pseudechis australis venomics: adaptation for a defense ... · 1 min with an isolation width of 4...

Published: March 22, 2011

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

ARTICLE

pubs.acs.org/jpr

Pseudechis australis Venomics: Adaptation for a Defense againstMicrobial Pathogens and Recruitment of Body TransferrinDessislava 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 andInflammation, 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 Energ�eticas e Nucleares, Av. Lineeu Prestes 2242, 05508-000 S~ao Paulo, Brazil^Department of Physics, IBILCE/UNESP, Crist�ov~ao Colombo 2265, CEP 15054-000, S~ao Jos�e do Rio Preto, SP Brazil#Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria

’ INTRODUCTION

Human envenoming by poisonous snakes is of public healthsignificance and a serious medical problem. Snake venoms arevery rich and incompletely explored sources of pharmacologi-cally important compounds. The best example for the develop-ment of pharmaceutical products based on investigations onvenom compounds is captopril, an inhibitor of the angiotensinI-converting enzyme (ACE inhibitor), used for treatment ofhypertension.1 The knowledge of the venom composition isnecessary for the improvement of antivenoms used for theneutralization of snakebite consequences, for quality control ofvenom preparations2 and for structure-based design of noveldrugs, especially for the blood pressure regulation and thetreatment of coagulopathy. Snake venom components have asignificant potential for clinical applications as diagnostic agents.3

The venomics of a large number of viperid snakes have beeninvestigated with respect to their pharmacological and medical

application.2,4�15 During the past decade, investigations werefocused on the molecular origin and evolution of the snakevenom proteome.16�23 Methods for the venom proteome anal-ysis with special attention to the structure, function and role ofmetalloproteinases in the viperid snakebite pathogenesis weredeveloped.24�28 Proteomic and transcriptomic approaches weresuccessfully combined in investigations on the venom composi-tions of South American snakes.29,30

Australian elapid snakes are among the most venomous in theworld.31 Their bites cause morbidity and in some casesmortality.32 However, in comparison to viperid snakes, consider-ably less information about the elapid venom proteome isavailable. Venom compositions of Naja naja atra,33 Pseudonajatextilis,34 Micrurus surinamensis (fish eating coral snake)35 and

Received: December 16, 2010

ABSTRACT: The venom composition of Pseudechis australis, awidely distributed in Australia reptile, was analyzed by 2-DE andmass spectrometric analysis. In total, 102 protein spots wereidentified as venom toxins. The gel is dominated by horizontaltrains of spots with identical or very similar molecular massesbut differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostaticcharge. The results demonstrate a highly specialized biosynth-esis of toxins destroying the hemostasis (P�III metallopro-teases, SVMPs), antimicrobial proteins (L-amino acid oxidases,LAAOs, and transferrin-like proteins, TFLPs), and myotoxins(phospholipase A2s, PLA2s). The three transferrin isoforms ofthe Australian P. australis (Elapidae snake) venom are highlyhomologous to the body transferrin of the African Lamprophisfuliginosus (Colubridae), an indication for the recruitment ofbody 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 enzymaticactivities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, andbiotechnological purposes.

KEYWORDS: snake venomics, Pseudechis australis, 2-D electrophoresis, electrospray mass spectrometry, venom transferrin,enzyme activity

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2441 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-DPAGE of Australian snake venoms were analyzed and novelproteins identified.37 Transcriptomic approaches were appliedfor analyzing venom gland genes of Oxyuranus scutellatus,38

Micrurus corallinus39 and Bungarus flaviceps.40

Snakes of the genus Pseudechis, also known as “Black Snakes”or “Bongani Sibanda”, are widespread in all Australian statesexcept for Tasmania. Nine species were recognized: P. australis(King brown snake or mulga snake), P. butleri (Spotted mulgasnake), P. collettii (Collett’s snake), P. guttatus (Blue-bellied blacksnake), P. papuanus (Papuan black snake), P. pailsi, P. porphyr-iacus (Red-bellied black snake), P. rossignolii (Papuan dwarf kingbrown) and P. weigeli (Pygmy mulga snake).41,42 P. australis isone of the longest venomous snakes in the world (2.5�3 m inlength) and is encountered in most Australian states except forVictoria and Tasmania.

In the present paper, we report the proteomic profile of thePseudechis australis venom. The venom components were ana-lyzed by 2-D gel electrophoresis and electrospray mass spectro-metry, and classified into protein families.

’MATERIALS AND METHODS

Collection of the VenomCrude venom, pooled from several specimens of Pseudechis

australis, was a kind gift of Dr. P. Mirtschin (Venom Supplies Pyt.Ltd., Australia). Snakes of both genders were milked and thelyophilized venom was stored at 4 �C.

2-D Gel Electrophoresis and Electrospray Mass Spectrom-etry

Two-dimensional electrophoresis was performed as describedpreviously.43 The venom was suspended in deionized water anddesalted using an Amicon-Ultra 0.5 mL filter (Millipore) with a10 kDa cutoff prior to the 2-D electrophoresis. 200 μg of the totalprotein were mixed with 135 μL DeStreak solution (GEHealthcare) and 0.5% IPG (immobilized pH gradient), pH 3 �10, in nonlinear (NL) buffer (v/v) (GE Healthcare, Uppsala,Sweden). The sample was agitated for 15 min at room tempera-ture and centrifuged for 30 min at 13 000 rpm to removeprecipitates. The equilibration was performed with the super-natant loaded on 7 cm Immobiline DryStrip pH 3�10 NL (GEHealthcare, Uppsala, Sweden). In the first dimension, proteinswere separated by an IPGphore electrophoresis unit overnight(GEHealthcare, Uppsala, Sweden). After isoelectric focusing thestrips were equilibrated for 15 min in equilibration buffercontaining 0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v),6 M urea, 4% sodium dodecyl sulfate and 2% dithioerythrithol.In a second equilibration step the strips were incubated with0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v), 6 M urea,4% sodium dodecylsulfate and 2.5% iodoacetamide for 15 min.The strips were stored at �20 �C until used in the seconddimension, performed on a 10% Tris-tricine-polyacrylamide gel(100 � 100 � 1.0 mm3).44

After electrophoresis both gels were stained overnight withcoomassie brilliant blue and destained as described previously.45

Gels were scanned and imported into the software Delta2Dsoftware package (Decodon, Greifswald, Germany).

Protein spots of interest were cut from the polyacrylamide gelsand digested overnight using trypsin (Sigma, Munich, Germany)according to the protocol from Shevchenko, modified in aprevious study.46 Peptides were desalted (ZipTip pipet tips,

Millipore) and reconstituted in 0.1% formic acid. Samples wereinjected by an autosampler and concentrated on a trappingcolumn (nanoAcquity UPLC column, C18, 180 μm � 2 cm,5 μm, Waters) with water containing 0.1% formic acid at flowrates of 15 μL/min. After 2 min the peptides were eluted onto aseparation column (nanoAcquity UPLC column, C18, 75 μm x10 cm, 1.75 μm, Waters). Chromatography was performed with0.1% formic acid in solvent A (100% water) and B (100% ACN).The peptides were eluted over 8 min with 20 � 80% solvent Bgradient using a nano-HPLC system (nanoAcquity, Waters)coupled to an LTQ-Orbitrap mass spectrometer (Thermo FisherScientific). The capillary voltage in MS andMS/MS experimentswas set to 2000 V. The collision gas was helium at a pressure of0.1 MPa, and the collision energy was 40 V. For an unbiasedanalysis, continuous scanning of eluted peptide ions was carriedout between 300 and 2000 m/z, automatically switching toMS/MS CID mode on ions exceeding an intensity of 5000.For MS/MS measurements, a dynamic precursor exclusion of1 min with an isolation width of 4 m/z was used.

Raw data were applied to a database search using ThermoProteome Discoverer software (v1.0 build 43) to carry out atandem ion search algorithm from the MASCOT house server(v2.2.1) by database comparison against all chordata entries inthe National Center for Biotechnology Information (NCBInrdatabase 2010) with 10 ppm tolerance for the precursor and0.8 Da for MS2 fragments. Further, trypsin with a maximum oftwo missed cleavages was selected and variable modifications,such as methionine oxidation and carbamidomethylation ofcysteine, were allowed. Peptides were considered to be identifiedby Mascot when a probability <0.05 (probability based ionthreshold scores >40) was achieved. Proteins were consideredto be identified if at least two peptides were identified. In somecases there is a theoretical possibility that non-P. australis venompeptides do not 100% mirror the corresponding fragmentsbecause similar tryptic peptides of the same mass can possessnonidentical sequences due to different sequential order of pairsof residues.

Enzymatic ActivitiesProteolytic activity was determined by the method of Johnson

et al.47 The venom was assayed using 1.2% casein solution inTris-HCl buffer, pH 7.4, at 37 �C. Undigested casein wasprecipitated with 0.5M perchloric acid and centrifuged. Digestedcasein in the supernatant was determined by measuring theabsorbance at 280 nm. Unit definition: One CTA unit liberatesfrom cow casein 0.1 micro equivalents of tyrosine for 1 min at37.5 �C. One CTA unit is equal to 0.096 proteolytic units as usedby SIGMA Chemical Corporation, St Louis, MO.

