Malaysian Journal of Biochemistry and Molecular Biology (2006) 14, 25-32

25

Proteomic Analysis of Snakehead Fish (Channa striata) Muscle Tissue

Lay-Harn Gam, Chiuan-Yee Leow and Saringat Baie
School of Pharmaceutical Sciences, University Sciences of Malaysia, 11800 USM, Penang, Malaysia.

Abstract
Snakehead fish, also known as Haruan, is recognized in Asia Pacific countries as a remedy for healing of wounds. The fish enhances dermal wound healing and reduces post-operative pain and discomfort. The efficacy of wild type snakehead fish has made it a common food served to women after childbirth or those who had undergone surgery. Due to high demands of snakehead fish, farming of the fish is now carried out commercially. However, the flesh of cultured snakehead fish has been said to produce different texture from the wild type fish. In this study, analysis of the protein composition of the flesh of snakehead fish was carried out. Wild type snakehead fish of different sizes and caught in different months of the year were compared. The data showed that fish of smaller sizes yielded higher protein content as compared to the bigger fish. However, protein profiles of the fish were similar for all the different months of catching. The major group of protein in snakehead fish was enzymes, followed by structural proteins. The protein profile displayed may be used as a reference for farming and culturing of snakehead fish. Keywords: Snakehead fish, proteomic

Introduction
Snakehead fish is an obligatory air-breather and predaceous fish that resides in swamps, slow-flowing streams and in crevices near riverbanks in Southern China. In taxonomy, it belongs to the family of Ophiocephalidae or Channidae [1]. The habitats of the fish are always infested by thick aquatic vegetation which expands over the entire water surface. Snakehead fish is consumed mainly as a remedy to help the healing of wounds after a clinical operation, road accident and caesarian. The biochemical analysis of its flesh was undertaken based on the knowledge that the fish contained -3 polysaturated fatty acids that regulate prostaglandin synthesis and also influence the immune system [2,3]. In addition, the amino acid composition in snakehead fish has also been analyzed and was reported to play a role in the process of wound healing [3]. The efficacies of wild type snakehead fish in the healing of wounds have been proven [4,5]. Due to high demand, snakehead fish has been cultured commercially. However, the tissue texture of cultured snakehead fish is different from the wild type snakehead fish. Therefore, the knowledge on the protein composition of wild type snakehead fish will be beneficial where it can be used as a reference to culture snakehead fish. In this study, the aqueous soluble protein profiles of different sizes of wild type snakehead fish were determined. The fish were caught in different months and at different places in the region of northern Malaysia. The data obtained represent the protein profile of the wild type fish, which can be used as a reference for culturing and farming of snakehead fish.

Materials and Methods

Preparation of Snakehead fish Muscle Tissue Three batches of snakehead fish caught in November 2002 (B1), January 2003 (B2) and April 2003 (B3), respectively were used in this study. The fish from each batch were further subdivided according to their lengths. All fishes were washed, beheaded, sliced and covered with ice to ensure freshness of the fish tissues. The fishs muscle tissue was then sliced into smaller pieces and placed in sterile universal bottles and kept at -20C prior to freeze-drying. Freeze dried snakehead fish muscle tissue was homogenized to powder form. Extraction of protein from snakehead fish muscle tissue was carried out on 1.0 mg of powdered fish muscle using 1 mL of 40 mM Tris (pH 8.8) extraction buffer. The sample mixture was then vortexed for 2 minutes and centrifuged at 12, 000 ( g for 30 min at room temperature and the supernatant was recovered. Protein Concentration Determination Protein concentration determination was carried out using the method described by Bradford [6]. Bovine Serum Albumin (BSA) was used to construct a standard protein concentration curve. The assay was performed in a 96 well plate. Protein concentration standards ranging from 0.1 - 1.4 mg BSA/mL were prepared. Five L of each of the protein standards were added to separate wells in the 96 well plates in triplicates. Five L of phosphate buffer was added to the blank wells. Fifty mg
Author for correspondence: Dr Lay-Harn Gam, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia. Fax no: 604-6570017 E-mail: layharn@usm.my

