Proteomic Analysis of Snakehead Fish (Channa striata) Muscle Tissue
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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 Materials and Methods
Snakehead fish is an obligatory air-breather and Preparation of Snakehead fish Muscle Tissue
predaceous fish that resides in swamps, slow-flowing
Three batches of snakehead fish caught in November
streams and in crevices near riverbanks in Southern China.
2002 (B1), January 2003 (B2) and April 2003 (B3),
In taxonomy, it belongs to the family of Ophiocephalidae
respectively were used in this study. The fish from each
or Channidae [1]. The habitats of the fish are always
batch were further subdivided according to their lengths.
infested by thick aquatic vegetation which expands over
All fishes were washed, beheaded, sliced and covered
the entire water surface.
with ice to ensure freshness of the fish tissues. The fish’s
muscle tissue was then sliced into smaller pieces and
Snakehead fish is consumed mainly as a remedy to
placed in sterile universal bottles and kept at -20°C prior
help the healing of wounds after a clinical operation, road
to freeze-drying. Freeze dried snakehead fish muscle
accident and caesarian. The biochemical analysis of its
tissue was homogenized to powder form. Extraction of
flesh was undertaken based on the knowledge that the fish
protein from snakehead fish muscle tissue was carried
contained ω-3 polysaturated fatty acids that regulate
out on 1.0 mg of powdered fish muscle using 1 mL of 40
prostaglandin synthesis and also influence the immune
mM Tris (pH 8.8) extraction buffer. The sample mixture
system [2,3]. In addition, the amino acid composition in
was then vortexed for 2 minutes and centrifuged at
snakehead fish has also been analyzed and was reported to
12, 000 ( g for 30 min at room temperature and the
play a role in the process of wound healing [3]. The
supernatant was recovered.
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, Protein Concentration Determination
the tissue texture of cultured snakehead fish is different Protein concentration determination was carried out
from the wild type snakehead fish. Therefore, the using the method described by Bradford [6]. Bovine
knowledge on the protein composition of wild type Serum Albumin (BSA) was used to construct a standard
snakehead fish will be beneficial where it can be used as a protein concentration curve. The assay was performed in
reference to culture snakehead fish. a 96 well plate. Protein concentration standards ranging
from 0.1 - 1.4 mg BSA/mL were prepared. Five µL of
In this study, the aqueous soluble protein profiles of each of the protein standards were added to separate
different sizes of wild type snakehead fish were wells in the 96 well plates in triplicates. Five µL of
determined. The fish were caught in different months phosphate buffer was added to the blank wells. Fifty mg
and at different places in the region of northern Malaysia.
The data obtained represent the protein profile of the Author for correspondence: Dr Lay-Harn Gam, School of
wild type fish, which can be used as a reference for Pharmaceutical Sciences, Universiti Sains Malaysia, 11800
culturing and farming of snakehead fish. USM, Pulau Pinang, Malaysia. Fax no: 604-6570017
E-mail: layharn@usm.myProteomic analysis of snakehead fish muscle tissue 26
of powdered tissue was dissolved in 1000 µl of buffer removed and the gel pieces were dried in a vacuum
(for the Tris buffer extract, 10 µl of extract was diluted in centrifuge. The gel pieces were swollen in digestion buffer
1000 µl of buffer), it was then centrifuged at 12,000 x g containing 50 mM NH4HCO3, 5 M CaCl2, and 12.5 ng/µl
for 30 min and the supernatant was recovered. Five µL of trypsin in an ice-cold bath. After 45 minutes, the
of the supernatant was then added to the wells. 250 µL of supernatant was removed and replaced with 10 µl of the
the Bradford Reagent was added to each plate well that same buffer but without trypsin to keep the gel pieces
contained standards and samples. The plate was then wet during enzymatic cleavage at 37 °C overnight.
shaken for approximately 30 seconds and incubated at Peptides were extracted from the gel matrix by adding 15
room temperature for 15 minutes. The absorbance was µl of 20 mM NH4HCO3, vortexed and incubated at room
measured at 595 nm. A standard curve was plotted using temperature for 10 minutes. The supernatant was
net absorbance versus protein concentration of each recovered after a brief spin. This was followed by adding
standard. The protein concentration of unknown samples (1 to 2 times the volume of gel pieces) 5% (v/v) formic
was determined by comparing the average A595 values acid in acetonitrile:water mixture (70:30), vortexed and
against the standard curve. incubated for 20 minutes at room temperature. It was
then spun down and the supernatant was recovered. These
Sodium Dodecyl Sulphate-Polyacrlyamide Gel steps were repeated 3 times. Pooled extracts were dried
Electrophoresis (SDS-PAGE) down in a vacuum centrifuge and stored at – 20°C.
