Fish Physiol Biochem DOI 10.

1007/s10695-013-9782-x

Application MALDI TOF on protein identication of vitellogenin in giant grouper (Epinephelus lanceolatus)
Ahmad Daud Om Saah Jasmani Nosrihah Ismail S. Y. Yeong A. B. Abol-Muna

Received: 28 February 2013 / Accepted: 2 March 2013 Springer Science+Business Media Dordrecht 2013

Abstract A new proteomics technology has been implemented to study the protein repertoires of developing oocytes of giant grouper (Epinephelus lanceolatus). Knowledge of the chemical composition and physiochemical properties of vitellogenin (Vtg) is necessary to interpret the functional and biological properties attributed during ovulation. Vtg, as a biomarker indicator in sex determination, has been analyzed to determine the sex and maturational status of sh in the absence of the gonad tissue. A male giant grouper was induced by 2 mg/kg of 17-estradiol (E2), and blood was sampled at days 0, 1, 3, 5, and 10. SDS-PAGE 1D electrophoresis was used to analyze Vtg protein, and Vtg identication was done with 4800 Plus MALDI TOF/TOFTM mass spectrophotometer
A. D. Om (&) Fisheries Research Institute (FRI) Tanjong Demong, Besut, Terengganu 22200, Malaysia e-mail: ahmaddaudom@yahoo.com S. Jasmani Institute of Tropical Aquaculture (AKUATROP), Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia N. Ismail Medical Biotechnology Laboratory, Faculty of Medicine, Universiti Malaya, Kuala Lumpur 50603, Malaysia S. Y. Yeong A. B. Abol-Muna Faculty of Fisheries and Aquaindustry, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu 21030, Malaysia

(Applied Biosystems/MDS SCIEX, USA). Meanwhile, MS/MS de novo sequencing identied the proteins by matching sequences of tryptic peptides to the known sequences of other species. Vtg was conrmed by MASCOT at 95 % signicant level, and molecular mass was 187 kDa. Protein resolved on SDS-PAGE as a double band of approximately the same mass as determined with MALDI-TOF. The N-terminal sequences and identication of Vtg were also determined. The potential of using MS methods to understand the structure and function of Vtg is discussed. Keywords Giant grouper MALDI-TOF 1D electrophoresis Vitellogenin Biomarkers

Introduction Development of gonad can be observed both at the morphological level by visual inspection of their size, color, and transparency and at molecular level by following changes in the protein and mRNA patterns accompanying their maturation. One- and two-dimensional gel electrophoresis (2-DE) and multidimensional protein identication technology combined with tandem mass spectrometry (MS/MS) are among the most powerful tools available for comparative proteomic studies. With these methodologies, facilities detection was important for the

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identication and characterization of protein participating in different cellular process, such as the oocytes maturation (Coonrod et al. 2002). De novo N-terminal sequencing of proteins is important, because the key information about the proteolytic processing such as the nature of modication or site of degradation might not be available otherwise. Vitellogenin (Vtg), a phospholipoglycoprotein, plays a role in maintaining the fertilization ability of the oocyte and allows subsequent normal development. Quantication of Vtg in blood is useful for different purposes. The reproductive status and degree of sexual maturation of shes can be assessed according to the levels of Vtg in plasma. Vtg functions as a nutritional source for the developing embryo, rather than as an important functional protein (Denslow et al. 1999). Therefore, Vtg is an ideal biomarker for female sex determination. Male normally do not synthesize Vtg; however, they will be induced to synthesize. The expression of this protein can also be induced in males under the effects of estrogenic compounds. Relying on this observation, some studies have used Vtg as biomarker of environmental endocrine disruption in many species. Therefore, the purpose of this study was to characterize giant grouper vitellogenin for eventual use in sex determination and as maturation indicator of this species as well as for reproductive studies in developing aquaculture.