Proteomic analysis of snakehead fish muscle tissue

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of powdered tissue was dissolved in 1000 l of buffer (for the Tris buffer extract, 10 l of extract was diluted in 1000 l of buffer), it was then centrifuged at 12,000 x g for 30 min and the supernatant was recovered. Five L of the supernatant was then added to the wells. 250 L of the Bradford Reagent was added to each plate well that contained standards and samples. The plate was then shaken for approximately 30 seconds and incubated at room temperature for 15 minutes. The absorbance was measured at 595 nm. A standard curve was plotted using net absorbance versus protein concentration of each standard. The protein concentration of unknown samples was determined by comparing the average A595 values against the standard curve. Sodium Dodecyl Sulphate-Polyacrlyamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE was performed as described by Laemmli [7]. Ten per cent polyacrlamide gel in a vertical slab gel apparatus (Hoefer) was used. Protein samples (Tris buffer extraction) were then loaded into the wells of polymerized gel. Electrophoresis was performed at a constant voltage of 200 volts when samples were in the stacking gel. When the dye front reached the resolving gel, voltage was increased to 245 volts. The run was stopped when the dye front was 2 to 3 mm away from the bottom edge of the gel. At completion of electrophoresis, the glass sandwich was disassembled. The stacking gel was discarded and the resolving gel was stained using Coomassie Blue. Molecular weights of the proteins were determined by comparing relative mobility of protein bands to the standard protein markers. The Coomassie Blue stained gel images were acquired and digitized using Versadoc Imaging Scanner. Protein bands intensity analysis was carried out using the Camag TLC Scanner 3 and the densitometry analysis was performed using the CATS software. In-Gel Digestion The polyacrylamide gel was washed thoroughly with 100 mM NH4HCO3. The protein bands were then excised from the gel. In-gel digestion using trypsin was performed according to Shevchenko, et al. [8] with slight modification. The gel pieces were first excised and shrunk by dehydration in acetonitrile. The solvent was then discarded and the gel pieces were dried in a vacuum centrifuge. A volume of 10 mM dithiotreitol (DTT) in 100 mM NH4HCO3 sufficient to cover the gel pieces was added and the protein was reduced for 1 hour at 56C. After cooling to room temperature, the DTT solution was replaced with a same volume of 55 mM iodoacetic acid in 100 mM NH4HCO3. After 45 minutes incubation at ambient temperature in the dark with occasional vortexing, the gel pieces were washed with 50-100 l of 100 mM NH4HCO3 for 10 minutes, dehydrated with acetonitrile, rehydrated in 100 mM NH4HCO3 and dehydrated in the same volume of acetonitrile. The liquid phase was

removed and the gel pieces were dried in a vacuum centrifuge. The gel pieces were swollen in digestion buffer containing 50 mM NH4HCO3, 5 M CaCl2, and 12.5 ng/l of trypsin in an ice-cold bath. After 45 minutes, the supernatant was removed and replaced with 10 l of the same buffer but without trypsin to keep the gel pieces wet during enzymatic cleavage at 37 C overnight. Peptides were extracted from the gel matrix by adding 15 l of 20 mM NH4HCO3, vortexed and incubated at room temperature for 10 minutes. The supernatant was recovered after a brief spin. This was followed by adding (1 to 2 times the volume of gel pieces) 5% (v/v) formic acid in acetonitrile:water mixture (70:30), vortexed and incubated for 20 minutes at room temperature. It was then spun down and the supernatant was recovered. These steps were repeated 3 times. Pooled extracts were dried down in a vacuum centrifuge and stored at 20C. HPLC-MS Analysis Mass spectrometric analysis was carried out using an ion trap mass spectrometer (Agilent, VL). The peptides were ionized using the electrospray soft ionization technique (ESI). The mass spectrometer was operated in a two-mode program consisting of full MS Scan and full MS/MS Scan, whereby, the most intense ion in the full MS Scan was isolated and subjected to full MS/MS Scan. The peptides resulting from the in-gel digestion was reconstituted in 50 L of ddH20. The peptides were separated on a reversed-phase column (1mm x 250 mm, 5 m, 300 A). The HPLC separation condition was at linear 5% B to 95 % B in 65 minutes at 20 l/min flow rate. The eluent of HPLC was directed to a mass spectrometer, which was interfaced with the HPLC. The parameters used for acquiring the MS data were: heated capillary temperature of 300 C, dry gas flow rate of 8.0 L/min and nebulizer gas pressure of 30.0 psi. The parameters set for the MS/MS Scan were collision energy (voltage) = 1.15 V, charge state = 2, minimum threshold = 5000 counts, and the isolation width = 2 m/z. The MS/ MS spectra were recorded in the automated MS to MS/ MS switching mode with an m/z dependent set. Sequence Database Search The MS/MS data were subjected to Mascot protein database search engine (www.matrixscience.com). The search engine contains the calculated spectra for all peptides in the National Centre for Biotechnology Information (NCBI) non-redundant sequences database [9]. The taxonomy and enzyme selected was Actinopterygii (Ray-Finned Fish) (29309 sequences) and Trypsin, respectively, whilst Fixed Modification was Carboxymethyl (C). The Peptide Mass Tolerance was set at 2 Da whereas 0.8 Da was set for the Fragment Mass (MS/MS) Tolerance. The data format was selected as Mascot Generic and only one missed cleavage was allowed. Instrument type set was ESI-TRAP i.e. Electrospray Ionization and Ion Trap Mass Spectrometer

