J. Phycol. 42, 113–120 (2006) r 2006 Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00182.

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PROTEOMICS OF THE RED ALGA, GRACILARIA CHANGII (GRACILARIALES, RHODOPHYTA)1
Pooi-Fong Wong
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

Li-Jing Tan
Laboratories of Organismal Biosystems, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 Osaka, Japan

Hannah Nawi
Malaysia Biotechnology Corporation, Level 23, Menara Naluri, 161, Jalan Ampang, 50450 Kuala Lumpur, Malaysia

and Sazaly AbuBakar2
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

The application of proteomics in alga research is still quite limited. The present report describes the establishment of the proteome of a red alga of economic importance, Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Initially, four protein extraction methods including direct precipitation by trichloroacetic acid/acetone, direct lysis using urea buffer, Tris buffer, and phenol/chloroform extraction were compared for their suitability to generate G. changii proteins for two-dimensional gel electrophoresis (2-DE). The phenol/chloroform protein extraction method gave the best 2-DE resolution of the proteins. Using these 2-DE gels and mass spectrometry, several proteins including pigment proteins, metabolic enzymes, and ion transporters were identified. These findings highlight the potential of using proteomic approaches for the investigation of G. changii protein function. Key index words: gracilaria changii; mass spectrometry; proteomics; rhodophyta; two-dimensional electrophoresis Abbreviations: 2-DE, two-dimensional electrophoresis; CHAPS, 3- [(3-cholamidopropy/dimethy-ammonia]-1-propanesulfonate; DTT, dithioethreitol; Exp, experimental; IEF, isoelectric focusing; IPG, immobilized pH gradients; MALDI-ToF, matrix-assisted laser desorption ionization-time of flight; MS, mass spectrometry; MS/MS, mass spectrometry/ mass spectrometry, TBP, tributylphosphine; TCA, trichloroacetic acid

INTRODUCTION

1 2

Received 20 May 2005. Accepted 31 October 2005. Author for correspondence: e-mail: sazaly@ummc.edu.my.