Phospholipase A2 activity was determined using the CaymanChemical Secretory PLA2 Assay kit (Ann Arbor, MI) containinga bee venom PLA2 as a standard. 1,2 � dithio analog ofdiheptanoyl phosphatidylcholine was used as a substrate. Therelease of free thiols upon the PLA2 catalyzed hydrolysis of thethioester bond at the sn-2 position was detected spectrophoto-metrically using 5,50-dithiobis(2-nitrobenzoic acid).

L-Amino acid oxidase activity was determined by the methodof Wellner et al.48 using L-phenylalanine as substrate. One unit ofactivity is the amount of enzyme required to give an absorbanceof 0.030 at 300 nm.

Alkaline phosphatase activity was measured by the method ofSulkowski et al.49 using p-nitrophenylphosphate as a substrate.



One unit of activity is defined as the amount of enzyme whichliberates 1 μmole of p-nitrophenol per min.

Acid phosphatase activity was determined by the method ofTu and Chua.50 o-Carboxyphenylphosphate (0.0036 M) wasused as a substrate and the initial rate of hydrolysis of thesubstrate at 25 �C was determined from the increase of theabsorbance at 300 nm due to the liberation of salicylic acid.Venom concentration was adjusted so that the increase of theabsorbance was linear for at least 5 min. One unit of acidphosphatase activity is equivalent to 1 μmole of the substratehydrolyzed per min.

’RESULTS

2-D Gel Electrophoresis of the Pseudechis australis VenomThe proteomic composition of the P. australis venom was

investigated by 2-D electrophoresis. The separated protein bandswere subjected to tryptic digestion and the venom componentswere identified by MS/MS and MASCOT search program(Figure 1, Tables 1 and 2). The oxidized methionine residuesare indicated by Mox. The oxidation of methionine is dueprobably to the procedure of harvesting and sample handling.The samples were dried in the presence of ambient air which isknown to cause oxidation of methionine. The gel ensureddetailed information about the components with molecularmasses 9�110 kDa and pI values between 3 and 10. A total of110 spots were detected and identified on the 2-D gel. Theisolated proteins were assigned to the following protein families:metalloproteases, phospholipases A2, L-amino acid oxidases,transferrin-like proteins, ecto-50-nucleotidases, nerve growthfactors and serine protease inhibitors. The major group is thatof the hemostasis-related SVMPs including 53% of the identifiedproteins. The isoforms of LAAOs comprise the second largestprotein family (20% of the identified toxins). In the third positionare PLA2s representing 18.5% of the identified venom compo-nents. The representatives of the other five protein familiescomprise 8.5% of the analyzed toxins. A characteristic featureof the 2-D gel is the presence of several multiple horizontal trains

of spots with identical or very similar molecular masses butdifferent isoelectric points.

P�III metalloproteasesP�III SVMPs are the most widely represented family of toxins

in the P. australis venom accounting for 53% of the identifiedtoxins (Figure 2). A group of high molecular mass enzymes wasidentified from spots of multiple horizontal trains in the upperleft part of the 2-D gel (Figure 1, Table 1). Spots 1�5 containP�III metalloproteases with molecular masses of 100�105 kDaand pI values between 4.7 and 5.2. A second group of multipleisoforms of P�III SVMPs is shown in the upper right panel of thegel (Figure 1). Again, horizontal trains of spots with identicalmolecular masses, but differing in the pI values were observed.Metalloproteases (60�85 kDa) were identified in spots 7�26,29, 30�35, 42, 44�47, 59�74, 87, and 88. The peptide analysesshowed a high degree of sequence similarity between the P�IIISVMPs from the P. australis venom and their counterparts fromthe other elapid Australian snakes: Austrelaps superbus (theLowland copperhead), Pseudechis porphyriacus (Red-belliedblack snake), Oxyuranus scutellatus (the Coastal taipan) andNotechis scutatus (Tables 1 and 2).

Proteins with molecular masses in the region of 33�43 kDaand pI values between 4.0 and 7.2 were isolated from spots36�41, 75�77 and 80. They form another group of processedP�III SVMPs (Figure 1). The molecular masses are character-istic for the medium size class II proteases, but sequencesimilarities with the P�III group suggest that these proteinsbelong to a group of processed P�III enzymes.

Antimicrobial Proteins: L-Amino Acid Oxidases and Trans-ferrin-Like Proteins

Multiple isoforms of L-amino acid oxidases were found in theprocessed spots of the 2-DE gel (20% of the identified proteins;Figure 1, Tables 1 and 2). Spots 47, 49, 51, 53, 55, 57, and 61 forma horizontal train in the pI range from 6.7 to 7.8. The proteinspossess similar molecular masses from 58 to 62 kDa. A secondtrain in the same pI interval is formed by spots 48, 50, 52, 54, 56,58 and 60, containing proteins with molecular masses of

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



Journal of Proteome Research ARTICLETable1.

