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
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.
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 fish’s
muscle tissue was then sliced into smaller pieces and
placed in sterile universal bottles and kept at -20°C 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.
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 ω-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.
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 µL of
each of the protein standards were added to separate
wells in the 96 well plates in triplicates. Five µL 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: [email protected]
Proteomic analysis of snakehead fish muscle tissue
of powdered tissue was dissolved in 1000 µl of buffer
(for the Tris buffer extract, 10 µl of extract was diluted in
1000 µl of buffer), it was then centrifuged at 12,000 x g
for 30 min and the supernatant was recovered. Five µL
of the supernatant was then added to the wells. 250 µL 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 56°C.
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 µl 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
26
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/µl
of trypsin in an ice-cold bath. After 45 minutes, the
supernatant was removed and replaced with 10 µl 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
µl 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 – 20°C.
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 µL of ddH20. The peptides were
separated on a reversed-phase column (1mm x 250 mm,
5 µm, 300 A). The HPLC separation condition was at
linear 5% B to 95 % B in 65 minutes at 20 µl/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
27
(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 fish’s 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.
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 0.364±0.001 mg
protein/mg tissue and 0.371±0.001mg 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
0.449±0.001 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
Protein Concentration from Haruan with Various Length
600.0
b
25.00
20.00
ab
ab
ab
b
b
ab
b
a
a
ab
a
ab
ab a
a
a
ab
a
ab
a
15.00
B1
B2
B3
10.00
b
500.0
ab
a
ab
5.00
Content (mg / g)
Concentration (mg / mL)
30.00
400.0
a
a
300.0
200.0
100.0
0.00
0.0
16
23
24
25
28
Length (cm)
29
30
38
B1
B2
B3
Batch Number
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
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
Accession number
Protein Name
Mw
pI
Function
Band 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
2
Proteomic analysis of snakehead fish muscle tissue
SWISS-PROT
Accession number
Q90333
Q6QUR3
Q7T2J3
Q76BG1
Protein Name
29
Mw
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)
pI
Function
Band number
4.40
8.39
6.44
8.09
Structural
Structural
Structural
Enzyme
10
9
2
9
8.42
Enzyme
8,9,10
7.23
Enzyme
10
7.74
8.63
Enzyme
Enzyme
10
10
8.54
Enzyme
10
8.09
Enzyme
10
6.67
Enzyme
8
7.31
Enzyme
10
6.49
Enzyme
10
6.67
Enzyme
10
6.91
Enzyme
10
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
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
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
6.47
Hypothetical protein
9
Proteomic analysis of snakehead fish muscle tissue
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 µg) 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 fish’s
length, respectively. Lanes 7 to 14 represent the protein
profiles of B3 snakehead fish at 16, 23, 24, 25, 28, 29, 30
30
and 38 cm fish’s 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.
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.
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
Proteomic analysis of snakehead fish muscle tissue
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.
25
20
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.
15
Acknowledgements
10
5
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er
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O
En
ol
as
e
Al
do
la
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se
eh
yd
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se
Is
om
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Number of Identified
Proteins
30
31
Enzymes
Figure 5: Various types of enzymes identified in Snakehead
fish muscle tissue.
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.
Proteomic analysis of snakehead fish muscle tissue
32
References
1.
2.
3.
4.
5.
6.
7.
8.
Qasim. The growth of the freshwater murrel, Ophiocephalus
punctatus Bloch. Hydrogiologica 1966; 27: 289-316.
Bowman WC and Rand MJ. Textbook of Pharmacology,
2nd Ed. Blackwell Sci. Pub., Oxford, London. 1980.
Mat Jais AM, McCulloch R and Croft K. Fatty Acid and
amino acid composition in Haruan as a Potential Role in
Wound Healing. Gen. Pharmac1994; 25: 947-950.
Baie S Hj, Sheikh KA. The wound healing properties of
Channa striatus-cetrimide cream-tensile strength
measurement. J. Ethnopharm. 2000a; 71: 93-100.
Baie S Hj, Sheikh KA. The wound healing properties of
Channa striatus-cetrimide cream-wound contraction and
glycosaminoglycan measurement. J. Ethnopharm. 2000b;
73: 15-30.
Bradford, MM. A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing
the principle of protein-dye binding. Analyt. Biochem.
1976; 72: 248-254.
Laemmli UK. Cleavage of Structural Proteins during the
Assembly of the Head of Bacteriophage T4. Nature. 1970;
227: 680-685.
Shevchenko A, Matthias W, Vorm O and Mann M.. Mass
spectrometric sequencing of protein from silver-stained
polyacrylamide gels. Anal. Chem. 1996;68: 850-858.
9.
10.
11.
12.
13.
14.
Perkin DN, Pappin DJC, Creasy DM and Cottrell JS.
Probability-based protein identification by searching
sequence databases using mass spectrometry data.
Electrophoresis. 1999; 20: 3551-3567.
Ng PKL, and Lim KKP. Snakeheads (Pisces: Channidae):
Natural history, biology and economic importance. In
Essays in Zoology. Paper Commemorating the 40 th
Annivesary of the Department of Zoology, National
University of Singapore (ed. Chou L. M. and. Ng P. N. L),
pp. 127-152. Department of Zoology, National University
Of Singapore, Singapore. 1990.
Folkvord A. and Ottera H. Effects of initial size distribution,
day length, and feeding frequency on growth, survival and
cannibalism in juvenile Atlantic cod (Gadus morhua L.).
Aquaculture. 1993; 114: 243-260.
Fountoulakis M, Tsangaris G, Oh J, Maris A and Lubec G.
Protein profile of the HeLa cell line. J. Chromato. A.
2004;1038: 247-265.
Afjehi-Sadat L, Shin J, Felizardo M, Lee K, Slavc I and
Lubec G. Detection of hypothetical proteins in 10 individual
human cell lines. Biochim. Biophy. Acta. 2005; 1747: 67-80.
Nakagawa T, Watabe S and Hanshimoto K. Identification
of three major components in fish sarcoplamic proteins.
Nippon Suisan Gakkaishi. 1988; 54 (6): 999-1004.