SDS-PAGE was performed as described by Laemmli
[7]. Ten per cent polyacrlamide gel in a vertical slab gel HPLC-MS Analysis
apparatus (Hoefer) was used. Protein samples (Tris buffer Mass spectrometric analysis was carried out using an
extraction) were then loaded into the wells of polymerized ion trap mass spectrometer (Agilent, VL). The peptides
gel. Electrophoresis was performed at a constant voltage were ionized using the electrospray soft ionization
of 200 volts when samples were in the stacking gel. technique (ESI). The mass spectrometer was operated in
When the dye front reached the resolving gel, voltage a two-mode program consisting of full MS Scan and full
was increased to 245 volts. The run was stopped when MS/MS Scan, whereby, the most intense ion in the full
the dye front was 2 to 3 mm away from the bottom edge MS Scan was isolated and subjected to full MS/MS Scan.
of the gel. At completion of electrophoresis, the glass
sandwich was disassembled. The stacking gel was The peptides resulting from the in-gel digestion was
discarded and the resolving gel was stained using reconstituted in 50 µL of ddH20. The peptides were
Coomassie Blue. Molecular weights of the proteins were separated on a reversed-phase column (1mm x 250 mm,
determined by comparing relative mobility of protein 5 µm, 300 A). The HPLC separation condition was at
bands to the standard protein markers. linear 5% B to 95 % B in 65 minutes at 20 µl/min flow
rate. The eluent of HPLC was directed to a mass
The Coomassie Blue stained gel images were acquired spectrometer, which was interfaced with the HPLC. The
and digitized using Versadoc Imaging Scanner. Protein parameters used for acquiring the MS data were: heated
bands intensity analysis was carried out using the Camag capillary temperature of 300 °C, dry gas flow rate of 8.0
TLC Scanner 3 and the densitometry analysis was L/min and nebulizer gas pressure of 30.0 psi. The
performed using the CATS software. parameters set for the MS/MS Scan were collision energy
(voltage) = 1.15 V, charge state = 2, minimum threshold
In-Gel Digestion = 5000 counts, and the isolation width = 2 m/z. The MS/
MS spectra were recorded in the automated MS to MS/
The polyacrylamide gel was washed thoroughly with
MS switching mode with an m/z dependent set.
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 Sequence Database Search
modification. The gel pieces were first excised and shrunk The MS/MS data were subjected to Mascot protein
by dehydration in acetonitrile. The solvent was then database search engine (www.matrixscience.com). The
discarded and the gel pieces were dried in a vacuum search engine contains the calculated spectra for all
centrifuge. A volume of 10 mM dithiotreitol (DTT) in peptides in the National Centre for Biotechnology
100 mM NH4HCO3 sufficient to cover the gel pieces was Information (NCBI) non-redundant sequences database
added and the protein was reduced for 1 hour at 56°C. [9]. The taxonomy and enzyme selected was
After cooling to room temperature, the DTT solution was Actinopterygii (Ray-Finned Fish) (29309 sequences) and
replaced with a same volume of 55 mM iodoacetic acid Trypsin, respectively, whilst Fixed Modification was
in 100 mM NH4HCO3. After 45 minutes incubation at Carboxymethyl (C). The Peptide Mass Tolerance was set
ambient temperature in the dark with occasional vortexing, at ± 2 Da whereas ± 0.8 Da was set for the Fragment
the gel pieces were washed with 50-100 µl of 100 mM Mass (MS/MS) Tolerance. The data format was selected
NH4HCO3 for 10 minutes, dehydrated with acetonitrile, as Mascot Generic and only one missed cleavage was
rehydrated in 100 mM NH4HCO3 and dehydrated in the allowed. Instrument type set was ESI-TRAP i.e.
same volume of acetonitrile. The liquid phase was Electrospray Ionization and Ion Trap Mass SpectrometerProteomic analysis of snakehead fish muscle tissue 27
(1100 Series, Agilent, Germany). Proteins’ functions and The phenomenon of cannibalism in snakehead fish
characteristic information were obtained from both the may be one of the reasons why muscle protein synthesis
PubMed (www.ncbi.nlm.nih.gov/entrez) and Swiss Prot activity in smaller snakehead fish is more active and
(www.expasy.ch/sprot/sprot-top.html). rapid as compared to the larger fish. The secretion of
myofibrillar protein and collagen were greater in the
Results and Discussion smaller fish for their movement to survive in the
present of larger predator. In contrast, the consistent
Snakehead fish has long been consumed as a source protein content amongst the larger fish shows the fishes
of dietary protein. It is also well known traditionally for of 25 to 38 cm lengths have achieved their full protein
its medicinal property for healing of wounds. In this capacity.
