolyzing a limited number of peptide bonds. possible identity with the two LAAOs, described in the paper The individual variations in the alkaline phosphatase activity mentioned above. It is known that LAAOs oligomerize in among the snake venoms, compared in Table 3, are not drastic water.51 Most probably, the aggregates dissociate under the and hardly can influence the total toxicity. conditions used for the 2-DE. The pathogen A. hydrophila, present in a high concentration in frogs which comprise a ’ significant part of the P. australis diet, was the most sensitive CONCLUDING REMARKS bacterium tested with venom LAAOs (LAO1 and LAO2) from The venom composition of P. australis demonstrates a highly the same snake.52 specialized biosynthesis of large quantities of antibacterial toxins The presence of three transferrin isoforms in the P. australis and proteins disrupting the hemostasis or exerting myotoxic venom demonstrates recruitment of a body protein into the effects. The results of the venom proteome analysis point to an snake venom. This result supports the theory that the snake adaptation of the venomic system for tissue destruction, blood venom toxins evolve from recruitment of body proteins into coagulation blockade and a defense against microbial pathogens the chemical arsenal of the snake.20 The high degree of sequence from the prey. The last hypothesis is supported by the obvious similarity between the body transferrin, found in the liver of the relationship between the presence of potent antimicrobial pro- African house snake Lamprophis fuginosus (a colubrid snake) and teins in the venom, its bactericidal effects and the bacterial the TFLPs in the venom of the Australian P. australis is surprising. contamination of the food used in the snake diet. The antibac- Both snakes inhabit different continents. Moreover, the trans- terial activity can also prevent bacterial infections from the buccal ferrins mentioned above have the same molecular masses as the cavity into the venom gland. To our knowledge, the body human protein. Transferrin is a blood plasma protein for iron transferrin is unknown as a recruited component of the elapid delivery to the tissues,76 associated with the innate immune or other snake venoms. A possible role of the venom transferrin system. It is produced mainly in the liver. A possible explanation could be strengthening of the antimicrobial effect. The high of the transferrin physiological role as a venom protein is degree of sequence homology between the body transferrin of connected with the metal binding properties of this protein. the Colubridae African house snake Lamprophis fuliginosus and þ The binding of Fe3 makes the environment unsuitable for the the transferrin-like proteins from the Australian snake Pseudechis

2461 dx.doi.org/10.1021/pr101248e |J. Proteome Res. 2011, 10, 2440–2464 Journal of Proteome Research ARTICLE australis (Elapidae) venom is surprising. Both snakes inhabit (8) Angulo, Y.; Escolano, J.; Lomonte, B.; Gutierrez, J. M.; Sanz, L.; distant regions with a very little likelihood for interbreeding. Calvete, J. J. Snake venomics of Central American pitvipers: clues for The horizontal spot s of proteins belonging to the predomi- rationalizing the distinct envenomation profiles of Artropoides nummifer and Atropoides picadoi. J. Proteome Res. 2008, 7, 708–719. nant families of SVMPs, PLA2s and LAAOs are a characteristic feature of the 2-D gel and suggest post-translational modifica- (9) Alape-Giron, A.; Sanz, L.; Escolano, J.; Florez-Díaz, M.; Madrigal, M.; tions of these enzymes. Sasa, M.; Calvete, J. J. Snake venomics of the lancehead pitviper Bothrops asper. Geographic, individual, and ontogenetic variations. J. Proteome Res. Pseudechis australis has a large venom output, up to 150 mg in 2008, 7, 3556–3571. one bite, and represents a rich source of pharmacologically active (10) Sanz, L.; Ayvazyan, N.; Calvete, J. J. Snake venomics of the compounds. Knowledge of the venomic composition reveals Armenian mountain vipers Macrovipera lebetina obtuse and Vipera raddei. possibilities for the preparation of a more efficient , for J. Proteomics 2008, 71, 198–209. adequate treatment of the consequences of the snakebite and (11) Tashima, A. K.; Sanz, L.; Camargo, A. C.; Serrano, S. M.; for the design of new medicines. Large quantities of proteins Calvete, J. J. Snake venomics of the Brazilian pitvipers Bothrops cotiara influencing the hemostasis or with antibacterial properties can be and Bothrops fonsecai. J. Proteomics 2008, 71, 473–485. fi (12) Sanz, L.; Escolano, J.; Ferretti, M.; Biscoglio, M. J.; Rivera, E.; obtained from this venom for medical, scienti c and biotechno- logical purposes. The venomic composition of P. australis is Crescenti, E. J.; Angulo, Y.; Lomonte, B.; Gutierrez, J. M.; Calvete, J. J. relevant to the pathologies associated with the snakebites, in Snake venomics of the South and Central American Bushmasters. Comparison of the toxin composition of Lachesis muta gathered from particular to the hemostatic disorders and myotoxicity. proteomic versus transcriptomic analysis. J. Proteomics 2008, 71, 46–60. ’ AUTHOR INFORMATION (13) Calvete, J. J.; Borges, A.; Segura, A.; Flores-Diaz, M.; Alape- Giron, A.; Gutierrez, J. M.; Diez, N.; De Sousa, L.; Kiriakos, D.; Sanchez, Corresponding Author E.; Faks, J. G.; Escolano, J.; Sanz, L. Snake venomics and antivenomics of *Tel.: þ494089984744. Fax: þ494089984747. E-mail: Christian. Bothrops colombiensis, a medically important pitviper of the Bothrops [email protected]. atrox-asper complex endemic to Venezuela: Contributing to its taxon- omy and snakebite management. J. Proteomics 2009, 72, 227–240. 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