Proteomic Analysis of Snakehead Fish (Channa striata ... - EJUM

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Proteomic analysis of snakehead fish muscle tissue30The protein profile of the aqueous soluble proteinextracted from various sizes snakehead fish muscle tissuesfrom B2 and B3 is shown in figure 3 (protein profile ofB1 is not shown; there was no variation between thethree batches). Each of the lanes was loaded with similaramounts (50 µg) of protein extracts from fish of differentlengths. Lanes 1 to 6 represent the protein profiles fromB2 snakehead fish at 23, 24, 25, 28, 29 and 30 cm fish’slength, respectively. Lanes 7 to 14 represent the proteinprofiles of B3 snakehead fish at 16, 23, 24, 25, 28, 29, 30and 38 cm fish’s length, respectively. The protein profilesof fish with different lengths and month of catches didnot differ significantly. Upon Coomassie Blue staining,protein bands, which were evenly distributed in the rangeof molecular masses from 10 kDa to 205 kDa weredetected. The relative intensity of the protein band ineach lane was evaluated using densitometry analysis(Figure 4). In addition to the similar protein profiledisplayed by all the fish, the relative intensity of theproteins is also similar. Thus, the non-variable features(protein profiles and bands intensity) shown by wild typesnakehead fish is beneficial for monitoring of the proteincomposition of cultured snakehead fish.The list of proteins that were identified in this study isshown in table 1. Approximately 43.5 % of the totalproteins identified in snakehead fish muscle tissue werebasic proteins. These basic proteins have theoretical pIvalues of between 7.03 and 10.47. Forty-five proteins or52.9 % of the total proteins were identified as acidicproteins. Their pI values were ranged from 4.40 to 6.98.pI of three of the identified proteins were not shown inthe database.Figure 3: SDS-PAGE aqueous soluble protein profile ofsnakehead fish muscle tissue proteins from batch2 (B2) and batch 3 (B3). Protein bands werestained with Coomassie Blue. Lanes 1-6 representprotein profiles from B2 fish with 23, 24, 25, 28,29 and 30 (cm) fish length, respectively. Lanes 7-14 represent protein profiles from fish of B3 with16, 23, 24, 25, 28, 29, 30 and 38 cm fish length,respectively. Lane M represents the proteinmarkers with molecular weights shown on theleft. The last lane on the right shows the labeledof protein bands which correspond to the proteinband number in Table 1.Figure 4: Three dimensional densitometric analysis of SDS-PAGE from Figure 3. Traces from 1 - 14 representthe different protein lanes in SDS-PAGE. Peakscorrespond to bands of SDS-PAGE. Trace Mrepresents protein markers.In general, there was a good correlation between theobserved and theoretical molecular weight (M r ) values ofthe identified proteins. However, thirteen proteins showedheterogeneity and were represented by more than oneband. These proteins include Aldolase A. (SWISS-PROTaccession number: Q8JH72), Brain glycogenphosphorylase Pygb (SWISS-PROT accession number:Q6PUS4), Complement factor Bf-1 (SWISS-PROTaccession number: Q9YGE7), Creatine kinase (EC 2.7.3.2)(SWISS-PROT accession number: S13164), Creatinekinase, brain (SWISS-PROT accession number: Q7ZU04),Creatine kinase M1-CK (SWISS-PROT accession number:Q9YI16), Creatine kinase muscle isoform 1 (SWISS-PROT accession number: Q7T1J3), Creatine kinase(Fragment) (SWISS-PROT accession number: Q98SS7),Fructose-bisphosphate aldolase B (EC 4.1.2.13) (SWISS-PROT accession number: ALFB_SPAAU), Muscle-typecreatine kinase CKM2 (SWISS-PROT accession number:Q8JH38), Pol-like protein (SWISS-PROT accessionnumber: Q76IM5), TYK2 tyrosine kinase (SWISS-PROTaccession number: Q9PWD1), Topoisomerase 2(fragment) (SWISS-PROT accession number: Q6DR47)and Triosephosphate isomerase 1b (SWISS-PROTaccession number: Q7T315). In this study, all the proteins’identities were successfully assigned except for theproteins bands 4, 5 and 16, which may indicate theirnovel nature.A total of 85 proteins were identified in snakeheadfish muscle tissue. About 73 % of the total identifiedproteins were classified as enzymes or enzyme subunitswith various catalytic activities (Figure 5). Six of theproteins identified were structural proteins. Other proteinswere found to be responsible for cellular activities suchas the ribosomal protein, transcription factor, transport