well designed to be more easy to handle the broodstocks. The stocking densities of broodstock were 24 kg/ton. Water management changes were 100 % on alternate days, and water quality management also follows the SOP. Water quality measurement was taken once a week by using YSI 5,000 device, which is monitored on dissolved oxygen, pH, salinity, and temperature. Broodstock were fed in one-day interval with tuna (albacore sp.) at feeding rate of 1 % of body weight. Induction of vitellogenin in male giant grouper Vitellogenin synthesis was induced in three male giant groupers by 17b-estradiol (E2) injection (Syndel Asia Sdn. Bhd.) . E2 was dissolved in peanut oil/acetone (9:1) (Sigma) and stored aliquoted at -30 C. Fish received intramuscular injection by 2 mg E2/kg body weight on the initial day. Blood was collected from the afferent lamentary artery (AFA) in the gill lament by heparinized needles into 1-ml plastic syringes and transferred into cryovials containing heparin 10 mM EDTA and 100 TIU/l aprotinin. Blood samples were obtained on days 0, 1, 3, 5, and 10 after injection and then centrifuged at 14,000 rpm on Eppendorf 5415D Microcentrifuge, Hamburg, Germany, for 3 min. The supernatant was collected and stored at -30 C prior to use. Electrophoresis of Vitellogenin

Materials and methods Fish broodstock Giant grouper broodstock, males (35.0 4.5 kg; body weight, 115 7.5 cm; total length) and females (natural) (20.0 3.15 kg; body weight, 95 3.12 cm; total length), reared in indoor berglass (FRP) tank (10 ton) at FRI Tanjong Demong, Besut, Terengganu, were used in this experiment. There were stocks in the tank since December 2009, obtained from the supplier in Penang and Langkawi. Broodstock management was based on standard operational protocol (SOP) of FRI Tg. Demong Hatchery Operation (2004). For the purpose of good management practice (GMP) of broodstock maintenance, the tank shape should be round, the tank depth should be between 0.75 meters, and the tank should be Vtg fractions were determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Laemmli 1970) in 6.5 % gel. Five microliters of thawed plasma with concentration of 1.0 mg/ml protein from both experimental and control sh is loaded onto a 6.5 % polyacrylamide SDS-PAGE, was diluted in 0.125 M Tris base, 6.5 % SDS, 20 % glycerol, 10 % mercaptoethanol (sample buffer), and boiled for 4 min. Electrophoresis was conducted using 6.5 % gels in 25 mM Trisbase, 192 mM glycine, 0.1 % SDS, pH 8.3, in polyacrylamide at a constant voltage of 120 V for the rst 15 min and 150 V for the following 45 min. PageRuler Protein Ladder was used as standard for molecular weight determination. The gels were xed and stained with 0.025 % Coomassie Brilliant Blue. The band corresponding to the induced Vtg was easily recognized by comparison of the

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protein banding from both control (positive-natural female and negative-natural male) sh. In-gel digestion procedure Protein bands from SDS-PAGE gels were washed thoroughly four times with Mili-Q water, and then, each band was diced into approximately 1-mm square (Fig. 1a) using surgical blades and placed into 2-ml plastic vials for enzymatic digestion and dried in a SpeedVac. An unstained piece of gel from one corner of gel slab was simultaneously processed for use as a blank. Briey, a plug of the protein of interest was transferred to a microfuge tube and washed with 50 ll of 50 mM ammonium bicarbonate, 50 % (v/v) acetonitrile (ACN) was added and incubated at 37 C for 20 min on a rotating mixer (Fig. 1b). Samples were destined with serial washes of 50 % (v/v) methanol and 5 % (v/v) acetic acid over a period of 24 h. These processes are critical for removing the excess SDS and salts in the gel pieces, which interfere in most MALDI and ESI analyses. This process was repeated until all stain had been removed. The plugs were then washed in 100 mM

ammonium bicarbonate, which was subsequently discarded. Iodoacetamide (IA) (150 ll, 55 mM) was added to each plug and incubated at 37 C. After 20 min, the supernatant was discarded, iodoacetamide (500 ll, 100 mM) was added to each tube, and incubation continued in the dark for 60 min. The gel was dehydrated using 50 ll of 100 % ACN, and incubation at 37 C was resumed for 15 min (Fig. 1c). The supernatant was removed from the dehydrated plug, which was allowed to air dry. Once dry, the gel was rehydrated in 50 mM ammonium bicarbonate (25 ll) containing trypsin (6 ng/ll trypsin in 50 mM ammonium bicarbonate), vortexed briey, spun down at 200 rpm for 1 min, and allowed for 15 min; then, 50 mM ammonium bicarbonate (50 ll) was added to each tube, and digestion was allowed to continue overnight at 37 C; the reaction was hated by the addition of 10 ll of formic acid. MALDI-TOF preparation MALDI sample preparation with the mixed matrix/ analyzed solution droplet dried in air on the sample plate (Fig. 1d). With this method, inhomogeneous