Assignm

ento

fthe

ProteinsIsolated

from

theSpotsof

the2-D-GelElectroph

oresisof

thePseudechisa

ustralisVenom

toProtein

Families

byMS/MSandMASC

OT

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

1asrin

111572527

Austrelapssuperbus

5.95

71183

942

593.8

2NDNAQLL

TGIK

P�III�

SVMP

861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

2asrin

111572527

Austrelapssuperbus

5.95

71183

149.6

2861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

3asrin

111572527

Austrelapssuperbus

5.95

71183

149.6

2861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

4asrin

111572527

Austrelapssuperbus

5.95

71183

122.9

2593.8

2NDNAQLL

TGIK

P�III�

SVMP

861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

5asrin

111572527

Austrelapssuperbus

5.95

71183

133.7

3861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

7asrin

111572527

Austrelapssuperbus

5.95

71183

211

2671.9

2RNDNAQLL

TGIK

P�III�

SVMP

1292

2AAKDDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756

Pseudechisporphyriacus

5.76

70518

151

2671.9

2RNDNAQLL

TGIK

P�III�

SVMP

955.9

2NGHPC

QNNQGYCYNGK

8asrin

111572527

Austrelapssuperbus

5.95

71183

211

2861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

151

2955.9

2NGHPC

QNNQGYCYNGK

P�III�

SVMP

593.8

2NDNAQLL

TGIK

9asrin

111572527

Austrelapssuperbus

5.95

71183

318

3861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

3DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

180

3955.9

2NGHPC

QNNQGYCYNGK

P�III�

SVMP

593.8

2NDNAQLL

TGIK

10asrin

111572527

Austrelapssuperbus

5.95

71183

267

3861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

154

3955.9

2NGHPC

QNNQGYCYNGK

P�III�

SVMP

593.8

2NDNAQLL

TGIK

11asrin

111572527

Austrelapssuperbus

5.95

71183

234

3861.7

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

130

3955.9

2NGHPC

QNNQGYCYNGK

P�III�

SVMP

593.8

2NDNAQLL

TGIK

12asrin

111572527

Austrelapssuperbus

5.95

71183

318

4861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

13asrin

111572527

Austrelapssuperbus

5.95

71183

314

3861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

14asrin

111572527

Austrelapssuperbus

5.95

71183

161

3671.9

2RNDNAQLL

TGIK

P�III�

SVMP

861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

15asrin

111572527

Austrelapssuperbus

5.95

71183

155

3671.9

2RNDNAQLL

TGIK

P�III�

SVMP

861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

16asrin

111572527

Austrelapssuperbus

5.95

71183

313

3861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

152

2593.8

2NDNAQLL

TGIK

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

17asrin

111572527

Austrelapssuperbus

5.95

71183

194

21156.9

2DDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

112

2955.9

2NGHPC

QNNQGYCYNGK

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

18asrin

111572527

Austrelapssuperbus

5.95

71183

227

21156.9

2DDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

118

2955.9

2NGHPC

QNNQGYCYNGK

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

19asrin

111572527

Austrelapssuperbus

5.95

71183

316

3861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

139

2593.8

2NDNAQLL

TGIK

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

20asrin

111572527

Austrelapssuperbus

5.95

71183

271

4861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

205

3593.8

2NDNAQLL

TGIK

P�III�

SVMP

490.3

3KRNDNAQLL

TGIK

P�III�

SVMP

21asrin

111572527

Austrelapssuperbus

5.95

71183

308

2861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

porphyriacase-1

145982756


5.76

70518

139

2593.8

2NDNAQLL

TGIK

690.3

2CPL

MTNQCLA

RP�

III�

SVMP

22asrin

111572527

Austrelapssuperbus

5.95

71183

190

21156.9

2DDCDLP

ESCTGQSA

ECPT

DR

861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

23asrin

111572527

Austrelapssuperbus

5.95

71183

266

31156.9

2DDCDLP

ESCTGQSA

ECPT

DR

861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

porphyriacase-1

145982756


5.76

70518

235

3955.9

2NGHPC

QNNQGYCYNGK

690.3

2CPL

MTNQCLA

R

24metalloproteinase

precursor

118151738

Dem

ansia

vestigata

5.55

68267

902

413.7

2KTVLL

PR

349.7

2TVLL

PR

25asrin

111572527

Austrelapssuperbus

5.95

71183

213

3861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

593.8

2NDNAQLL

TGIK



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

26asrin

111572527

Austrelapssuperbus

5.95

71183

174

3861.7

2AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

593.8

2NDNAQLL

TGIK

porphyriacase-1

145982756


5.76

70518

922

593.8

NDNAQLL

TGIK

P�III�

SVMP

690.3

CPL

MTNQCLA

R

29porphyriacase-1

145982756


5.76

70518

613

9981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

735.4

2KRNDNAQLL

TGIK

australease-1

145982758

Pseudechisaustralis

5.45

71238

272

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

asrin

111572527

Austrelapssuperbus

5.95

71183

256

4861

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

735.9

2KRNDNAQLL

TGIK

scutatease-1

145982766

Notechisscutatus

4.99

68020

100

2732.8

2CPIMTNQCIALK

P�III�

SVMP

636.9

3NGHPC

QNNQGYCYNGK

30asrin

111572527

Austrelapssuperbus

5.95

71183

160

2861

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

593.8

2NDNAQLL

TGIK

porphyriacase-1

145982756


5.76

70518

135

2593.8

2NDNAQLL

TGIK

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

31L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

663

6549.6

3RFD

EIVGGFD

QLP

RLA

AO

553.8

2RPL

EECFR

australease-1

145982758

Pseudechisaustralis

5.45

71238

597

9981.7

AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

436

6981.7

AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

939.9

LQHEA

QCDSG

ECCER

32L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

1375

17746.9

2FD

EIVGGFD

QLP

RLA

AO

981

2IQ

QNAED

VRVTYQTPA

K

L-am

ino-acidoxidaseprecursor

12391680

Oxyuranus

scutellatus

scutellatus

8.99

59032

595

7608.8

2FW

EADGIH

GGK

LAAO

630.3

2SD

DLF

SYEK

R

australease-1

145982758

Pseudechisaustralis

5.45

71238

422

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

323

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

939.9

2LQ

HEA

QCDSG

ECCER

L-am

ino-acidoxidase

126035677

Najaatra

8.44

51406

281

4608.8

2FW

EADGIH

GGK

LAAO

509.3

2VTLL

EASE

R

33L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

953

15746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

asrin

111572527

Austrelapssuperbus

5.95

71183

289

4861

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

porphyriacase-1

145982756


5.76

70518

162

2690.3

2CPL

MTNQCLA

RP�

III�

SVMP

593.8

2NDNAQLL

TGIK



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

34asrin

111572527

Austrelapssuperbus

5.95

71183

141

4861

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

735.9

2KRNDNAQLL

TGIK

porphyriacase-1

145982756


5.76

70518

128

6735.9

2KRNDNAQLL

TGIK

P�III�

SVMP

955.9

2NGHPC

QNNQGYCYNGK

35L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

988

13746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

porphyriacase-1

145982756


5.76

70518

252

2981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

36asrin

111572527

Austrelapssuperbus

5.95

71183

414.3

5861

3AAKDDCDLP

ESCTGQSA

ECPT

DR

P�III�

SVMP

1156.9

2DDCDLP

ESCTGQSA

ECPT

DR

australease-1

145982758

Pseudechisaustralis

5.45

71238

356.3

5981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

690.3

2CPL

MTNQCLA

R

porphyriacase-1

145982756


5.76

70518

290.7

5981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

955.9

2NGHPC

QNNQGYCYNGK

37australease-1

145982758

Pseudechisaustralis

5.45

71238

305.4

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

707.3

2DDPD

YGMVEA

GTK

38australease-1

145982758

Pseudechisaustralis

5.45

71238

572.5

8981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

281.4

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

39australease-1

145982758

Pseudechisaustralis

5.45

71238

564

8981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

307.4

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

40australease-1

145982758

Pseudechisaustralis

5.45

71238

606

10981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

334.7

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

891.7

3DDCDLP

ESCTGQSA

ECPT

DSF

QR

41australease-1

145982758

Pseudechisaustralis

5.45

71238

516.4

8981.3

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

891.7

3DDCDLP

ESCTGQSA

ECPT

DSF

QR

porphyriacase-1

145982756


5.76

70518

355.7

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

891.7

3DDCDLP

ESCTGQSA

ECPT

DSF

QR

42L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

514.8

4552.2

2SD

DIFSY

EKLA

AO

728.8

2EA

DYEE

FLEIAK

porphyriacase-1

145982756


5.76

70518