Proteomic analysis of snakehead fish muscle tissue

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(1100 Series, Agilent, Germany). Proteins functions and characteristic information were obtained from both the PubMed (www.ncbi.nlm.nih.gov/entrez) and Swiss Prot (www.expasy.ch/sprot/sprot-top.html).

Results and Discussion

Snakehead fish has long been consumed as a source of dietary protein. It is also well known traditionally for its medicinal property for healing of wounds. In this study, the proteins extracted from the fishs muscle tissue were analyzed. Different sizes of wild type snakehead fish caught at different seasons were used in the study. The data obtained provide useful information on the nutritional and medicinal properties of snakehead fish. Culturing of snakehead fish has been carried out in Malaysia due to the high demand of the fish. As a large proportion of fish muscle tissue is made up of proteins, cultured fish muscle tissue texture can be monitored by comparing their protein profile with those of the wild type fish. In order to study the protein profile of snakehead fish, fishes of different sizes that were caught at different seasons were analyzed. Three batches of snakehead fish caught in November (B1), January (B2) and March (B3), respectively were used in this study. From each batch, eight different lengths of snakehead fish were used, which were at 16 cm, 23 cm, 24 cm, 25 cm, 28 cm, 29 cm, 30 cm and 38 cm lengths. In B1, it was found that smaller fish (16 cm and 23 cm) contained significantly higher protein content (P<0.05) than the larger fish (Figure 1). The same observation also occurred in B2 and B3. However, only the 16 cm fish showed significantly higher protein content (P<0.05) than the larger fish in these two batches of fishes. Generally, the protein content for the fish length from 25 to 38 cm did not differ significantly with respect to their sizes in all the three batches of the fish analyzed.
Protein Concentration from Haruan with Various Length
30.00
Concentration (mg / mL)
b ab ab b a ab a a ab ab b b a ab ab ab a a a ab a ab a

The phenomenon of cannibalism in snakehead fish may be one of the reasons why muscle protein synthesis activity in smaller snakehead fish is more active and rapid as compared to the larger fish. The secretion of myofibrillar protein and collagen were greater in the smaller fish for their movement to survive in the present of larger predator. In contrast, the consistent protein content amongst the larger fish shows the fishes of 25 to 38 cm lengths have achieved their full protein capacity. The problem of cannibalism is believed to be the cause of the low survival of smaller fishes in culturing of snakehead fish [10]. The alternative approach would be to provide adequate food [11] or partially control cannibalism by grading fishes into approximately similar size group. Figure 2 illustrates the protein contents of the three different batches of snakehead fish. It was found that snakehead fish from B1 and B2 did not vary significantly in their protein concentration (P>0.05). Fishes from both of these batches contained on average 0.3640.001 mg protein/mg tissue and 0.3710.001mg protein/mg tissue, respectively. However, snakehead fish from B3 showed significantly higher protein content than B1 and B2 (P<0.05). Snakehead fish from B3 contained on average 0.4490.001 mg protein/mg tissue. Different in protein concentration in the three batches of snakehead fish (Figure 2) can be explained by the seasons when the fish were caught. In Malaysia, the weather condition for November (B1), January (B2) and March (B3) are dry, extremely dry and rainy, respectively. Our data have shown that the fish that were caught during rainy season yielded significantly higher protein compared to those that were caught during the dry seasons. During rainy season food availability is much more abundant for carnivore fish type, such as the snakehead fish.
Protein Content from haruan B1, B2 and B3
600.0
Content (mg / g)

25.00 20.00 15.00 10.00 5.00 0.00

ab

500.0
B1 B2 B3

b a a

400.0 300.0 200.0 100.0 0.0

16

23

24

25 28 Length (cm)

29

30

38

B1

B2 Batch Number

B3

Figure 1: Comparison of the protein concentration (50 mg of dry tissue) of Snakehead fish muscles tissue from three batches of fish. Protein concentration was analyzed using Bradford method. Different alphabet annotation represents significant difference (p<0.05) between the different batches of fish. Statistical analysis was carried out using analysis of variance (ANOVA).