Fig. 1 Schematic outline of protein identication steps by mass spectrometric (MALDI-TOF) analysis

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sample distribution and sweet spots can be expected. For peptide and protein samples, each compound was prepared to 1 nmol/ll as a stock concentration then diluted in 50 % ACN to desired concentration for analysis. Matrix of matrix ACHCA (alpha-cyano-cinnamic acid) was dissolved in 50 % ACN to nal compound of 50 nmol/ll. Each compound and matrix was mixed as equal volume and deposited onto a 0.7 ll sample plate area. The mixture was allowed to air dry or vacuum dry prior to analysis. Samples to be analyzed for nominal molecular weight were exchanged into low-salt (\20 mM) buffers, then spotted onto the MALDI target with an equivolume amount of MALDI matrix (Sinapinic acid in 50 % ACN/0/1 % TFA), and allowed to air dry. Spectra were acquired for 2,500 shots with an accelerating voltage of 25,000 V. Calibration with external standards resulted in typical mass accuracies of 0.1 % (Calibration mix of Mass Standard Kit ABSCIEX TOF/TOF). Analysis on Vtg molecular weight was performed with 4800 Plus MALDI TOF/ TOFTM mass spectrophotometer (Applied Biosystems/MDS SCIEX, USA) (Fig. 1e). Fractions of tryptic peptides were desalted using Zip-Tips (Millipore). Peptides were eluted from the Zip-Tips (Millipore) with 0.1 % TFA-acetonitrile ACN (50:50). Samples were mixed in a 1:1 ratio with a saturated solution of a-cyano-4-hydroxycinnamic acid in ACN/ water/triuoroacetic acid (50:49:1, v/v/v).

Fig. 2 Vitellogenin band appeared in positive control (natural female) and negative control (natural male) of giant grouper

Molecular weight calculation and identication of vitellogenin The relative molecular massof the Vtg and its derived peptide mass ngerprints were searched in the corresponding organisms database or the Swiss-Prot, inhouse database by using the MASCOT search engine with a probability-based scoring algorithm (Fig. 1f) (http: www.matrixscience.com) and a free-access internet search engine tools. The interpretation of the MS/MS spectra was performed with BioAnalyst, the sequencing algorithm provided with the GPS Explorer Software from ABSCIEX. The product ion spectra obtained from the MALDI-MS/MS of the tryptic in-gel digestion of giant grouper Vtg were analyzed using a computerbased sequencing algorithm called Lutesk.

Fig. 3 Vitellogenin band appeared in male giant grouper after induced with E2 on days 3, 5, and 10

Result Control and E2-induced plasma samples were analyzed in parallel by SDS-PAGE. After staining with Coomassie Blue, the Vtg band was easily distinguished by comparing the intensity of the bands between both samples. This is a routine analytical technique, which permits the perusal of qualitative and semi-quantitative results, obtained in protein biochemistry experiments. The extracellular SDS-PAGE proles of giant grouper Vtg are shown in Figs. 2 and 3. Molecular weight of Vtg band determined by the internal marker

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Fish Physiol Biochem Fig. 4 MALDI-MS mass ngerprinting generated by in-gel 1D electrophoresis tryptic digestion of vitellogenin a male giant grouper after induced with E2, b natural male giant grouper, and c natural female giant grouper

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ranged from 250 to 130 kDa. The appearance of Vtg was conrmed in natural female giant grouper but not in natural male giant grouper. Figure 3 presents E2induced Vtg in male giant grouper, and Vtg band was detected at similar band position with natural female and male giant groupers (Fig. 2) on days 3, 5, and 10 after injection. The bands corresponding to Vtg were excised and subjected to in-gel digestion with trypsin, and the

Fig. 5 Signicant level of 95 % condant Vtg, where P is a probability that observed match is a random event

results were analyzed by MALDI-MS. The MALDIMS spectrum obtained for giant grouper Vtg is shown in Fig. 4ac. The experimental protocol used in this study generated good-quality spectra enabling easy identication of Vtg. Vitellogenin MS spectra from natural female giant grouper (Fig. 4b) were similar in relative intensity of the spectra in induced male of E2 experiment (Fig. 4c). However, the MS spectra of natural male (Fig. 4a) was not appeared in mass ngerprinting, and it is revealed that natural male giant grouper does not synthesis Vtg (Fig. 4b) as long as no bands were also detected in natural male (Fig. 2). It was conrmed that MS spectra of Vtg were quite reproducible in terms of detection of abundant ions (2,292 m/z). The major peaks from this spectrum were used for protein identication using the PMF search in MASCOT. Database references were referred to Laeur et al. (1995). The proteins from database showed a statistically signicant hit from the list of peaks (Fig. 5), which is 34, and indicated extensive homology (p \ 0.05). By matching the peptide masses generated following the trypsin digestion with those available in the database, Table 1 shows the most