study, the proteins extracted from the fish’s muscle tissue
were analyzed. Different sizes of wild type snakehead The problem of cannibalism is believed to be the
fish caught at different seasons were used in the study. cause of the low survival of smaller fishes in culturing of
The data obtained provide useful information on the snakehead fish [10]. The alternative approach would be
nutritional and medicinal properties of snakehead fish. to provide adequate food [11] or partially control
Culturing of snakehead fish has been carried out in cannibalism by grading fishes into approximately similar
Malaysia due to the high demand of the fish. As a large size group.
proportion of fish muscle tissue is made up of proteins,
cultured fish muscle tissue texture can be monitored by Figure 2 illustrates the protein contents of the three
comparing their protein profile with those of the wild different batches of snakehead fish. It was found that
type fish. snakehead fish from B1 and B2 did not vary significantly
in their protein concentration (P>0.05). Fishes from both
In order to study the protein profile of snakehead fish, of these batches contained on average 0.364±0.001 mg
fishes of different sizes that were caught at different protein/mg tissue and 0.371±0.001mg protein/mg tissue,
seasons were analyzed. Three batches of snakehead fish respectively. However, snakehead fish from B3 showed
caught in November (B1), January (B2) and March (B3), significantly higher protein content than B1 and B2
respectively were used in this study. From each batch, (PProteomic analysis of snakehead fish muscle tissue 28
Table 1: List of water soluble proteins detected in Haruan’s muscle tissue. Band number are refer to indication mentioned
in Figure 3.
SWISS-PROT
Protein Name Mw pI Function Band number
Accession number
KIBOA/ P00570 Adenylate kinase (EC 2.7.4.3) 21761 8.94 Enzyme 14
Q804Y1 Aldolase (Fragment) 17427 8.73 Enzyme 10
Q8JH72 Aldolase A. 40223 8.27 Enzyme 2,8,9,10
Q7ZW73 Aldolase b, fructose-bisphosphate 39700 8.75 Enzyme 10
Q6PUS4 Brain glycogen phosphorylase Pygb 97916 6.11 Enzyme 2,7
Q9YGE7 Complement factor Bf-1 85034 5.90 Enzyme 1,6
Q7ZU04 Creatine kinase, brain 43178 5.49 Enzyme 7,9
Q804Z1 Creatine kinase 43231 6.29 Enzyme 9
Q804Z2 Creatine kinase 43032 6.32 Enzyme 9
Q9I8I6 Creatine kinase (EC 2.7.3.2) 46859 8.73 Enzyme 9
S13164 Creatine kinase (EC 2.7.3.2) 43267 6.20 Enzyme 7,9,10
Q98SS7 Creatine kinase (Fragment) 29125 8.89 Enzyme 7,9
Q9DFM2 Creatine kinase (Fragment) 21139 5.79 Enzyme 9
Q7T1J1 Creatine kinase brain isoform (Fragment) 42608 5.89 Enzyme 9
Q7T306 Creatine kinase CKM3 43115 6.29 Enzyme 9
Q9YI16 Creatine kinase M1-CK 42983 6.21 Enzyme 2,9
Q9YI15 Creatine kinase M2-CK 43133 6.22 Enzyme 9
Q9YI14 Creatine kinase M3-CK 43185 6.25 Enzyme 9
Q7T1J0 Creatine kinase mitochondrial isoform precursor 47108 8.50 Enzyme 9
Q7T1J3 Creatine kinase muscle isoform 1 42713 6.32 Enzyme 9,10
Q7T1J2 Creatine kinase muscle isoform 2 42888 6.44 Enzyme 9
Q7ZZM5 Enolase (Fragment) 28757 8.15 Enzyme 6
Q6TH14 (AAQ97775) Enolase 1 (AY398342 NID) 47848 - Enzyme 6
Q6GQM9 Enolase 2 47160 4.77 Enzyme 6
O57518 Fructose-1, 6-bisphosphate aldolase 39957 6.21 Enzyme 10
Q76BF6 Phosphoglycerate kinase (Fragment) 41657 6.04 Enzyme 9
Q8AY84 Phosphoglycerate kinase (Fragment) 11317 4.67 Enzyme 9
Q6NXD1 PKM 2 protein 58598 6.36 Enzyme 5
Q803D2 Platelet-activating factor acetylhydrolase, isoform 47080 6.97 Enzyme 7
Ib, alpha subunit b.