The Rhodophyta is a large, morphologically diverse group of algae consisting of more than 700 genera and 6000 species (Chapman et al. 1998). Among these red algae, the genus Gracilaria (Gracilariales) in particular has become an important commercial agarophyte because it contributes to about 70% of the raw materials required for the production of hydrocolloid agar (McHugh 2002). There are more than 40 different species of Gracilaria distributed worldwide (Phang 1994). At least 20 species are present in Malaysia with G. changii (Xia et Abbott) Abbott, Zhang et Xia being one of the most abundant (Phang et al. 1996, Lim and Phang 2004). This relatively bushy alga is purple to dark brown in color, and is distinguished by abrupt constrictions at the base of the lateral branches, forming a slender stipe (Phang 1994). The agar found in the cell walls of this alga is used in food and fertilizers, and in industrial, scientific, and medical applications (Indergaard 1983). In Malaysia, G. changii is also used as food, as a local delicacy, and as a food source for shellfish aquaculture (Phang and Lewmanomont 2001). In comparison with many common vegetables, the presence of high levels of fiber, minerals, and omega fatty acids, and moderate concentrations of lipids and proteins, makes G. changii suitable as a potential health food (Norziah and Chio 2000). Combined with its high tolerance to harsh conditions and a wide range of salinities, cultivation of this species shows great economic potential (Phang 1998). Taxonomic difficulties associated with a shortage of knowledge of its reproductive features and a high degree of morphological variability have hampered significant progress in our understanding of G. changii, particularly with respect to specific protein functions. The application of proteomic tools has helped to unravel the functions of many proteins in a number of
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thaliana were presented by Kamo et al. 4% CHAPS. and resolubilized in 40 mM Tris buffer pH 8. and mutant characterization were also made possible using proteomics technologies (Thiellement et al. 0. Komatsu et al. Epiphytes.000 V/h at step and hold mode. For lysis buffer protein extraction. Approximately 100 mg of the resulting powder was placed into a 1. except for step 4. the seaweed powders were mixed with buffer containing 40 mM Tris-HCl pH 8. 4–7. 1991. dried. 2001. higher plants including Arabidopsis thaliana L.000g for 1 h at 41 C and the supernatant was reserved. and kept frozen at À 701 C until required. In algal research. followed by 0. (1995) and in subsequent proteomic studies. (Heynh. and 2 mM tributylphosphine (TBP) (Bio-Rad Laboratories). then. and electrophoresed at a constant current of 15 mA for 1 h. the sample was sedimented at 40. TRI reagent (Molecular Research Center Inc.375 M Tris-HCl. For Tris buffer protein extraction. Comparisons between species and strains of healthy and diseased plants could enable identification of strainspecific proteins or disease-associated proteins.5 mA for 1 h. the frozen seaweed was ground to a fine powder with a mortar and pestle in liquid nitrogen. Selangor.000g at 41 C. Following the incubation. Protein pellets were washed. the sample was centrifuged at 40. A total of 100 and 500 mg of protein was used for the 7 and 17–18 cm IPG strips. and finally 20 mA for 6. changii proteome. Cincinnati. 0. Endonuclease (0. Uppsala. Comparative analysis for detection of genetic diversity of different strains. Proteins were precipitated in TCA overnight at À 201 C. The mixture was sonicated as above. relies on the critical component of proteomics. Sweden) and pI 5–8.0007% of bromophenol blue. Proteins were extracted using Tris buffer. . and 2 mM TBP. For protein sample preparation. Rockford. The seaweeds were carefully chosen. The proteins profiles of at least five different tissues (seed. which involves high-resolution separation of proteins using two-dimensional electrophoresis and identification of proteins using mass spectrometry (MS).5 h per gel. Oryza sativa L. or phenol/chloroform.000 V/h. the powder was mixed with a pre-chilled ( À 201 C) solution of 10% TCA in acetone with 20 mM dithioethreitol (DTT). labeled. 2003). Several extractions were performed to obtain sufficient proteins for 2-DE. MO. The former would require that representative proteins extracted from tissues and the proteins are solubilized in buffer appropriate for isoelectric focusing (IEF) and MS.) (Kamo et al. The gel strips were then rinsed in water and equilibrated for 2 min in SDS-PAGE running buffer. 40 mM tris.8. 6% CHAPS. in which 4000 V was applied for 16. defense mechanism proteins. (Higginbotham et al. The pellet was then dried in a vacuum concentrator and resolubilized in a buffer (50 mL/mg of dried pellet) containing 9. USA). WONG ET AL. and proteins were extracted following the protocol recommended by the manufacturer (Fig. After IEF. west coast of Peninsula Malaysia. 2004. 7 cm and 18 cm (Amersham Biosciences. The suspension was then centrifuged at 20. for 7 cm strips similar parameters were used. 2% SDS. 5 mM MgCl2 and sonicated in an ice water bath for 15–30 min. The seaweed G. (Koller et al. 8000 V for 4000 V/h. removing those suspected of having endoparasitic infections. The gels were stained with either silver stain (Blum et al. USA). 0.5% DTT. After the equilibration. 2% SDS. however. IL. held in place by 1% agarose gel. USA).A. MATERIALS AND METHODS Sample Preparation. The gel strip was positioned on top of a vertical 15% polyacrylamide gel. Hercules.375 M TrisHCl.8 containing 8 M urea. 7 and 17 cm (BioRad Laboratories)] were rehydrated with the protein samples overnight at 50 V at 201 C in a total volume of 125 mL (7 cm strips) and 350 mL (17–18 cm strips) of rehydrating buffer (8 M urea. 1. 20% glycerol. In the present study. St.000g for 10 min at 41 C. comparison of different genotypes for phylogenetic analysis. as shown schematically in Fig. Sigma Aldrich. proteomics studies on subcellular compartment protein expressions such as the transmembrane thykaloids and chloroplast of the green algae Chlamydomonas reinhardtii P. Sodium dodecyl sulfate was added to the supernatant to a final concentration of 1%. -F. 1999. Proteins were focused with IPG phor (Amersham Biosciences). 1). lysis buffer. in late February 2003. 0. respectively. 0. and the resulting supernatant was collected. 2002. The samples were then separately sonicated in a water bath sonicator and the individual specimens were separated into single use packs. The resulting protein pellet was washed twice with icecold acetone containing 20 mM DTT. 2004). cell wall of Haematococcus pluvialis Flotow (Wang et al. The success of these studies. methods for extraction of G. Porubleva et al. and 65 mM DTT) followed by equilibration buffer II (6 M urea. CA. 2% CHAPS. Komatsu and Tanaka 2005). root. leaf. 500 V for 500 V/h and 1000 V for 1000 V/h at gradient mode. Louis. 2005) have been reported. The outcome of such studies would provide a better understanding of the biosynthetic pathways useful perhaps for crop quality improvement.114 P. 4% w/v 3.5 mL tube for a single extraction. 2000). The immobilized pH gradients (IPG) strips [pI 3–10. changii proteins and separations by two-dimensional electrophoresis (2-DE) were optimized to enable establishment of the G.[(3-cholamidopropy/dimethy-ammonia]-1propanesulfonate (CHAPS) (Pierce. The reagent was added to the seaweed powder (1 mL/100 mg). Dangeard (Yamaguchi et al. This was followed by an initial attempt to annotate the proteome using MS. and Zea mays L. The mixture was then centrifuged at 20. changii was collected from the mangroves of Morib. 2002). 0. Mechin et al.5 M urea. stem. 2004). 2001. 20% glycerol and 260 mM iodoacetamide) for 15 min each at room temperature. excess liquid on the gel strips was blotted dry with a hand towel. specific sets of proteins involved in the different germination stages including the seed storage proteins. IEF was performed at 201 C using the following parameters for 17–18 cm strips: 200 V for 200 V/h. Gallardo et al. 18 mM DTT. and callus) of A.02 unit Á mL À 1) was then added to the sample and it was incubated overnight at 41 C. 2002. Stauber et al.2% Bio-Lyte 4–7 (Bio-Rad Laboratories. and mitochondria of the colorless alga Polytomella species (van Lis et al. USA) containing phenol and guanidine-isothiocyanate was used for phenol/chloroform protein extraction.02 unit Á mL À 1 endonuclease (E8263. Two-dimensional electrophoresis. Jacobs et al. For protein precipitation using TCA/acetone. 17. catabolic enzymes.. 1.8% ampholytes of pH 3–10 (Amersham Biosciences). the IPG strips were equilibrated in equilibration buffer I (6 M urea. 2002) were identified.000g for 30 min at 41 C to obtain the supernatant. the seaweed powder were treated with 8 M urea. OH. trichloroacetic acid (TCA)/acetone precipitation.25% SDS. 2001. sand and silt were removed by successive washing in sterile seawater using a soft brush with a final rinse in distilled water. 1995. and proteins with novel roles in germination (Gallardo et al. and 8000 V for 40.