Table 1 Peptide mass ngerprint (PMF) of the trypsin digestion of male giant grouper Vtg induced with E2 Observed m/z 835.52 898.58 939.61 1,089.60 1,106.62 1,120.64 1,130.76 1,464.86 1,770.00 1,793.02 1,918.05 2,231.20 2,253.33 2,274.32 2,486.60 3,097.85 3,366.87
a

Calculated m/z 834.47 897.58 938.61 1,088.59 1,105.53 1,119.53 1,129.69 1,463.79 1,769.15 1,791.75 1,916.92 2,229.97 2,252.17 2,272.96 2,485.36 3,096.43 3,365.83

D m/z 1.05 1.01 1.00 1.01 1.09 1.11 1.07 1.07 0.85 1.27 1.13 1.23 1.16 1.36 1.24 1.42 0.04

Sequence K.ALHPELR.M K.IAARLNIK.E K.VVENPLLR.E K.NPALSESTDR.I K.ISWGEQCRK.Y K.CQEETKNLR.G K.FLELVQLLR.Ia R.TKQSLEFLEIEK.E R.ITPLLPTKVLVIPIR.R R.RNSSSSSSSSSSSESR.S K.NSISFAHSWILPAESCR.D K.NKMPSSCYQVAAQDCTDELK.F R.SPATIHPDVAAACSAAMKILGTK.L R.TSSASSLASFFSDSSSSSSSSSSDR.R K.ALLLSGINFHYAKPVLAAEMRR.I R.NSMLDSSELLPMEEEDVEPIPEYKFR.R R.ILPTVAGIPMELSLYSAAVAAASVEIKPNTSPR.L

Position 543549 880887 423430 692701 1,3101,318 205213 331339 256267 926941 1,0791,096 1,6011,617 1,4611,480 595617 1,1851,207 768789 952977 790822

Match N.M N.M N.M N.M N.M N.M M N.M N.M N.M N.M N.M N.M N.M N.M N.M N.M

Only this sequence matched with ions score 41. N.M indicates not matched with sequence homology, and M indicates matched with sequence homology. Nominal mass of Vtg was 187,805 Da and calculated pI value was 8.93

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Fish Physiol Biochem Fig. 6 Amino acid sequence (1,704) in singleletter code of giant grouper vitellogenin peptide showing sequence coverage after trypsin digestion in underline bold letters. The shaded sequence displays the peptide sequence matched with sequence homology with reference