Q76IM5 Pol-like protein 149432 9.28 Enzyme 11,12
Q7SXV3 Pygb protein (Fragment) 60118 7.30 Enzyme 7
Q8JJC2 Pyruvate kinase 58767 6.35 Enzyme 5
Q8QGU8 Pyruvate kinase 58582 7.96 Enzyme 5
Q7M558 Replicase/ helicase/ endonuclease 350347 8.68 Enzyme 9
BAD04856 (Q76B34) Reverse transcriptase 132804 - Enzyme 14
Q7T040 Solble guanylyl cylase alpha2 subunit 90214 7.54 Enzyme 10
Q8UW40 ST7 protein 58059 7.03 Enzyme 14
Q6DR47 Topoisomerase 2 (Top 2A protein) 178147 8.93 Enzyme 2,5,8,10,11,
12, 13, 14, 15
Q76BE1 Triose phosphate isomerase (Fragment) 25178 6.00 Enzyme 11
Q7T315 Triosephospahte isomerase 1b 27100 6.90 Enzyme 11,12
Q90XF8 Triosephosphate isomerase B 26476 7.60 Enzyme 12
Q9PWD1 TYK2 tyrosine kinase 129986 8.38 Enzyme 7,9
Q7ZU23 Actin, alpha 1, skeletal muscle 42304 5.23 Structural 2
Q6TNW2 Actinin, alpha 2. 104086 5.23 Structural 7
Q6DHS1 Actin, alpha 2, smooth muscle, aorta 42374 5.23 Structural 2Proteomic analysis of snakehead fish muscle tissue 29
SWISS-PROT
Protein Name Mw pI Function Band number
Accession number
Q90333 Fast skeletal myosin light chain 3 16794 4.40 Structural 10
Q6QUR3 Myosin heavy chain (Fragment) 23564 8.39 Structural 9
Q7T2J3 Skeletal muscle actin (Fragment) 43041 6.44 Structural 2
Q76BG1 Fructose-bisphosphate aldolase A 36509 8.09 Enzyme 9
(Fragment)
ALFB_SPAAU Fructose-bisphosphate aldolase B 40069 8.42 Enzyme 8,9,10
(EC 4.1.2.13)
Q90Z48 Glyceraldehyde phosphate dehydrogenase 36425 7.23 Enzyme 10
(EC 1.2.1.12)
Q8AWX8 Glyceraldehyde-3-phosphate dehydrogenase 36244 7.74 Enzyme 10
Q8JIQ0 Glyceraldehyde-3-phosphate dehydrogenase 36069 8.63 Enzyme 10
(EC 1.2.1.12)
Q9PTW5 Glyceraldehyde-3-phosphate dehydrogenase 36192 8.54 Enzyme 10
(EC 1.2.1.12)
LDHA_ SPHAG L-lactate dehydrogenase A chain 36650 8.09 Enzyme 10
(EC 1.1.1.27)
LDHA_CHAAC L-lactate dehydrogenase A chain 36261 6.67 Enzyme 8
(EC 1.1.1.27)
LDHA_CYPCA L-lactate dehydrogenase A chain 36413 7.31 Enzyme 10
(EC 1.1.1.27)
LDHA_ELEMC L-lactate dehydrogenase A chain 36387 6.49 Enzyme 10
(EC 1.1.1.27)
LDHA_HARAN L-lactate dehydrogenase A chain 36200 6.67 Enzyme 10
(EC 1.1.1.27)
LDHA_BRARE L-lactate dehydrogenase A chain 36382 6.91 Enzyme 10
(EC 1.1.1.27)
Q9PV91 Muscle creatine kinase 43041 6.44 Enzyme 2
Q90X19 Muscle-specific creatine kinase 43030 6.32 Enzyme 9
Q8JH39 Muscle-type creatine kinase CKM1 43351 6.98 Enzyme 9
Q8JH38 Muscle-type creatine kinase CKM2 42985 6.44 Enzyme 1,7,9
Q9DFL9 Nuclease diphosphate kinase B 17218 6.82 Enzyme 15
Q9PTF3 Nucleoside diphosphate kinase- Z3 19562 7.68 EnzEnzymeyme 15
Q8QFU1 Phosphoglucose isomerase -2 62173 6.82 Enzyme 5
Q8QFT1 Phosphoglucose isomerase-2 (EC 5.3.1.9) 62166 7.85 Enzyme 5
Q90YR3 40S ribosomal protein S11 18610 10.47 Ribosomal 10
Q7ZV05 Similar to 40S ribosomal protein S11 18568 10.47 Ribosomal 10
Q8QGQ9 Teashirt-like zinc finger protein (Fragment) 95735 8.03 Transcription factor 9
Q9I8L6 T-box transcription factor 49606 7.78 Transcription factor 13
Q8UWF2 Glutamate receptor subunit 1B (Fragment) 62737 8.10 Transport 5
JC4956/ Q90W12 Vitellogenin precursor 184710 9.07 Calcium ion binding 5
Q9PVM6 Elongation factor 1 alpha 50743 9.16 Translation Factor 7
Q7T1U2 Tmc2-related protein 2 (Fragment) 75970 5.99 Translation Factor 7
Q90XI6 RAG2 (Fragment) 17314 9.01 DNA-RNA -binding 7
Q98TT9 GDNF family receptor alpha-1a 54506 8.45 Signal transduction 10
Q7SYD3 zgc: 67559 protein (Hypothetical protein) 104082 5.09 Hypothetical protein 7
Q6DG54 Zgc:92037 58897 6.88 Hypothetical protein 5
AAH59437 zgc: 73059 (BC059437 NID) 46671 - Hypothetical protein 9
AAH59571 (Q6PBV4) zgc: 73229 protein (BC059571 NID) 29926 5.61 Hypothetical protein 7
Q6NXB1 Hypothetical protein zgc:77002 34254 5.44 Hypothetical protein 12
Q7ZZ46 SI:dZ249N21.1.3 (Novel protein similar 455282 5.32 Hypothetical protein 9
to human titin (TTN) (Fragment)