299.5

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

671.9

2RNDNAQLL

TGIK

43L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

583.4

7728.8

2EA

DYEE

FLEIAK

LAAO

607.8

2DVNLA

SQKPS

R



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

44L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

996.2

13746.9

2FD

EIVGGFD

QLP

RLA

AO

824.9

2RFD

EIVGGFD

QLP

R

transferrin

108792441

Lamprophisfuliginosus

6.4

78321

291.7

5755.9

2LK

QEC

FSQQQSK

transferrin

684.4

2CGLV

PILT

EIPR

489.2

2GSG

GEG

GLS

EK

483.2

2LF

GSQ

GTQK

574.8

2DFP

ELICVR

porphyriacase-1

145982756


5.76

70518

220.9

4690.3

2CPL

MTNQCLA

RP�

III�

SVMP

626.2

3LQ

HEA

QCDSG

ECCER

australease-1

145982758

Pseudechisaustralis

5.45

71238

516.4

8707.3

2DDPD

YGMVEA

GTK

P�III�

SVMP

690.3

2CPL

MTNQCIAR

45L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

680.9

5824.9

2RFD

EIVGGFD

QLP

RLA

AO

607.8

2DVNLA

SQKPS

R

australease-1

145982758

Pseudechisaustralis

5.45

71238

372.6

5981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

707.3

2DDPD

YGMVEA

GTK

porphyriacase-1

145982756


5.76

70518

311.8

5690.3

2CPL

MTNQCIAR

P�III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

transferrin

108792441


6.4

78321

268

4684.4

2CGLV

PILT

EIPR

transferrin

755.9

2LK

QEC

FSQQQSK

489.2

2GSG

GEG

GLS

EK

574.8

2DFP

ELICVR

46L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

658.1

7824.9

2RFD

EIVGGFD

QLP

RLA

AO

607.8

2DVNLA

SQKPS

R

porphyriacase-1

145982756


5.76

70518

316.6

6690.3

2CPL

MTNQCIAR

P�III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

transferrin

108792441


6.4

78321

175.9

5755.9

2LK

QEC

FSQQQSK

transferrin

684.4

2CGLV

PILT

EIPR

635.3

2QEC

FSQQQSK

489.2

2GSG

GEG

GLS

EK

483.2

2LF

GSQ

GTQK

47L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

814.3

9824.9

2RFD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

porphyriacase-1

145982756


5.76

70518

241.7

5690.3

2CPL

MTNQCIAR

P�III�

SVMP

626.2

3LQ

HEA

QCDSG

ECCER

48L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

737.7

8824.9

2RFD

EIVGGFD

QLP

RLA

AO

553.8

2RPL

EECFR

49L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

667.7

8746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK



Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

50L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

667.7

6607.8

2DVNLA

SQKPS

RLA

AO

536.8

2IQ

QNAED

VR

51L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

835.1

11662.3

2DGWYVNLG

PMR

LAAO

668.8

2EQ

IQALC

YPS

K

52L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

687.5

5607.8

2DVNLA

SQKPS

RLA

AO

746.9

2FD

EIVGGFD

QLP

R

53L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

686

6607.8

2DVNLA

SQKPS

RLA

AO

536.8

2IQ

QNAED

VR

54L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

820.7

10746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

55L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

835.1

7824.9

2RFD

EIVGGFD

QLP

RLA

AO

607.8

2DVNLA

SQKPS

R

porphyriacase-1

145982756


5.76

70518

135.3

2682.8

2FS

SCSV

QEH

QR

P�III�

SVMP

939.9

2LQ

HEA

QCDSG

ECCER

56L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

918

12746.9

2FD

EIVGGFD

QLP

RLA

AO

668.8

2EQ

IQALC

YPS

K

57L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

944

13728.8

2EA

DYEE

FLEIAK

LAAO

608.8

2FW

EADGIH

GGK

porphyriacase-1

145982756


5.76

70518

982.5

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

1338.1

2DDCDLP

ESCTGQSA

ECPT

DSF

QR

58L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

510.4

3607.8

2DVNLA

SQKPS

RLA

AO

454.2

2VTYQTPA

K

59L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

965.6

13746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

porphyriacase-1

145982756


5.76

70518

478.2

9981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

1338

2DDCDLP

ESCTGQSA

ECPT

DSF

QR

australease-1

145982758

Pseudechisaustralis

5.45

71238

422.3

7981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

1338

2DDCDLP

ESCTGQSA

ECPT

DSF

QR

textilease-1

145982770

Pseudonajatextilis

4.98

68626

115.5

2578.2

2CGDGMVCSN

RP�

III�

SVMP

636.9

3NGHPC

QNNQGYCYNGK

60L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

1284.8

17746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

L-am

ino-acidoxidase

123916680

Oxyuranus

scutellatus

8.99

59032

648.2

8669.3

2EG

WYVNLG

PMR

LAAO

854.9

2NEK

EGWYVNLG

PMR

porphyriacase-1

145982756


5.76

70518

284.3

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

61L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

857

10746.9

2FD

EIVGGFD

QLP

RLA

AO

728.8

2EA

DYEE

FLEIAK

porphyriacase-1

145982756


5.76

70518

565.1

9981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

1338

2DDCDLP

ESCTGQSA

ECPT

DSF

QR

australease-1

145982758

Pseudechisaustralis

5.45

71238

494.5

9981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

scutellatease-1

145982762

Oxyuranus

scutellatus

4.89

68542

143.7

2586.2

2CGDGMVCSN

RP�

III�

SVMP

636.9

3NGHPC

QNNQGYCYNGK

62australease-1

145982758

Pseudechisaustralis

5.45

71238

152

2981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

63australease-1

145982758

Pseudechisaustralis

5.45

71238

169.6

3981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

22GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

147

3690.3

3CPL

MTNQCLA

RP�

III�

SVMP

981.7

AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

64australease-1

145982758

Pseudechisaustralis

5.45

71238

181.7

3981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

149.4

3981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

620.8

2AYVGTLC

SLEK

65australease-1

145982758

Pseudechisaustralis

5.45

71238

159.5

2690.3

2CPL

MTNQCLA

RP�

III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

ecto-5

0 -nucleotidase

211926754

Gloydiusblomhoffi

brevicaudus

8.65

64441

153.4

3653.9

2QVPV

VQAYAFG

K50-nucleotidase

726

3GDSSNHSSGNLD

ISIVGDYIK


476.3

2VGIIGYTTK

porphyriacase-1

145982756

5.76

70518

134.2

2515.7

2CGMLY

CVK

P�III�

SVMP

620.8

2AYVGTLC

SLEK

66australease-1

145982758

Pseudechisaustralis

5.45

71238

321.4

3981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

67australease-1

145982758

Pseudechisaustralis

5.45

71238

725.1

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

305.2

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

68australease-1

145982758

Pseudechisaustralis

5.45

71238

598.8

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

254.6

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR



Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

69australease-1

145982758

Pseudechisaustralis

5.45

71238

601.6

7981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

240.7

5690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

ecto-5

0 -nucleotidase

211926754

Gloydiusblomhoffi

brevicaudus

8.65

64441

125.4

2653.2

2QVPV

VQAYAFG

K50-nucleotidase

726

3GDSSNHSSGNLD

ISIVGDYIK

70australease-1

145982758

Pseudechisaustralis

5.45

71238

486

8981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

192.1

4690.3

2CPL

MTNQCLA

RP�

III�

SVMP

981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

ecto-5

0 -nucleotidase

211926754

Gloydiusblomhoffi

brevicaudus

8.65

64441

183

5778

3GDSSNHSSGNLD

ISIVGDYIKR

50-nucleotidase

1211.1

2FH

ECNLG

NLICDAVIYNNVR

71australease-1

145982758

Pseudechisaustralis

5.45

71238

592.3

9981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

208.3

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

939.9

2LQ

HEA

QCDSG

ECCER

72australease-1

145982758

Pseudechisaustralis

5.45

71238

734.7

10981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

257.1

7981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

939.9

2LQ

HEA

QCDSG

ECCER

scutellatease-1

145982762

Oxyuranus

scutellatus

4.89

68542

189.9

3677.9

2YIELY

VVVDNK

P�III�

SVMP

741.9

2KYIELY

VVVDNK

73australease-1

145982758

Pseudechisaustralis

5.45

71238

738

12981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

277.5

7981.7

32AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

939.9

2LQ

HEA

QCDSG

ECCER

scutellatease-1

145982762

Oxyuranus

scutellatus

4.89

68542

201.6

3677.9

2YIELY

VVVDNK

P�III�

SVMP

741.9

KYIELY

VVVDNK

74australease-1

145982758

Pseudechisaustralis

5.45

71238

581

10981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

959

2GGPG

VNLS

PDICFT

INQK

porphyriacase-1

145982756


5.76

70518

215.3

6981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMP

939.9

2LQ

HEA

QCDSG

ECCER

75australease-1

145982758

Pseudechisaustralis

5.45

71238

471.8

7981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

P�III�

SVMPfragment

959

2GGPG