10

20

30

40

50

MKAVVLALTL AFVAGQNFAP EFAAGKTYVY KYEALILGGL PEEGLARAGL 60 KISTKLLLSA 110 LAIPIKFEYT 70 80 90 100

ADQNTYMLKL VEPELSEYSG 120 130

IWPKDPAVPA TKLTAALAPQ 140 YRGILNILQL 150 NIKKTHKVYD 200

NGVVGKVFAP EEVSTLVLNI 160 170

180

190

LQEVGTQGVC KTLYSISEDA 210

RIEN I LLTKT RDLSNCQERL NKDIGLAYTE 230 240 250

220

KCDKCQEETK NLRGTTTLSY VLKPVADAVM ILKAYVNELI QFSPFSEANG 260 270 280 290 300

AAQMRTKQSL EFLEIEKEPI PSVKAEYRHR GSLKYEFSDE LLQTPLQLIK 310 320 330 340 350

ISDAPAQVAE VLKHLATYNI EDVHENAPLK FLELVQLLRI ARYEDLEMYW 360 370 380 390 EKFMAEEITI 440 400 AEAAQAFITA 450

NQYKKMSPHR HWFLDTIPAT GTFAGLRFIK 410 420 430 DKVVENPLLR 470

VHMVTADPEV IKLFESLVDS 460 CPVELIKPIQ

EVVFLGYGTM VNKYCNKTVD 480 490 500

QRLSDAIAKN EEENIILYIK 510 520 RNIAKKESRM 570

VLGNAGHPSS FKSLTKIMPI 530 540 550

HGTAAVSLPM TIHVEAIMAL 560 LSCIVLFETS

VQELALQLYM DKALHPELRM 580 590 600

PSMGLVTTVA NSVKTEENLQ VASFTYSHMK SLSRSPATIH 610 620 630 640 650

PDVAAACSAA MKILGTKLDR LSLRYSKAVH VDLYNSSLAV GAAATAFYIN 660 670 680 NIEGLQELIL 730 FFGQEIGFAN 780 690 KNPALSESTD 740 IDKPMIDKAV 790 800 750 700

DAATFMPKSF VAKTKGFIAG STAEVLEIGA 710 RITKMKRVIK ALSEWRSLPT 760 KFGKELFIQE YGREALKALL 801 720 SKPLASVYVK 770

LSGINFHYAK FVLAAEMRRI LPTVAGIPME 820 830 840 850

LSLYSAAVAA ASVEIKPNTS PRLSADFDVK TLLETDVELK AEIRPMVAMD 860 870 880 890 900

TYAVMGLNTD IFQAALVARA KLHSVVPAKI 910 920

AARLNIKEGD FKLEALPVDV 930 940 950

PENITSMNVT TFAVARNIEE PLVERITPLL 960 970

PTKVLVPIPI RRHTSKLDPT 980 990 1000

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Fish Physiol Biochem Fig. 6 continued

RNSMLDSSEL LPMEEEDVEP 1010 IPEYKFRRFA 1020 KKYCAKHIGV GLKACFKFAS 1030 1040 1050

QNGASIQDIV LYKLAGSHNF SFSVTPIEGE 1060 1070 1080

VVERLEMEVK VGAKAAEKLV 1090 1100

KRINLSEDEE TEEGGPVLVK LNKILSSRRN 1110 SSSSSSSSSSS SRKIDLAART 1160 SSRRSSSSSSS SSSSSSSSSSS 1210 SSSSSDRRSK 1260 1220 1120 NSSSSSSSSRN 1170 RRVNSTRSSS 1230

SSSSSSSSSSSS SSSESRSSRS 1130 1140 1150

SSSSSSSSSSSS SSSSSSSSSSS 1180 SSSRTSSASS 1240 1190 LASFFSDSSS 1250 1200

EVMEKFQRLH KKMVASGSSA SSVNEAIYKEK KYLGEEAVV 1270 1280 1290 1300 NWRICADAVV 1340 SWERLPSTLK 1390 TIDIITKTPM 1440 1450 1400 1350

AVILRAVKAD KRMVGYQLGF YLDKPNARVQ IIVANISSSDS 1310 1320 1330

LSKHKVTTKI SWGEQCRKYS TNVTGETGIV SSSPAARLRV 1360 RYGKMVNKYV PVKILSDLIH 1410 SSSVYNVTMHL PMCIPIDEIK 1460 1370 TKRENSTRNI 1420 GLSPEFDEVID 1470 1380 SVIAVATSEK 1430

KIHFMVSKAA AAECSFVEDT 1480 1490 1500

LYTFNNRSYK NKMPSSCYQV AAQDCTDELK FMVLLRKDSS EQHHINVKIS 1510 EIDIDMFPKD 1520 1530 TQQLPLKIKT 1580 1540 KRRGLAVYAP 1590 1600 1550

DNVTVKVNEM EIPPPACLTA 1560 1570

SHGLQEVYFD RKTWRIKVAD WMKGKTCGLC GKADGEIRQE YHTPNGRVAK 1610 NSISFAHSWI 1620 1630 1640 DSTCFSVEPV 1690 1700 1650

LPAESCRDAS ECRLKLESVQ LEKQLTIHGE 1660 1670 CLASDPQTSV 1680

PRCLPGCLPV

KTTPVTVGFS

YDRSVDLRQT TQAHLACSCN

TKCS

matching peptide of giant grouper Vtg. It was conrmed that molecular weight of giant grouper Vtg was 187,805 Da and the peptide sequence was FLELVQLLR, and it matched only with sequence homology with reference sequence. The top and bottom spectra correspond to a m/z ranges of 804 to 3,750. A conceptual translation of the open reading frame resulted in a 1,704 amino acid protein sequence (Fig. 6). A signal peptide was predicted (underlined)

by aligning the giant grouper sequence with the N-terminal sequence of several other piscine Vtg. It shows that FLELVQLLR was the sequence homology for giant grouper Vtg and the only single sequence matched with references in database. Meanwhile, de novo peptide sequencing with MS/MS of giant grouper Vtg yields peptide ions that showed high homology to 6 sequences from several other sh species, including Plaice, Flounder, Medaka, Mummichog, Eelpout, and Goby (Table 2).