Figure 2: Protein content of Snakehead fish from B1, B2 & B3. The average protein content of all the fish according to the fish length. Different alphabet annotation represents significance different (p<0.05) between the different batches of fish. Statistical analysis was carried out using analysis of variance (ANOVA).

Proteomic analysis of snakehead fish muscle tissue

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Table 1: List of water soluble proteins detected in Haruans muscle tissue. Band number are refer to indication mentioned in Figure 3. SWISS-PROT Accession number KIBOA/ P00570 Q804Y1 Q8JH72 Q7ZW73 Q6PUS4 Q9YGE7 Q7ZU04 Q804Z1 Q804Z2 Q9I8I6 S13164 Q98SS7 Q9DFM2 Q7T1J1 Q7T306 Q9YI16 Q9YI15 Q9YI14 Q7T1J0 Q7T1J3 Q7T1J2 Q7ZZM5 Q6TH14 (AAQ97775) Q6GQM9 O57518 Q76BF6 Q8AY84 Q6NXD1 Q803D2 Q76IM5 Q7SXV3 Q8JJC2 Q8QGU8 Q7M558 BAD04856 (Q76B34) Q7T040 Q8UW40 Q6DR47 Q76BE1 Q7T315 Q90XF8 Q9PWD1 Q7ZU23 Q6TNW2 Q6DHS1 Protein Name Adenylate kinase (EC 2.7.4.3) Aldolase (Fragment) Aldolase A. Aldolase b, fructose-bisphosphate Brain glycogen phosphorylase Pygb Complement factor Bf-1 Creatine kinase, brain Creatine kinase Creatine kinase Creatine kinase (EC 2.7.3.2) Creatine kinase (EC 2.7.3.2) Creatine kinase (Fragment) Creatine kinase (Fragment) Creatine kinase brain isoform (Fragment) Creatine kinase CKM3 Creatine kinase M1-CK Creatine kinase M2-CK Creatine kinase M3-CK Creatine kinase mitochondrial isoform precursor Creatine kinase muscle isoform 1 Creatine kinase muscle isoform 2 Enolase (Fragment) Enolase 1 (AY398342 NID) Enolase 2 Fructose-1, 6-bisphosphate aldolase Phosphoglycerate kinase (Fragment) Phosphoglycerate kinase (Fragment) PKM 2 protein Platelet-activating factor acetylhydrolase, isoform Mw 21761 17427 40223 39700 97916 85034 43178 43231 43032 46859 43267 29125 21139 42608 43115 42983 43133 43185 47108 42713 42888 28757 47848 47160 39957 41657 11317 58598 47080 149432 60118 58767 58582 350347 132804 90214 58059 178147 25178 27100 26476 129986 42304 104086 42374 pI 8.94 8.73 8.27 8.75 6.11 5.90 5.49 6.29 6.32 8.73 6.20 8.89 5.79 5.89 6.29 6.21 6.22 6.25 8.50 6.32 6.44 8.15 4.77 6.21 6.04 4.67 6.36 6.97 9.28 7.30 6.35 7.96 8.68 7.54 7.03 8.93 6.00 6.90 7.60 8.38 5.23 5.23 5.23 Function Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Structural Structural Structural Band number 14 10 2,8,9,10 10 2,7 1,6 7,9 9 9 9 7,9,10 7,9 9 9 9 2,9 9 9 9 9,10 9 6 6 6 10 9 9 5 7 11,12 7 5 5 9 14 10 14 2,5,8,10,11, 12, 13, 14, 15 11 11,12 12 7,9 2 7 2

Ib, alpha subunit b.