VNLS

PDICFT

INQK

P�III�

SVMPfragment

porphyriacase-1

145982756


5.76

70518

267.1

5981.7

3AAKDDCDLP

ESCTGQSA

ECPT

DSF

QR

939.9

2LQ

HEA

QCDSG

ECCER



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

76L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

750.8

11746.9

2FD

EIVGGFD

QLP

RLA

AOfragment

1037.5

3VVVVGAGMAGLS

AAYVLA

GAGHQVTLL

EASE

R

CPL

MTNQCLA

R

australease-1

145982758

Pseudechisaustralis

5.45

71238

343.9

6690.3

2GGPG

VNLS

PDICFT

INQK

P�III�

SVMPfragment

959

2

77L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

879.8

131037.5

3VVVVGAGMAGLS

AAYVLA

GAGHQVTLL

EASE

RLA

AOfragment

NEK

DGWYVNLG

PMR

847.9

2GGPG

VNLS

PDICFT

INQK

australease-1

145982758

Pseudechisaustralis

5.45

71238

235.6

4959

2LQ

HEA

QCDSG

ECCER

P�III�

SVMPfragment

626.2

3

78L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

828

13746.9

2FD

EIVGGFD

QLP

RLA

AOfragment

1037.5

3VVVVGAGMAGLS

AAYVLA

GAGHQVTLL

EASE

R

SDDLF

SYEK

R

L-am

ino-acidoxidase

precursor

123916680

Oxyuranus

scutellatus

scutellatus

8.99

59032

374.8

6630.3

2SD

DIFSY

EKLA

AOfragment

552.2

2VTLL

EASE

R

L-am

ino-acidoxidase

126035677

Najaatra

8.44

51406

197

3509.3

2VTYQTPA

KLA

AOfragment

454.2

2

79venom

nervegrow

thfactor

183288314

Pseudechisaustralis

5.75

27138

326.3

41004

2GNTVTVEVDVNLN

NEV

YK

nervegrow

thfactor

874.9

2LW

NSY

CTTTQTFV

KPL

A2

phospholipaseA2isozym

e

PA-10A

129397

Pseudechisaustralis

8.52

13018

85.4

2848.9

2NLIQFS

NMIQ

CANK

478.5

3VHDDCYDQAGKK

80L-am

ino-acidoxidase

123916679

Pseudechisaustralis

6.26

59049

351.2

5746.9

2FD

EIVGGFD

QLP

RLA

AOfragment

728.8

2EA

DYEE

FLEIAK

australease-1

145982758

Pseudechisaustralis

5.45

71238

303

6959

2GGPG

VNLS

PDICFT

INQK

P�III�

SVMPfragment

707.3

2DDPD

YGMVEA

GTK

82phospholipaseA2isozym

e

PA-3

129447

Pseudechisaustralis

6.84

13941

286.2

5904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1056

2LT

LYSW

DCTGNVPICSP

K


e

HI-2009

295841609

Pseudechisaustralis

7.76

15760

244.1

4904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1136

2ITWYSW

DCTEN

VPT

CNPK


e

PA-12C

129471

Pseudechisaustralis

8.84

13798

190.1

2833.9

2NLIQFG

NM

oxIQ

CANK

PLA2

590.9

3CCQTHDNCYEQ

AGK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

181.8

3904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1142

2ITWYSW

DCTEN

VPT

CNPK


ePA

-5129452

Pseudechisaustralis

7.53

13914

160.3

31069.5

2LT

LYSW

DCTGNVPICNPK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK


e

PA-13

129474

Pseudechisaustralis

8.52

14002

95.8

21086.5

2LT

WYSW

DCTGDAPT

CNPK

PLA2

501.3

2GTPV

DEL

DR



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily


ePA

-3129447

Pseudechisaustralis

6.84

13941

321.1

4833.9

2NLIQFG

NM

oxIQ

CANK

PLA2

904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR


ePA

-5129452

Pseudechisaustralis

7.53

13914

267

51069.5

2LT

LYSW

DCTGNVPICNPK

PLA2

1070.1

3GSR

PSLD

YADYGCYCGWGGSG

TPV

DEL

DR

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

256.1

3904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

761.3

2APY

NDANWNID

TK

PA-18precursor

71066782

Pseudechisaustralis

7.87

16718

244.7

41063.5

2LT

LYSW

DCTGNVPT

CNPK

PLA2

894.9

2TEC

KDFA

CACDAAAAK


ePA

-13

129474

Pseudechisaustralis

8.52

14002

95.8

21086.5

2LT

WYSW

DCTGDAPT

CNPK

PLA2

667.6

3AAWHYLD

YGCYCGPG

GR

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

256.1

3904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

761.3

2APY

NDANWNID

TK


ePA

-12C

129471

Pseudechisaustralis

8.84

14798

197.7

31070.8

3GSR

PSLD

YADYGCYCGWGGSG

TPV

DEL

DR

PLA2

885.8

2CCQTHDNCYEQ

AGK

PA-19precursor

71066784

Pseudechisaustralis

8.6

16900

160.6

3884.9

2CCQVHDNCYEQ

AGK

PLA2

903.7

3HYMDYGCYCGWGGSG

TPV

DEL

DR


ePA

-1G

129477

Pseudechisaustralis

5.61

13815

147.6

2763.3

2ATYNDANWNID

TK

PLA2

825.9

2NLIQFG

NMIQ

CANK


ePA

-13

129474

Pseudechisaustralis

8.52

14002

479.5

6793.8

2CKDFV

CACDAAAAK

PLA2

1086.5

2LT

WYSW

DCTGDAPT

CNPK


ePA

-10A

129397

Pseudechisaustralis

8.52

13816

439.4

4840.9

2NLIQFS

NM

OXIQ

CANK

PLA2

1069.5

2LT

LYSW

DCTGNVPICNPK


ePA

-11

129415

Pseudechisaustralis

8.74

13755

435.1

51070.8

3GSR

PSLD

YADYGCYCGWGGSG

TPV

DEL

DR

PLA2

844.9

2CCQVHDNCYEQ

AGK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

412

6904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-3129447

Pseudechisaustralis

6.84

13941

351.1

5904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1056

2LT

LYSW

DCTGNVPICSP

K


ePA

-5129452

Pseudechisaustralis

7.53

13914

349.9

41069.5

2LT

LYSW

DCTGNVPICNPK

PLA2

1070.1

3GSR

PSLD

YADYGCYCGWGGSG

TPV

DEL

DR


ePA

-9C

129454

Pseudechisaustralis

7.94

14087

296

3653.8

2VHDEC

YGEA

VK

PLA2

793.8

2CKDFV

CACDAAAAK

PA-19precursor

71066784

Pseudechisaustralis

8.6

16900

282.9

4884.9

2CCQVHDNCYEQ

AGK

PLA2

903.7

3HYMDYGCYCGWGGSG

TPV

DEL

DR

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

280

4938.9

2TEC

KDFT

CACDAEA

AK

PLA2

1136

2ITWYSW

DCTEN

VPT

CNPK

PA-18precursor

71066782

Pseudechisaustralis

7.87

16718

248.2

41063.5

2LT

LYSW

DCTGNVPT

CNPK

PLA2

894.9

2TEC

KDFA

CACDAAAAK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

246.3

4904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1142

2ITWYSW

DCTEN

VPT

CNPK



Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

87australease-1

145982758

Pseudechisaustralis

5.45

71238

299.4

6690.3

2CPL

MTNQCLA

RP�

III�

SVMP

707.3

2DDPD

YGMVEA

GTK

88australease-1

145982758

Pseudechisaustralis

5.45

71238

306.1

6707.3

2DDPD

YGMVEA

GTK

P�III�

SVMP

626.2

3LQ

HEA

QCDSG

ECCER

90phospholipaseA2

295841613

Pseudechisaustralis

8.78

15724

224.8

4611.3

2IVCDCDAAVAK

PLA2

611.7

2AHDDCYGEA

GK


ePA

-12A

129458

Pseudechisaustralis

8.84

13758

153.5

3590.2

3CCQVHDNCYEQ

AGK

PLA2

619.3

2CTGNVPT

CNSK


ePA

-11

129415

Pseudechisaustralis

8.74

13755

150.6

3590.2

3CCQVHDNCYEQ

AGK

PLA2

619.3

2CTGNVPT

CNSK

92phospholipaseA2

295841613

Pseudechisaustralis

8.78

15724

119.5

2450.9

3AHDDCYGEA

GKK

PLA2

611.3

2IVCDCDAAVAK

94phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

186.1

3938.9

2TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK

phospholipaseA2

295841595

Pseudechisaustralis

16498

144.8

2528.2

2AFICNCDR

PLA2

725.3

2GTPV

DEL

DRCCK

95PA

-17precursor

71066780

Pseudechisaustralis

6.77

16768

122.1

2679.8

2DFT

CACDAEA

AK

PLA2

590.9

2CCQTHDNCYEQ

AGK


ePA

-5129452

Pseudechisaustralis

7.53

13914

120.8

2939.9

2TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK

PLA-1

precursor

71066734

Pseudechisaustralis

5.43

16929

100.5

2528.2

2AFICNCDR

PLA2

590.9

3CCQVHDNCYEQ

AGK

96phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

192

3626.3

3TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK

97phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

192

3626.3

3TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

205.4

3679.8

2DFT

CACDAEA

AK

PLA2

633.6

3CCQTHDNCYEQ

AGKK

PLA-3

precursor

71066722

Oxyuranus

microlepidotus

5.23

16878

68.9

2528.2

2AFICNCDR

PLA2

590.9

3CCQVHDNCYEQ

AGK

98phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

345.3

5761.4

2APY

NDANWNID

TK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

268.8

4633.6

3CCQTHDNCYEQ

AGKK

PLA2

679.8

2DFT

CACDAEA

AK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

258.3

3761.3

2APY

NDANWNID

TK

PLA2

632.9

3CCQTHDNCYEQ

AGKK

phospholipaseA2

24638107

Notechisscutatus

scutatus

5.05

14252

190.3

3761.3

2APY

NDANWNID

TK

PLA2

601.8

2GGSG

TPV

DEL

DR

phospholipaseA257

24638081

Lapemishardwickii

516932

98.4

2528.2

2AFICNCDR

PLA2

692.3

2TAAICFA

GAPY

NK



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

99phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

276.2

5938.9

2TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK


ePA

-1G

129477

Pseudechisaustralis

5.61

13815

256.4

3763.3

2ATYNDANWNID

TK

PLA2

922.9

2AEC

KDFV

CACDAEA

AK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

212.9

4633.6

3CCQTHDNCYEQ

AGKK

PLA2

679.8

2DFT

CACDAEA

AK

PLA-3

precursor

71066794


8.48

15825

170

3678.8

2DFV

CACDAEA

AK

PLA2

501.2

2GTPV

DEL

DR

PLA-1

precursor

71066734

Oxyuranus

microlepidotus

5.43

16929

100.5

3528.2

2AFICNCDR

PLA2

590.9

3CCQVHDDCYGEA

EK

100

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

257.2

5938.9

2TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK

101


ePA

-3129477

Pseudechisaustralis

6.84

13941

545.1

7904.4

3HYM

OXDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1056

2LT

LYSW

DCTGNVPICSP

K

PA-16precursor

71066778

Pseudechisaustralis

6.77

16650

463.3