Q7ZV29 zgc: 56252 (Similar to phosphoglycerate 45126 6.47 Hypothetical protein 9
kinase 1)Proteomic analysis of snakehead fish muscle tissue 30
The protein profile of the aqueous soluble protein and 38 cm fish’s length, respectively. The protein profiles
extracted from various sizes snakehead fish muscle tissues of fish with different lengths and month of catches did
from B2 and B3 is shown in figure 3 (protein profile of not differ significantly. Upon Coomassie Blue staining,
B1 is not shown; there was no variation between the protein bands, which were evenly distributed in the range
three batches). Each of the lanes was loaded with similar of molecular masses from 10 kDa to 205 kDa were
amounts (50 µg) of protein extracts from fish of different detected. The relative intensity of the protein band in
lengths. Lanes 1 to 6 represent the protein profiles from each lane was evaluated using densitometry analysis
B2 snakehead fish at 23, 24, 25, 28, 29 and 30 cm fish’s (Figure 4). In addition to the similar protein profile
length, respectively. Lanes 7 to 14 represent the protein displayed by all the fish, the relative intensity of the
profiles of B3 snakehead fish at 16, 23, 24, 25, 28, 29, 30 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
Figure 3: SDS-PAGE aqueous soluble protein profile of band. These proteins include Aldolase A. (SWISS-PROT
snakehead fish muscle tissue proteins from batch accession number: Q8JH72), Brain glycogen
2 (B2) and batch 3 (B3). Protein bands were phosphorylase Pygb (SWISS-PROT accession number:
stained with Coomassie Blue. Lanes 1-6 represent Q6PUS4), Complement factor Bf-1 (SWISS-PROT
protein profiles from B2 fish with 23, 24, 25, 28, accession number: Q9YGE7), Creatine kinase (EC 2.7.3.2)
29 and 30 (cm) fish length, respectively. Lanes 7- (SWISS-PROT accession number: S13164), Creatine
14 represent protein profiles from fish of B3 with kinase, brain (SWISS-PROT accession number: Q7ZU04),
16, 23, 24, 25, 28, 29, 30 and 38 cm fish length,
Creatine kinase M1-CK (SWISS-PROT accession number:
respectively. Lane M represents the protein
markers with molecular weights shown on the
Q9YI16), Creatine kinase muscle isoform 1 (SWISS-
left. The last lane on the right shows the labeled PROT accession number: Q7T1J3), Creatine kinase
of protein bands which correspond to the protein (Fragment) (SWISS-PROT accession number: Q98SS7),
band number in Table 1. Fructose-bisphosphate aldolase B (EC 4.1.2.13) (SWISS-
PROT 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
Figure 4: Three dimensional densitometric analysis of SDS- proteins were classified as enzymes or enzyme subunits
PAGE from Figure 3. Traces from 1 - 14 represent with various catalytic activities (Figure 5). Six of the
the different protein lanes in SDS-PAGE. Peaks proteins identified were structural proteins. Other proteins
correspond to bands of SDS-PAGE. Trace M were found to be responsible for cellular activities such
represents protein markers. as the ribosomal protein, transcription factor, transportProteomic analysis of snakehead fish muscle tissue 31
protein, calcium ion binding protein, DNA/RNA-binding In addition to sarcoplasmic proteins, myofibrillar
protein and signal transduction protein, which made up a protein or structural protein is also made up the major
minor constituent that consist of less than 2.4 % of the group of protein identified in snakehead fish. There were
total protein detected. Moreover, a series of hypothetical a total of six different myofibrillar proteins detected;
proteins or unknown gene products (about 8.2 % of the they were actin (alpha 1, skeletal muscle), actinin (alpha
total proteins) were also identified in this study. Generally, 2), actin (alpha 2, smooth muscle, aorta), fast skeletal
hypothetical proteins are still considered as a group of myosin light chain 3, myosin heavy chain (fragment) and