Pol-like protein Pygb protein (Fragment) Pyruvate kinase Pyruvate kinase Replicase/ helicase/ endonuclease Reverse transcriptase Solble guanylyl cylase alpha2 subunit ST7 protein Topoisomerase 2 (Top 2A protein) Triose phosphate isomerase (Fragment) Triosephospahte isomerase 1b Triosephosphate isomerase B TYK2 tyrosine kinase Actin, alpha 1, skeletal muscle Actinin, alpha 2. Actin, alpha 2, smooth muscle, aorta

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SWISS-PROT Accession number Q90333 Q6QUR3 Q7T2J3 Q76BG1

Protein Name

Mw

pI 4.40 8.39 6.44 8.09 8.42 7.23 7.74 8.63 8.54 8.09 6.67 7.31 6.49 6.67 6.91 6.44 6.32 6.98 6.44 6.82 7.68 6.82 7.85 10.47 10.47 8.03 7.78 8.10 9.07 9.16 5.99 9.01 8.45 5.09 6.88 5.61 5.44 5.32 6.47

Function Structural Structural Structural Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme Enzyme EnzEnzymeyme Enzyme Enzyme Ribosomal Ribosomal Transcription factor Transcription factor Transport Calcium ion binding Translation Factor Translation Factor DNA-RNA -binding Signal transduction Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein

Band number 10 9 2 9 8,9,10 10 10 10 10 10 8 10 10 10 10 2 9 9 1,7,9 15 15 5 5 10 10 9 13 5 5 7 7 7 10 7 5 9 7 12 9 9

Fast skeletal myosin light chain 3 16794 Myosin heavy chain (Fragment) 23564 Skeletal muscle actin (Fragment) 43041 Fructose-bisphosphate aldolase A 36509 (Fragment) ALFB_SPAAU Fructose-bisphosphate aldolase B 40069 (EC 4.1.2.13) Q90Z48 Glyceraldehyde phosphate dehydrogenase 36425 (EC 1.2.1.12) Q8AWX8 Glyceraldehyde-3-phosphate dehydrogenase 36244 Q8JIQ0 Glyceraldehyde-3-phosphate dehydrogenase 36069 (EC 1.2.1.12) Q9PTW5 Glyceraldehyde-3-phosphate dehydrogenase 36192 (EC 1.2.1.12) LDHA_ SPHAG L-lactate dehydrogenase A chain 36650 (EC 1.1.1.27) LDHA_CHAAC L-lactate dehydrogenase A chain 36261 (EC 1.1.1.27) LDHA_CYPCA L-lactate dehydrogenase A chain 36413 (EC 1.1.1.27) LDHA_ELEMC L-lactate dehydrogenase A chain 36387 (EC 1.1.1.27) LDHA_HARAN L-lactate dehydrogenase A chain 36200 (EC 1.1.1.27) LDHA_BRARE L-lactate dehydrogenase A chain 36382 (EC 1.1.1.27) Q9PV91 Muscle creatine kinase 43041 Q90X19 Muscle-specific creatine kinase 43030 Q8JH39 Muscle-type creatine kinase CKM1 43351 Q8JH38 Muscle-type creatine kinase CKM2 42985 Q9DFL9 Nuclease diphosphate kinase B 17218 Q9PTF3 Nucleoside diphosphate kinase- Z3 19562 Q8QFU1 Phosphoglucose isomerase -2 62173 Q8QFT1 Phosphoglucose isomerase-2 (EC 5.3.1.9) 62166 Q90YR3 40S ribosomal protein S11 18610 Q7ZV05 Similar to 40S ribosomal protein S11 18568 Q8QGQ9 Teashirt-like zinc finger protein (Fragment) 95735 Q9I8L6 T-box transcription factor 49606 Q8UWF2 Glutamate receptor subunit 1B (Fragment) 62737 JC4956/ Q90W12 Vitellogenin precursor 184710 Q9PVM6 Elongation factor 1 alpha 50743 Q7T1U2 Tmc2-related protein 2 (Fragment) 75970 Q90XI6 RAG2 (Fragment) 17314 Q98TT9 GDNF family receptor alpha-1a 54506 Q7SYD3 zgc: 67559 protein (Hypothetical protein) 104082 Q6DG54 Zgc:92037 58897 AAH59437 zgc: 73059 (BC059437 NID) 46671 AAH59571 (Q6PBV4) zgc: 73229 protein (BC059571 NID) 29926 Q6NXB1 Hypothetical protein zgc:77002 34254 Q7ZZ46 SI:dZ249N21.1.3 (Novel protein similar 455282 to human titin (TTN) (Fragment) Q7ZV29 zgc: 56252 (Similar to phosphoglycerate 45126 kinase 1)