61056

2LT

LYSW

DCTGNVPICSP

KPL

A2

950.4

2CCQTHDNCYGEA

EKK

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

419.4

61136

2ITWYSW

DCTEN

VPT

CNPK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK

venom

nervegrow

thfactor

83288314

Pseudechisaustralis

5.75

27138

263

2874.9

2LW

NSY

CTTTQTFV

KNerve

grow

thfactor

682.3

2ALT

MEG

NQASW

RPL

A2

PLA-2

precursor

71066792


15919

260.6

3904.4

3HYM

OXDYGCYCGWGGSG

TPV

DEL

DR

822.8

2CKDFV

CACDAEA

AK

102


ePA

-3129477

Pseudechisaustralis

6.84

13941

636.6

9922.9

2AEC

KDFV

CACDAEA

AK

PLA2

1056

2LT

LYSW

DCTGNVPICSP

K

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

500

61136

2ITWYSW

DCTEN

VPT

CNPK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

423.6

51356

2HYMDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1136

2ITWYSW

DCTEN

VPT

CNPK

PA-16precursor

71066778

Pseudechisaustralis

6.77

16650

375

51056

2LT

LYSW

DCTGNVPICSP

KPL

A2

950.4

2CCQTHDNCYGEA

EKK


ePA

-12C

129471

Pseudechisaustralis

8.84

13798

272.1

3825.9

2NLIQFG

NMIQ

CANK

PLA2

885.8

2CCQTHDNCYEQ

AGK


ePA

-13

129474

Pseudechisaustralis

8.52

14002

114.7

2649.8

2DFV

CACDAAAAK

PLA2

501.2

2GTPV

DEL

DR



Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

103

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

641.5

81136

2ITWYSW

DCTEN

VPT

CNPK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK


ePA

-3129477

Pseudechisaustralis

6.84

13941

542.5

7904.4

3HYM

OXDYGCYCGWGGSG

TPV

DEL

DR

PLA2

922.9

2AEC

KDFV

CACDAEA

AK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

481.3

6904.4

3HYM

oxDYGCYCGWGGSG

TPV

DEL

DR

PLA2

793.8

2CKDFV

CACDAAAAK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

476.3

51356

2HYMDYGCYCGWGGSG

TPV

DEL

DR

PLA2

1136

2ITWYSW

DCTEN

VPT

CNPK


ePA

-10A

129397

Pseudechisaustralis

8.52

13816

430.7

6840.9

2NLIQFS

NM

OXIQ

CANK

PLA2

1069.5

2LT

LYSW

DCTGNVPICNPK


ePA

-5129452

Pseudechisaustralis

7.53

13914

359.7

61069.5

2LT

LYSW

DCTGNVPICNPK

PLA2

939.9

2TEC

KDFT

CACDAEA

AK


ePA

-12C

129471

Pseudechisaustralis

8.84

13798

291.7

3825.9

2NLIQFG

NMIQ

CANK

PLA2

885.8

2CCQTHDNCYEQ

AGK

PA-18precursor

71066782

Pseudechisaustralis

7.87

16718

276.4

51063.5

2LT

LYSW

DCTGNVPT

CNPK

PLA2

894.9

2TEC

KDFA

CACDAAAAK


ePA

-13

129474

Pseudechisaustralis

8.52

14002

251.2

4649.8

2DFV

CACDAAAAK

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-9C

129454

Pseudechisaustralis

7.94

14087

235.5

3793.8

2CKDFV

CACDAAAAK

PLA2

649.8

2DFV

CACDAAAAK

104


ePA

-5129452

Pseudechisaustralis

7.53

13914

520

7840.9

2NLIQFS

NMIQ

CANK

PLA2


ePA

-10A

1069.5

2LT

LYSW

DCTGNVPICNPK

PA-18precursor

129397

Pseudechisaustralis

8.52

13816

485.1

6840.9

2NLIQFS

NMIQ

CANK

PLA2

1069.5

2LT

LYSW

DCTGNVPICNPK

phospholipaseA2

71066782

Pseudechisaustralis

7.87

16718

366.4

51063.5

2LT

LYSW

DCTGNVPT

CNPK

PLA2

894.9

2TEC

KDFA

CACDAAAAK


ePA

-3295841609

Pseudechisaustralis

7.76

15937

322.6

5761.3

2APY

NDANWNID

TK

PLA2


ePA

-9C

938.9

2TEC

KDFT

CACDAEA

AK

phospholipaseA2

129477

Pseudechisaustralis

6.84

13941

310.6

4833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

763.4

2ATYNDANWNID

TK

PA-19precursor

129454

Pseudechisaustralis

7.94

14087

304.6

4653.8

2VHDEC

YGEA

VK

PLA2

793.8

2CKDFV

CACDAAAAK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

354.9

3761.3

2APY

NDANWNID

TK

PLA2

793.8

2CKDFV

CACDAAAAK

phospholipaseA257

71066784

Pseudechisaustralis

8.6

16900

244.9

3625.6

3TEC

KDFT

CACDAEA

AK

PLA2

679.8

2DFT

CACDAEA

AK

24638107

Notechisscutatus

scutatus

5.05

14252

185.2

3761.3

2APY

NDANWNID

TK

PLA2

601.8

2GGSG

TPV

DEL

DR

24638081

Lapemishardwickii

516932

113.2

2528.2

2AFICNCDR

PLA2

692.3

2TAAICFA

GAPY

NK



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

105

PA-18precursor

71066782

Pseudechisaustralis

7.87

16718

298.2

51063.5

2LT

LYSW

DCTGNVPT

CNPK

PLA2

894.9

2TEC

KDFA

CACDAAAAK


ePA

-5129452

Pseudechisaustralis

7.53

13914

295.6

4840.9

2NLIQFS

NMIQ

CANK

PLA2

939.9

2TEC

KDFT

CACDAEA

AK

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

285

4761.3

2APY

NDANWNID

TK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK


ePA

-10A

129397

Pseudechisaustralis

8.52

13816

279.6

5840.9

2NLIQFS

NMIQ

CANK

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-12C

129471

Pseudechisaustralis

8.84

13798

221.2

3825.9

2NLIQFG

NMIQ

CANK

PLA2

592.3

2FV

CACDAAAAK


ePA

-9C

129454

Pseudechisaustralis

7.94

14087

194.5

4653.8

2VHDEC

YGEA

VK

PLA2

793.8

2CKDFV

CACDAAAAK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

190.2

3761.3

2APY

NDANWNID

TK

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-3129477

Pseudechisaustralis

6.84

13941

188.1

3833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

484.2

2VHDDCYGEA

EKK

phospholipaseA2

24638107

Notechisscutatus

scutatus

5.05

14252

143.6

2761.3

2APY

NDANWNID

TK

PLA2

601.8

2GGSG

TPV

DEL

DR

106


ePA

-9C

129454

Pseudechisaustralis

7.94

14087

340.2

5793.8

2CKDFV

CACDAAAAK

PLA2

675.8

2CTEN

VPICDSR


ePA

-10A

129397

Pseudechisaustralis

8.52

13816

308.8

4793.8

2CKDFV

CACDAAAAK

PLA2

718.3

2VHDDCYDQAGKK


ePA

-3129477

Pseudechisaustralis

6.84

13941

291.5

4833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

904.4

3HYMDYGCYCGWGGSG

TPV

DEL

DR


ePA

-13

129474

Pseudechisaustralis

8.52

14002

281

3649.8

2DFV

CACDAAAAK

PLA2

793.8

2CKDFV

CACDAAAAK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

241.7

3904.4

3HYMDYGCYCGWGGSG

TPV

DEL

DR

PLA2

793.8

2CKDFV

CACDAAAAK

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15760

215.6

3904.4

3HYMDYGCYCGWGGSG

TPV

DEL

DR

PLA2

654.3

2VHDDCYDQAGK


ePA

-12A

129458

Pseudechisaustralis

8.84

13758

209.5

3635.8

2SF

VCACDAAAAK

PLA2

833.9

2NLIQFG

NM

OXIQ

CANK


ePA

-11

129415

Pseudechisaustralis

8.74

13755

205.1

3833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

635.8

2SF

VCACDAAAAK



Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

107


ePA

-13

129474

Pseudechisaustralis

8.52

14002

485.4

7793.8

2CKDFV

CACDAAAAK

PLA2

1086.5

2LT

WYSW

DCTGDAPT

CNPK

phospholipaseA2

295841613

Pseudechisaustralis

8.78

15724

449.2

61067.5

2KGCYPV

LTLY

SWEC

TEK

PLA2

1003.5

2GCYPV

LTLY

SWEC

TEK


ePA

-11

129415

Pseudechisaustralis

8.74

13755

364.3

5833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

844.9

2CCQVHDNCYEQ

AGK


ePA

-12A

129458

Pseudechisaustralis

8.84

13758

355.7

5833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

884.9

2CCQVHDNCYEQ

AGK


ePA

-9C

129454

Pseudechisaustralis

7.94

14087

325.2

3653.8

2VHDEC

YGEA

VK

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-10A

129397

Pseudechisaustralis

8.52

13816

298.8

5793.8

2CKDFV

CACDAAAAK

PLA2

718.3

2VHDDCYDQAGKK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

278.3

4761.3

2APY

NDANWNID

TK

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-3129477

Pseudechisaustralis

6.84

13941

246.7

4833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

763.3

2ATYNDANWNID

TK

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15760

197.4

3761.3

2APY

NDANWNID

TK

PLA2

654.3

2VHDDCYDQAGK

venom

nervegrow

thfactor

83288328


6.94

26736

163.9

2682.3

2ALT

MEG

NQASW

RNerve

grow

thfactor

633.3

2ID

TACVCVISK

PLA2

PLA-4

precursor

71066796


8.33

15667

130.8

2501.2

2GTPV

DEL

DR

619.3

2CTGNVPT

CNSK

108

PA-20precursor

71066788

Pseudechisaustralis

8.59

16783

538.5

71003.5

2GCYPV

LTLY

SWEC

TEK

PLA2

1067.5

2KGCYPV

LTLY

SWEC

TEK


ePA

-13

129474

Pseudechisaustralis

8.52

14002

428

51086.5

2LT

WYSW

DCTGDAPT

CNPK

PLA2

680.3

3IH

DDCYIEAGKDGCYPK


ePA

-11

129415

Pseudechisaustralis

8.74

13755

411.8

4833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

844.9

2CCQVHDNCYEQ

AGK


ePA

-12A

129458

Pseudechisaustralis

8.84

13758

406.8

5833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

884.8

2CCQVHDNCYEQ

AGK


ePA

-12C

129471

Pseudechisaustralis

8.84

13798

406.3

4885.8

2CCQTHDNCYEQ

AGK

PLA2

826.3

2CTGNAPT

CNSK

PGCK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

325.8

4761.3

2APY

NDANWNID

TK

PLA2

793.8

2CKDFV

CACDAAAAK

PA-19precursor

71066784

Pseudechisaustralis

8.6

16900

224.5

3884.9

2CCQVHDNCYEQ

AGK

PLA2

625.6

3TEC

KDFT

CACDAEA

AK


ePA

-9C

129454

Pseudechisaustralis

7.94

14087

221.2

3778.4

2APY

NKDNYNID

TK

PLA2

793.8

2CKDFV

CACDAAAAK

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15760

188.6

2761.3

2APY

NDANWNID

TK

PLA2

938.9

2TEC

KDFT

CACDAEA

AK


ePA

-1G

129477

Pseudechisaustralis

5.61

13815

157

2763.4

2ATYNDANWNID

TK

PLA2

833.9

2NLIQFG

NM

OXIQ

CANK



Table1.