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Discussion Proteomic studies are now becoming powerful tools for the discovery of new proteins involved in the developmental processes. To date, several proteomic studies have been conducted to quantify the reproductive status and degree of sexual maturation of the shes (Cohen and Banoub 2011). Proteomic approaches are also feasible ways to identify spe pez et al. 2002) and organ-specic cies-specic (Lo (Sun et al. 2003) peptides in some species. Relevant studies on reproductive status and degree of sexual maturation of the sh have correlated with vitellogenin characteristics and can be assessed through proteomic studies. Vitellogenin (Vtg) was produced at specic tissue (liver) as a yolk protein precursor undergoes processing into smaller molecules and subsequently accumulates in developing oocytes as vitellin, it is essential to attain complete knowledge of structure of the Vtg molecule itself. Vtg, a phospholipoglycoprotein, plays a role in maintaining the fertilization ability of the oocyte and allows subsequent normal development. Vtg functions as a nutritional source for the developing embryo, rather than as an important functional protein (Denslow et al. 1999). Vtg can be dened as a set of amino acids arranged in a specic sequence to yield a dened activity and has specic characteristic to have a degree of homology or sequence similarity with other protein. It can be identied and characterized through its peptide mass ngerprint (PMF) (Henzel et al. 1993), in the case of organisms with fully sequenced genome, or by analysis of the fragmentation spectra of such peptide (PFF, peptide fragment ngerprinting or even de novo sequencing) obtained through tandem MS (Chapovetsky et al. 2007). One- and two-dimensional gel electrophoresis rg et al. 2005) and multidi(2-DE) (reviewed in Go mensional protein identication technology combined with tandem mass spectrometry (MS/MS) (reviewed in Aebersold and Mann 2003) are among the most powerful tools available for comparative proteomic studies. Matrix-assisted laser desorption/ionization mass systems (MALDI-MS) is a soft ionization technique in mass spectrometry, allowing the analysis of biomolecules (such as proteins and peptide), which tend to be fragile and fragment when ionized by more conventional ionization methods. The ionization is

Table 2 Comparison of MS/MS spectra of giant grouper vitellogenin to vitellogenin sequences from other species using the Mascot search engine Matching sequences Plaice Pleuronectus plactessa E. Flounder Platichthys esus Medaka Oryzias latipes Mummichog Fundulus heteroclitus Y. goby Acanthogobies avimanus Eelpout Zoarces viviparus Rainbow trout Oncorhynchus mykiss F. minnow Pimephales promelas J. Sillago, smelt Sillago japonica Whitesh Corogonus lavaretus Giant grouper Epinephelus lanceolatus (in this experiments) No. of sequences: 1. PIHELAVEAKLAK, 2. FLELIQLLR, 3. TLYAITEDEK, 4. THYVISEDAK 5. TEGI/LQEALLK 6. THYVISEDAK X X X X X X X X X X X X 1 X 2 X 3 4 5 6

triggered by laser beam. A matrix is used to protect the biomolecules from being destroyed by direct laser beam and to facilitate vaporization and ionization. The molecular mass values of the trypsinized peptides obtained by MALDI-MS are then used to identify the predicated proteins using Web-based search engines such as MASCOT. The present study demonstrates that molecular mass of giant grouper was 187 kDa and is close to 184 kDa for brook trout (Salvelinus fontinalis; Schafhauser-Smith and Benfey 2002), 180 kDa for Atlantic halibut (Hippoglossus hippoglossus; Norberg 1995), 180 kDa for sea bass (Dicentrarchus labrax L; Mananos et al. 1994), 180 kDa for seabream (Sparus aurata; Mosconi et al. 1998), and 170 kDa for barn ounder (Verasper mosri; Matsubara et al. 1999).