Proteomic analysis of snakehead fish muscle tissue

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The protein profile of the aqueous soluble protein extracted from various sizes snakehead fish muscle tissues from B2 and B3 is shown in figure 3 (protein profile of B1 is not shown; there was no variation between the three batches). Each of the lanes was loaded with similar amounts (50 g) of protein extracts from fish of different lengths. Lanes 1 to 6 represent the protein profiles from B2 snakehead fish at 23, 24, 25, 28, 29 and 30 cm fishs length, respectively. Lanes 7 to 14 represent the protein profiles of B3 snakehead fish at 16, 23, 24, 25, 28, 29, 30

and 38 cm fishs length, respectively. The protein profiles of fish with different lengths and month of catches did not differ significantly. Upon Coomassie Blue staining, protein bands, which were evenly distributed in the range of molecular masses from 10 kDa to 205 kDa were detected. The relative intensity of the protein band in each lane was evaluated using densitometry analysis (Figure 4). In addition to the similar protein profile displayed by all the fish, the relative intensity of the proteins is also similar. Thus, the non-variable features (protein profiles and bands intensity) shown by wild type snakehead fish is beneficial for monitoring of the protein composition of cultured snakehead fish. The list of proteins that were identified in this study is shown in table 1. Approximately 43.5 % of the total proteins identified in snakehead fish muscle tissue were basic proteins. These basic proteins have theoretical pI values of between 7.03 and 10.47. Forty-five proteins or 52.9 % of the total proteins were identified as acidic proteins. Their pI values were ranged from 4.40 to 6.98. pI of three of the identified proteins were not shown in the database. In general, there was a good correlation between the observed and theoretical molecular weight (Mr) values of the identified proteins. However, thirteen proteins showed heterogeneity and were represented by more than one band. These proteins include Aldolase A. (SWISS-PROT accession number: Q8JH72), Brain glycogen phosphorylase Pygb (SWISS-PROT accession number: Q6PUS4), Complement factor Bf-1 (SWISS-PROT accession number: Q9YGE7), Creatine kinase (EC 2.7.3.2) (SWISS-PROT accession number: S13164), Creatine kinase, brain (SWISS-PROT accession number: Q7ZU04), Creatine kinase M1-CK (SWISS-PROT accession number: Q9YI16), Creatine kinase muscle isoform 1 (SWISSPROT accession number: Q7T1J3), Creatine kinase (Fragment) (SWISS-PROT accession number: Q98SS7), Fructose-bisphosphate aldolase B (EC 4.1.2.13) (SWISSPROT accession number: ALFB_SPAAU), Muscle-type creatine kinase CKM2 (SWISS-PROT accession number: Q8JH38), Pol-like protein (SWISS-PROT accession number: Q76IM5), TYK2 tyrosine kinase (SWISS-PROT accession number: Q9PWD1), Topoisomerase 2 (fragment) (SWISS-PROT accession number: Q6DR47) and Triosephosphate isomerase 1b (SWISS-PROT accession number: Q7T315). In this study, all the proteins identities were successfully assigned except for the proteins bands 4, 5 and 16, which may indicate their novel nature. A total of 85 proteins were identified in snakehead fish muscle tissue. About 73 % of the total identified proteins were classified as enzymes or enzyme subunits with various catalytic activities (Figure 5). Six of the proteins identified were structural proteins. Other proteins were found to be responsible for cellular activities such as the ribosomal protein, transcription factor, transport

Figure 3: SDS-PAGE aqueous soluble protein profile of snakehead fish muscle tissue proteins from batch 2 (B2) and batch 3 (B3). Protein bands were stained with Coomassie Blue. Lanes 1-6 represent protein profiles from B2 fish with 23, 24, 25, 28, 29 and 30 (cm) fish length, respectively. Lanes 714 represent protein profiles from fish of B3 with 16, 23, 24, 25, 28, 29, 30 and 38 cm fish length, respectively. Lane M represents the protein markers with molecular weights shown on the left. The last lane on the right shows the labeled of protein bands which correspond to the protein band number in Table 1.