Con

tinu

ed

spot

no.

protein

accession

code

homology

with

a

proteinfrom

pIMW

Mascot

score

matched

peptides

a

peptide

ion

m/z

zMS/MSderived

sequence

proteinfamily

109

mulgin-2

82201571

Pseudechisaustralis

6.04

9549

212.6

31069.4

2TCLE

FIYGGCEG

NDNNFK

Serin

eproteinase

inhibitor

698.8

2FC

ELPP

DSG

SCK

110

phospholipaseA2

295841609

Pseudechisaustralis

7.76

15937

394.1

6938.9

2TEC

KDFT

CACDAEA

AK

PLA2

1136

2ITWYSW

DCTEN

VPT

CNPK


ePA

-12C

129471

Pseudechisaustralis

8.84

13798

351

5885.8

2CCQTHDNCYEQ

AGK

PLA2

826.3

2CTGNAPT

CNSK

PGCK

phospholipaseA2

295841605

Pseudechisaustralis

8.48

15937

318

5761.3

2APY

NDANWNID

TK

PLA2

793.8

2CKDFV

CACDAAAAK


ePA

-10A

129397

Pseudechisaustralis

8.52

13816

316.5

5793.8

2CKDFV

CACDAAAAK

PLA2

718.3

2VHDDCYDQAGKK

PA-17precursor

71066780

Pseudechisaustralis

6.77

16768

265.3

4679.8

2DFT

CACDAEA

AK

PLA2

1136

2ITWYSW

DCTEN

VPT

CNPK


ePA

-3129447

Pseudechisaustralis

6.84

13941

252.8

4833.9

2NLIQFG

NM

OXIQ

CANK

PLA2

763.3

2ATYNDANWNID

TK


ePA

-11

129415

Pseudechisaustralis

8.74

13755

252.7

3844.9

2CCQVHDNCYEQ

AGK

PLA2

833.9

2NLIQFG

NM

OXIQ

CANK

PA-19precursor

71066784

Pseudechisaustralis

8.6

16900

231.9

3884.9

2CCQVHDNCYEQ

AGK

PLA2

679.8

2DFT

CACDAEA

AK


ePA

-13

129474

Pseudechisaustralis

8.52

14002

196.7

4649.8

2DFV

CACDAAAAK

PLA2

793.8

2CKDFV

CACDAAAAK

PA-18precursor

71066782

Pseudechisaustralis

7.87

16718

136.1

3894.9

2TEC

KDFA

CACDAAAAK

PLA2

661.8

2VHDDCYGEA

EKaHeretherealnumberof

thematched

peptides

isshow

n.Onlyrepresentativepeptidesequencesareshow

ninthenextcolumn.



53�58 kDa. LAAOs with pI values in the acidic region, from 3.7to 4.3 and molecular masses of 56�65 kDa were identified inspots 32, 33, and 35 (Figure 1, Table 1). Peptides from proteinswith molecular weight of 80 kDa (spots 42�46) showedsequence similarity to other snake venom LAAOs. However,the molecular weight of these proteins is not characteristic of themonomeric oxidases, since these proteins oligomerize.51 Alldetected isoforms have a sequence similarity to both LAAOsisolated previously from the P. australis venom.52 Peptides fromthe proteins in spots 32 and 60 have a sequence similarity to theLAAOs from the Oxyuranus scutellatus scutellatus and Naja atravenoms.

Isoforms of transferrin-like proteins were identified from spots44, 45, and 46. The three proteins are in a horizontal in the pIrange of 7.5�7.8 (Figure 1). They have the same molecularmasses of 80 kDa and different isoelectric points, which suggestspossible post-translational modifications. All but one sequencesof the isolated tryptic peptides (Table 1) are 100% identical to

the respective segments in the body transferrin of the Africanhouse snake Lamprophis fuliginosus.53 The exception is a se-quence that has partial identity to the respective segment of thebody protein. The snake plasma protein and the three venomTFLPs possess identical molecular weights. This result suggestsrecruitment of body transferrin into the snake venom. Compar-ison of the five P. australis peptides with the sequence of humanplasma transferrin54 showed identity of the sequencesCGLVPXL and LFGSXXT.

Phospholipases A2

The proteomic analysis showed a large diversity of PLA2s inthe venomics of P. australis (Figure.1, Table 1). These enzymesrepresent 18.5% of all identified toxins (Figure 2). Acidic andbasic monomeric PLA2s were isolated from spots 90, 94 � 108and 110. These spots form a long of proteins with molecularweights of approximately 16 kDa in the pI interval from 5 to 9,again due probably to post-translational modifications. Theidentified enzymes likely belong to Group I since elapid snakevenom PLA2s are members of this group. Most probably, 13 ofthe proteins correspond to those isolated from the P. australisvenom PLA2s labeled as Pa-1G, Pa-3, Pa-5, Pa-9c, Pa-10A, Pa-11,Pa-12A, Pa-12c, Pa-13, Pa-16, Pa-17, Pa-18, and Pa-19.55�58 Theothers are isoforms of these enzymes, which have not beencharacterized before, with different pI values and/or molecularmasses. It should be mentioned that PLA2s isolated from spots90�100 are acidic proteins with pI values between 3.6 and 6.6(Figure 1). Usually, the acidic PLA2s are neither catalyticallyactive (or possess very low enzymatic activity) nor neurotoxic.59

However, Pa-1G is an exception to this rule and it is the firstacidic phospholipase A2 with high neurotoxicity (0.13 μgs/gbody wt).55 One peculiarity of the P. australis venomics is thehigh content of acidic PLA2s, while the single chain phospholi-polytic enzymes from other Australian elapids are almost exclu-sively basic.31 Basic PLA2s are highly homologous in their amino

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

1�5; 7�26; 29, 30�35; asrin Austrelaps superbus P�III metalloprotease

36�41; 42, 44�47; 59�74; porphyricase-1 Pseudechis porphyriacus P�III metalloprotease

75�77, 80, 87, 88 australease-1 Pseudechis australis P�III metalloprotease

scutatease �1 Notechis scutatus P�III metalloprotease

scutellatease Oxyuranus scutellatus P�III metalloprotease

32, 33, 35, 47�58; 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,

Pa-13, Pa-16, Pa-17, Pa-18, Pa-19

Pseudechis australis phospholipase A2

PLA2 isoforms Oxyuranus microlepidotus phospholipase A2

Notechis scutatus scutatus phospholipase A2

Lapemis hardwickii phospholipase A2

Pseudechis porphyriacus phospholipase A2

44�46 transferrin Lamprophis fuliginosus transferrin

65, 69, 70 ecto-50-nucleotidase Gloydius b. brevicaudus 50-nucleotidase79, 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

Figure 2. Percent of toxin sequences found in the Pseudechis australisvenom proteome.




acid sequences and, at the same time, differ considerably in theircatalytic properties and toxicity. Thus, Pa-11 is enzymatically 30times more active than Pa-13 and considerably more toxic thanthe second protein.57 Pa-13 showed no lethal activity at a doseof 7.4 μg/g mouse56 and Pa10A is another lethal PLA2.

60

Isoforms with higher molecular masses were isolated fromspots 82�84, 86 (basic proteins with molecular masses of32�34 kDa) and 92 (also basic protein with molecular massof 45�46 kDa). The enzymes from the first group are dimersbecause their molecular masses correspond to that of two-chaincomplexes. The protein in spot 92 could be an isoform of amultichain PLA2. Australian snakes contain such proteins; forexample, the 45.6 kDa taipoxin, the principle toxin of the O. s.scutellatus venom.31 The identified PLA2 isoforms showedsequence homology to phospholipolytic enzymes from thevenoms of Oxyuranus microlepidotus, Notechis scutatus scutatus,Lapemis hardwickii, and Pseudechis porphyriacus.

Other ProteinsA serine protease inhibitor was isolated from spot 109 of the

2-D gel (Figure 1, Tables 1 and 2). This is a basic polypeptide of9 kDa molecular mass and pI value of 7.4. It is homologous tomulgin-2, a 9.2 kDa protein with serine-type endopeptidaseinhibitor activity (GenBank: AAT4540.1). Three isoforms ofecto-50-nucleotidase with pI values between 8.5 and 8.7 wereidentified in the spots 65, 69, and 70. Spots 79, 101, and 107contain isoforms of venom nerve growth factor (Figure 1,Tables 1 and 2).

Enzymatic ActivitiesWe determined phospholipase A2, proteinase, L-amino acid

oxidase, alkaline phosphatase and acidic phosphatase activities ofthe Pseudechis australis venom. The results are presented inTable 3. The enzymatic activities of the Elapidae snake P. australisvenom are compared with the respective activities of Viperidaesnakes. The data are comparable because the activities weredetermined using the same methods and equipment. The venomPLA2 activity of the Elapidae snake is considerably higher thanthat of Bothrops alternatus, Crotalus d. terrificus, Vipera a. ammo-dytes, and Vipera a. meridionalis, but similar (even less) than thephospholipolytic activity of the Daboia russelli siamensis venom(Table 3). The venom proteinase activity is similar to that of theB. alternatus venom, but considerably higher than that of theother Viperidae snakes. Both P. australis and Vipera a. ammodytesvenoms show the highest LAAO activity. The alkaline phospha-tase activity of the mulga venom is similar to the activities of theother snakes. No acidic phosphatase activity was detected.

’DISCUSSION

P�III Metalloproteases, Phospholipases A2 and 50-Nucleo-tidases in Relation to the Pharmacological Activities of theP. australis Venom

Australian elapid snakes are among the most toxic in theworld.31 The major pathological effects of the P. australisenvenomation are severe disruption of hemostasis,31,61 muscledamage and necrosis.62 Mulga is a member of the nonprocoa-gulant group of elapid snakes.31 The coagulopathy should beattributed to the P�III SVMPs which predominate in thevenomics of P. australis (53% of all identified toxins). The classIII metalloproteases are composed of metalloprotease, disinte-grin-like and cysteine-rich domains.28 The metalloprotease do-main is responsible for the degradation of matrix proteins whilethe nonprotease domains exert anticoagulant effects.63 Thecysteine-rich domain inhibits the collagen-stimulated plateletaggregation.63 The P�III-enzymes induce also muscle damageand myonecrosis.64 In this way the metalloproteases contributesignificantly to the pathogenesis of the P. australis inducedenvenomation.