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One approach is to create the so-called sequence ladder of an isolated peptide that can be read following MALDI-MS analysis. These sequence ladders can be obtained either chemically or enzymatically. Chemical ladder sequencing is mainly based upon Edman degradation. Through MALDI-MS, the sequences of peptide could be obtained with mass difference between consecutive ions in the obtained spectrum corresponding to masses of amino acids and thus lead to peptide sequencing. Sequence ladder with MALDI-MS technique is rapid compared to conventional Edman sequencing method, because a reaction yield is not required and the one-step readout is very fast (within\1 min). An Edman degradation rate of 10 residues/hour has been achieved by the use of an automated instrument (Wang et al. 1994). The results of the present study facilitate a better understanding on vitellogenin as biomarker indicator in sex determination of giant grouper (Epinephelus lanceolatus). This information can be used for improving the production of giant grouper for broodstock management.

Conclusion This study demonstrated that MALDI-TOF can be used successfully for the identication of giant grouper peptide Vtg.
Acknowledgments This study is funded by the Ministry of Science, Technology and Innovation, Malaysia, under Intensied Research in Priority Areas (E-Science Fund, 004-07-05-06).

References
Aebersold R, Mann M (2003) Mass spectrometry-based proteomics. Nature 422:198207 Chapovetsky V, Gattegno T, Admon A (2007) Proteomic analysis of the developing sh oocyte. In: Babin PJ et al (ed) The Fish Oocyte: From basic studies to biotechnological applications, pp 99111 Cohen AM, Banoub JH (2011) Application of mass spectrometry for the analysis of vitellogenin, a unique biomarker for xenobiotics compounds. In: Banoub J (ed) Detection of biological agents for the prevention of bioterrorism. Springer, Berlin, pp 301318

Coonrod SA, Wright PW, Herr JC (2002) Oolemmal proteomics. J Reprod Immunol 53:5565 Denslow ND, Chow MC, Kroll KJ, Green L (1999) Vitellogenin as a biomarker of an exposure for estrogen or estrogen mimics. Ecotoxicology 8:385398 rg A, Weiss W, Dunn MJ (2005) Current two-dimensional Go electrophoresis technology for proteomics. Proteomics 5:826827 Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci 90:50115015 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:6885 Laeur GJ, Byren BM, Kanungo J, Nelson LD, Greenberg RM, Wallace RA (1995) Fundulus heteroclitus vitellogenin: the deduced primary structure of a piscine precursor to noncrystalline, liquid-phase yolk protein. J Mol Evol 41:505 521 pez JL, Marina A, Alvarez G, Va zquez J (2002) Application Lo of proteomics for fast identication of species-specic peptides from marine species. Proteomics 2:16581665 Mananos E, Zanuy S, Le Menn F, Carrilllo M, Nunez J (1994) Sea bass (Dicentrachus labras L.) vitellogenin. I-Induction, purication and partial characterization. Comp Biochem Physiol 107B:205216 Matsubara T, Ohkubo N, Andoh T, Sulivan C, Hara A (1999) Two forms of Vitellogenin, yielding two distinct lipovitellins, play different roles during oocyte maturation and early development of barn ounder, Verasper mosri, a marine teleost that spawns pelagic eggs. Dev Biol 213: 1832 Mosconi G, Carnevali O, Carletta R, Nabissi M, PolzonetteMagni AM (1998) Gilthead sea bream (Sparus aurata) vitellogenin: purication, partial characterization, and validation of an enzyme-linked immunosorbent assay (ELISA). Gen Comp Endocrinol 110:252261 Norberg B (1995) Atlantic halibut (Hippoglossus hippoglossus) vittelogenin: induction, isolation and partial characterization. Fish Physiol Biochem 14:13 Schafhauser-Smith D, Benfey TJ (2002) The purication and development of a quantitative enzyme linked immunosorbent assay (ELISA) for the measurement of vitellogenin in diploid and triploid brook trout (Salvelinus fontinalis). Fish Physiol Biochem 24:287298 Standard operational protocol (SOP) of Marine Finsh Hatchery (Malay Edition). FRI Tg. Demong Hatchery Operation (2004). In-house publication, 168 p Sun B, Pankhurst NW, Watts M (2003) Development of an enzyme-linked immunoassay for vitellogenin measurement in green- back ounder Rhombosolea tapirina. Fish Physiol Biochem 29:1321 Wang R, Chait BT, Kent SBH (1994) Protein ladder sequencing: towards automation. Technique in protein chemistry. Academic Press, New York, pp 1926

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