Figure 4: Three dimensional densitometric analysis of SDSPAGE from Figure 3. Traces from 1 - 14 represent the different protein lanes in SDS-PAGE. Peaks correspond to bands of SDS-PAGE. Trace M represents protein markers.

Proteomic analysis of snakehead fish muscle tissue

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protein, calcium ion binding protein, DNA/RNA-binding protein and signal transduction protein, which made up a minor constituent that consist of less than 2.4 % of the total protein detected. Moreover, a series of hypothetical proteins or unknown gene products (about 8.2 % of the total proteins) were also identified in this study. Generally, hypothetical proteins are still considered as a group of proteins that have no indication about their existence at the protein level. Most of them have been only described at the nucleic acid level as well as predicted from cDNA sequences but were never been identified by protein chemical method so far [12,13]. The major group of enzymes identified belonged to sarcoplasmic proteins, which is mainly composed of enzymes associated with energy-producing metabolism [14]. The identified sarcoplasmic proteins were found to responsible for the glycolysis activity and ATP hydrolysis. Among the enzymes, kinases are the most frequently identified proteins. It was revealed that twenty-seven proteins or 31.8 % of the total identified proteins were categorized as kinases. The proportional of major enzymes found in snakehead fish muscle tissue is shown in Figure 5. These enzymes include kinases, aldolase, dehydrogenase, isomerase, enolase and others. By number, six proteins (7.1 %) were responsible for aldolase activity. Ten proteins (11.8 %) were classified as dehydrogenase and another six proteins (7.1 %) were known as isomerase. Three proteins (3.5 %) were derived from enolase family. A series of glycolytic enzymes were identified in this study. These proteins were Phosphoglucose isomerase-2, Aldolase (also known as Fructose 1,6-biphosphate aldolase), Triosephosphate isomerase, Glyceraldehyde-3-phosphate dehydrogenase, Phosphoglycerate kinase, Enolase, Pyruvate kinase and L-lactate dehydrogenase.
30
Number of Identified Proteins

In addition to sarcoplasmic proteins, myofibrillar protein or structural protein is also made up the major group of protein identified in snakehead fish. There were a total of six different myofibrillar proteins detected; they were actin (alpha 1, skeletal muscle), actinin (alpha 2), actin (alpha 2, smooth muscle, aorta), fast skeletal myosin light chain 3, myosin heavy chain (fragment) and skeletal muscle actin (fragment). Other than these major proteins, some minor proteins such as Complement factor Bf-1, Brain glycogen phosphorylase Pygb, Pol-like protein, Platelet-activating factor acetylhydrolase (isoform Ib, alpha subunit b), Pygb protein (Fragment), Replicase/ helicase/endonuclease, Reverse transcriptase, Solble guanylyl cylase alpha2 subunit, ST7 protein and many more (as listed in Table 1) were also found in snakehead fish muscle tissue. These proteins were detected as low abundant proteins in snakehead fish muscle tissue. The list of protein identified in snakehead fish muscle tissue (Table 1) shown that the glycolytic and ATP metabolism are the main activities of the fish muscle tissue. Both of these metabolic specializations are essentially required for power locomotor in fish. The high abundance of these two groups of enzyme together with myofibrillar proteins suggests that snakehead fish muscle is composed mainly of white muscle tissue.

Conclusion
The protein profiles of snakehead fish of different sizes that were caught in different months of the year were compared. The results showed that all the fishes have similar protein profiles, where each protein band consisted of identical proteins. Furthermore, the relative intensity of protein bands of all the fishes analyzed is also similar. In view of high demands of snakehead fish, culturing of the fish is the only solution. The present data can be used as a reference for obtaining cultured snakehead fish most similar in fish muscle protein composition to the wild type fish.

25 20 15 10 5 0
Ki na se Al do la D se eh yd ro ge na se Is om er as e En ol as e th er s

Acknowledgements
We would like to thank Universiti Sains Malaysian short term grant for providing financial support to carry out this project. We also want to extend our gratitude to National Poison Centre, USM for providing infrastructure for analysis of proteins. Last but not least we appreciate the PubMed, Swiss Prot and also the MatrixScience that supply free protein software for protein identification.

Enzymes

Figure 5: Various types of enzymes identified in Snakehead fish muscle tissue.

Proteomic analysis of snakehead fish muscle tissue

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References
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