The other feature of the investigated venom composition isthe absence of serine proteases including enzymes with throm-bin-like activity. The severe disruption of hemostasis caused bythe P. australis bites proceeds without fibrinolysis,31 which is inline with the lack of serine proteases/fibrinogenases in the venomproteome.

The severe coagulopathic effect, caused by the P�III metallo-proteases, is strengthened by the high quantities of PLA2s, thethird largest group of toxins in the venomics of P. australis.Anticoagulant phospholipases A2 can bind and block factors ofthe coagulation cascade.31 A hemotoxic PLA2, potent inhibitor ofthe platelet aggregation, was isolated from the venom of anotherAustralian elapid, Austrelaps superbus.65 It is homologous to anenzyme from the P. australis venom (Table 1).

The king brown snake venom caused rhabdomyolysis fol-lowed by myoglobinuria and nephropathy.62 Neurotoxicity canbe supposed due to the presence of PLA2s in the venom.However, myotoxicity is the major pharmacological effect fol-lowing the P. australis bites.66 This can be explained by a strongand direct myotoxic action of a large quantity of PLA2s on themuscles. Myotoxicity is independent of the enzymatic activity.67

Analysis of the structure�function relationships and crystal-lographic investigations on snake venom PLA2s demonstratedthat the C-terminal part of the polypeptide chain, an exposedhydrophobic surface and interfacial surface charge are importantstructural determinants of the myotoxicity.68,69 Investigations ofthe action of five P. australis venom PLA2s on nerves and musclesdemonstrated that the predominant pharmacological effect is

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

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 0aData from ref 43 and references therein.



myotoxicity.70 However, neuromuscular effects of some isolatedking brown snake venom phospholipases A2 have beenobserved.60,71 These toxins produce muscle paralysis by reducingacetylcholine release60 or act postsynaptically to depress themuscle contractility.71 The high content of PLA2s is in accor-dance with the myotoxic effects of the P. australis snakebites.66

P�III metalloproteases also contribute significantly to themyotoxicity.

50-Nucleotidases inhibit the platelet aggregation via increasedadenosine signaling72 acting as anticoagulants. In this way thethree isoforms described in Table 1 strengthen additionally thecoagulopathic effects of the P. australis venom.

Adaptation of the P. australis venom for defense againstmicrobial pathogens. Recruitment of body transferrin intothe snake venom

The venomics of mulga snake reveal a high content ofantibacterial proteins, LAAOs and transferrin-like proteins(22.5% of the identified proteins). Potent antibacterial activityof the P. australis venom was demonstrated against Gram-positive and Gram-negative bacteria.52 This snake feeds uponfrogs containing the Aeromonas hydrophila,73 a heterotrophic,Gram-negative bacterium widespread among amphibians andfish. The pathogen is toxic to many organisms and can survive inaerobic and anaerobic environments. The P. australis venomshowed the highest antibacterial activity toward A. hydrophilaamong 21 tested Elapidae snake venoms,52 which correlates withfeatures of the snake diet. The presence of a large diversity ofLAAO isoforms in the venomics of the king brown snake (20% ofthe identified proteins) can account for the bactericidal effects ofthe venom because these enzymes are active against variousbacteria.52,74,75 L-Amino acid oxidases exert their antibacterialeffect through the hydrogen peroxide liberated after the oxidativedeamination of amino acids. Two L-amino acid oxidases, LAO1and LAO2, were isolated from the venom of P. australis.52 Bothenzymes possess subunit molecular masses of 56 kDa and formaggregates of 142 kDa. The correlation of the subunit molecularmasses with those of the proteins from spots 32 and 35 suggests apossible identity with the two LAAOs, described in the papermentioned above. It is known that LAAOs oligomerize inwater.51 Most probably, the aggregates dissociate under theconditions used for the 2-DE. The pathogen A. hydrophila,present in a high concentration in frogs which comprise asignificant part of the P. australis diet, was the most sensitivebacterium tested with venom LAAOs (LAO1 and LAO2) fromthe same snake.52

The presence of three transferrin isoforms in the P. australisvenom demonstrates recruitment of a body protein into thesnake venom. This result supports the theory that the snakevenom toxins evolve from recruitment of body proteins intothe chemical arsenal of the snake.20 The high degree of sequencesimilarity between the body transferrin, found in the liver of theAfrican house snake Lamprophis fuginosus (a colubrid snake) andthe TFLPs in the venom of the Australian P. australis is surprising.Both snakes inhabit different continents. Moreover, the trans-ferrins mentioned above have the same molecular masses as thehuman protein. Transferrin is a blood plasma protein for irondelivery to the tissues,76 associated with the innate immunesystem. It is produced mainly in the liver. A possible explanationof the transferrin physiological role as a venom protein isconnected with the metal binding properties of this protein.The binding of Fe3þ makes the environment unsuitable for the

bacterial survival, that is, transferrin has a bactericidal effect.Transferrins play a major role in iron transport and defenseagainst microbial pathogens.53 One of the reasons for theincorporation of TFLPs into the snake venom could be strength-ening of the antimicrobial effect of the venom.

Enzymatic ActivitiesThe snake venom enzymatic activities contribute considerably

to the total toxic effect. For this reason they are an importantcharacteristic of the venom. The relatively high PLA2 activity ofthe king brown snake venom is in agreement with its destructiveeffects on the body tissues. PLA2s hydrolyze membrane phos-pholipids and liberate lysophospholipids and fatty acids, includ-ing arachidonate. In this way they exert pathological effects onthe prey. The damage of biological membranes leads to changesin the permeability to ions and drugs.77 On the other handlysophospholipids are involved in cell lysis77 and arachidonate isa precursor of mediators of inflammation such as thromboxanes,prostaglandins and leukotriens.78 The catalytic activity of PLA2

leads to a serious disturbance of important physiological pro-cesses in the prey. Taking into consideration the relatively highcontent of these enzymes in P. australis, it can be concluded thatphospholipolytic enzymes play an important role in the lifethreatening effects caused by the mulga snakebite.

The high LAAO activity corresponds to the large quantity ofthese enzymes in the investigated venom. The catalytic activity ofthese enzymes results in the formation of the highly cytotoxichydrogen peroxide, which accounts for the strong antimicrobialeffect of the P. australis venom. L-Amino acid oxidases inducenecrotic and apoptotic cell death.75 Probably, this effect is usedby the snake as a defense against pathogens from the prey.Of course, LAAOs contribute to the total toxicity of the venomaimed at the killing of the small animals used as food.

The proteolytic activity of the P. australis venom is higher thanthat of a number of Viperidae snake venoms but, in principle, it isnot as high as it can be expected from the large quantity ofmetalloproteases. Most probably, the low level of this activity isdue to the high specificity of the P�III metalloproteases in thevenom, hydrolyzing a limited number of peptide bonds.

The individual variations in the alkaline phosphatase activityamong the snake venoms, compared in Table 3, are not drasticand hardly can influence the total toxicity.

’CONCLUDING REMARKS

The venom composition of P. australis demonstrates a highlyspecialized biosynthesis of large quantities of antibacterial toxinsand proteins disrupting the hemostasis or exerting myotoxiceffects. The results of the venom proteome analysis point to anadaptation of the venomic system for tissue destruction, bloodcoagulation blockade and a defense against microbial pathogensfrom the prey. The last hypothesis is supported by the obviousrelationship between the presence of potent antimicrobial pro-teins in the venom, its bactericidal effects and the bacterialcontamination of the food used in the snake diet. The antibac-terial activity can also prevent bacterial infections from the buccalcavity into the venom gland. To our knowledge, the bodytransferrin is unknown as a recruited component of the elapidor other snake venoms. A possible role of the venom transferrincould be strengthening of the antimicrobial effect. The highdegree of sequence homology between the body transferrin ofthe Colubridae African house snake Lamprophis fuliginosus andthe transferrin-like proteins from the Australian snake Pseudechis



australis (Elapidae) venom is surprising. Both snakes inhabitdistant regions with a very little likelihood for interbreeding.

The horizontal spot s of proteins belonging to the predomi-nant families of SVMPs, PLA2s and LAAOs are a characteristicfeature of the 2-D gel and suggest post-translational modifica-tions of these enzymes.

Pseudechis australis has a large venom output, up to 150 mg inone bite, and represents a rich source of pharmacologically activecompounds. Knowledge of the venomic composition revealspossibilities for the preparation of a more efficient antivenom, foradequate treatment of the consequences of the snakebite andfor the design of new medicines. Large quantities of proteinsinfluencing the hemostasis or with antibacterial properties can beobtained from this venom for medical, scientific and biotechno-logical purposes. The venomic composition of P. australis isrelevant to the pathologies associated with the snakebites, inparticular to the hemostatic disorders and myotoxicity.

’AUTHOR INFORMATION

Corresponding Author*Tel.: þ494089984744. Fax: þ494089984747. E-mail: [email protected].

Author Contributions†These authors have contributed equally to this work.

’ACKNOWLEDGMENT

This work was supported by a grant from the DeutscheForschungsgemeinschaft (project BE 1443-18-1 and BE1443) andfrom FAPESP/CNPq/CAPES. We are grateful to Dr. P. Mirtschin(Venom Supplies, Pyt. Ltd., Australia) for providing us with thevenom sample.

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pseudechis australis venomics: adaptation for a defense ... · 1 min with an isolation width of 4...

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