proteins that have no indication about their existence at skeletal muscle actin (fragment). Other than these major
the protein level. Most of them have been only described proteins, some minor proteins such as Complement factor
at the nucleic acid level as well as predicted from cDNA Bf-1, Brain glycogen phosphorylase Pygb, Pol-like
sequences but were never been identified by protein protein, Platelet-activating factor acetylhydrolase (isoform
chemical method so far [12,13]. Ib, alpha subunit b), Pygb protein (Fragment), Replicase/
helicase/endonuclease, Reverse transcriptase, Solble
The major group of enzymes identified belonged to guanylyl cylase alpha2 subunit, ST7 protein and many
sarcoplasmic proteins, which is mainly composed of more (as listed in Table 1) were also found in snakehead
enzymes associated with energy-producing metabolism fish muscle tissue. These proteins were detected as low
[14]. The identified sarcoplasmic proteins were found to abundant proteins in snakehead fish muscle tissue.
responsible for the glycolysis activity and ATP hydrolysis.
Among the enzymes, kinases are the most frequently The list of protein identified in snakehead fish muscle
identified proteins. It was revealed that twenty-seven tissue (Table 1) shown that the glycolytic and ATP
proteins or 31.8 % of the total identified proteins were metabolism are the main activities of the fish muscle
categorized as kinases. The proportional of major tissue. Both of these metabolic specializations are
enzymes found in snakehead fish muscle tissue is shown essentially required for power locomotor in fish. The
in Figure 5. These enzymes include kinases, aldolase, high abundance of these two groups of enzyme together
dehydrogenase, isomerase, enolase and others. By with myofibrillar proteins suggests that snakehead fish
number, six proteins (7.1 %) were responsible for aldolase muscle is composed mainly of white muscle tissue.
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
Conclusion
from enolase family. A series of glycolytic enzymes The protein profiles of snakehead fish of different
were identified in this study. These proteins were sizes that were caught in different months of the year
Phosphoglucose isomerase-2, Aldolase (also known as were compared. The results showed that all the fishes
Fructose 1,6-biphosphate aldolase), Triosephosphate have similar protein profiles, where each protein band
isomerase, Glyceraldehyde-3-phosphate dehydrogenase, consisted of identical proteins. Furthermore, the relative
Phosphoglycerate kinase, Enolase, Pyruvate kinase and intensity of protein bands of all the fishes analyzed is
L-lactate dehydrogenase. also similar. In view of high demands of snakehead fish,
culturing of the fish is the only solution. The present
30
data can be used as a reference for obtaining cultured
snakehead fish most similar in fish muscle protein
Number of Identified
25
composition to the wild type fish.
20
Proteins
15
10 Acknowledgements
5 We would like to thank Universiti Sains Malaysian
0 short term grant for providing financial support to carry
out this project. We also want to extend our gratitude
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to National Poison Centre, USM for providing
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infrastructure for analysis of proteins. Last but not
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Enzymes
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least we appreciate the PubMed, Swiss Prot and also
Figure 5: Various types of enzymes identified in Snakehead the MatrixScience that supply free protein software for
fish muscle tissue. protein identification.Proteomic analysis of snakehead fish muscle tissue 32
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