Proteomic analysis of snakehead fish muscle tissue31protein, calcium ion binding protein, DNA/RNA-bindingprotein and signal transduction protein, which made up aminor constituent that consist of less than 2.4 % of thetotal protein detected. Moreover, a series of hypotheticalproteins or unknown gene products (about 8.2 % of thetotal proteins) were also identified in this study. Generally,hypothetical proteins are still considered as a group ofproteins that have no indication about their existence atthe protein level. Most of them have been only describedat the nucleic acid level as well as predicted from cDNAsequences but were never been identified by proteinchemical method so far [12,13].The major group of enzymes identified belonged tosarcoplasmic proteins, which is mainly composed ofenzymes associated with energy-producing metabolism[14]. The identified sarcoplasmic proteins were found toresponsible for the glycolysis activity and ATP hydrolysis.Among the enzymes, kinases are the most frequentlyidentified proteins. It was revealed that twenty-sevenproteins or 31.8 % of the total identified proteins werecategorized as kinases. The proportional of majorenzymes found in snakehead fish muscle tissue is shownin Figure 5. These enzymes include kinases, aldolase,dehydrogenase, isomerase, enolase and others. Bynumber, six proteins (7.1 %) were responsible for aldolaseactivity. Ten proteins (11.8 %) were classified asdehydrogenase and another six proteins (7.1 %) wereknown as isomerase. Three proteins (3.5 %) were derivedfrom enolase family. A series of glycolytic enzymeswere identified in this study. These proteins werePhosphoglucose isomerase-2, Aldolase (also known asFructose 1,6-biphosphate aldolase), Triosephosphateisomerase, Glyceraldehyde-3-phosphate dehydrogenase,Phosphoglycerate kinase, Enolase, Pyruvate kinase andL-lactate dehydrogenase.Number of IdentifiedProteins302520151050KinaseAldolaseDehydrogenaseIsomeraseEnzymesEnolaseOthersFigure 5: Various types of enzymes identified in Snakeheadfish muscle tissue.In addition to sarcoplasmic proteins, myofibrillarprotein or structural protein is also made up the majorgroup of protein identified in snakehead fish. There werea total of six different myofibrillar proteins detected;they were actin (alpha 1, skeletal muscle), actinin (alpha2), actin (alpha 2, smooth muscle, aorta), fast skeletalmyosin light chain 3, myosin heavy chain (fragment) andskeletal muscle actin (fragment). Other than these majorproteins, some minor proteins such as Complement factorBf-1, Brain glycogen phosphorylase Pygb, Pol-likeprotein, Platelet-activating factor acetylhydrolase (isoformIb, alpha subunit b), Pygb protein (Fragment), Replicase/helicase/endonuclease, Reverse transcriptase, Solbleguanylyl cylase alpha2 subunit, ST7 protein and manymore (as listed in Table 1) were also found in snakeheadfish muscle tissue. These proteins were detected as lowabundant proteins in snakehead fish muscle tissue.The list of protein identified in snakehead fish muscletissue (Table 1) shown that the glycolytic and ATPmetabolism are the main activities of the fish muscletissue. Both of these metabolic specializations areessentially required for power locomotor in fish. Thehigh abundance of these two groups of enzyme togetherwith myofibrillar proteins suggests that snakehead fishmuscle is composed mainly of white muscle tissue.ConclusionThe protein profiles of snakehead fish of differentsizes that were caught in different months of the yearwere compared. The results showed that all the fisheshave similar protein profiles, where each protein bandconsisted of identical proteins. Furthermore, the relativeintensity of protein bands of all the fishes analyzed isalso similar. In view of high demands of snakehead fish,culturing of the fish is the only solution. The presentdata can be used as a reference for obtaining culturedsnakehead fish most similar in fish muscle proteincomposition to the wild type fish.AcknowledgementsWe would like to thank Universiti Sains Malaysianshort term grant for providing financial support to carryout this project. We also want to extend our gratitudeto National Poison Centre, USM for providinginfrastructure for analysis of proteins. Last but notleast we appreciate the PubMed, Swiss Prot and alsothe MatrixScience that supply free protein software forprotein identification.
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Proteomic Analysis of Snakehead Fish (Channa striata ... - EJUM

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