Sophia-Antipolis Cedex. 1. trypsindigested. mass tolerance of 0.com) and ProFound (http://www. images were filtered to remove the background noise and all gels were aligned. Schematic diagram of the protein extraction methods for Gracilaria changii. Japan).65 or lower indicates that the candidate is likely to be a random match (Zhang and Chait 2000).htm) to query against the NCBI protein database of Eukaryotes (Profound) or Other Eukaryotes (MASCOT). the peptides were mixed with a saturated matrix (a-cyano-4hydroxycinnamic acid (LaserBio Labs. normalized. For MS analysis.5 mL) was spotted onto the sample plate for MS. A score of 1. MASCOT uses a probability-based MOWSE score. Profound uses Bayesian theory to rank the protein sequences in the database by their probability of occurrence. Protein identifications were then performed using MASCOT (http://www. 1987) or hot Coomassie Blue (Westermeier and Naven 2002). A Z score is used as an indicator of the quality of the search results where a Z score corresponds to the percentile of the search in the random match population. The candidate protein is ranked first together with the family of similar protein and belonging to the alga family. The stained gels were then imaged with an Epson 1600 Pro Scanner (Nagano. Peptide masses were derived following ionization using matrix-assisted laser desorption ionization-time of flight MS (MALDIToF) (Amersham Biosciences). and the peptides were extracted using an automated spot digester (Amersham Biosciences). 1999). The following parameters were considered during the search: monoisotopic masses.05).5% trifluoroacetic acid and 50% acetonitrile) in a 1:1 ratio. The proteins are ranked with decreasing probability of being a random match to the experimental data (Perkins et al.unb.05).5 or 1 Da. France) prepared in 0.1.PROTEOME OF GRACILARIA CHANGII 115 FIG. The gel plugs were destained. oxidation of methionine. the hit must have the highest score or must be significant (Po0. Images from triplicate gels of the 17–18 cm strips of pH 4–7 and 5–8 were analyzed quantitatively for differentially expressed proteins using PDQUEST version 7. To denote a protein as unambiguously identified.1 (Bio-Rad Laboratories). Peptide Mass Fingerprinting. Prior to protein spots detection.matrixscience. The total score is the absolute probability that the observed match is a random event. The matrix-peptides mixture (0. and one missed cleavage for trypsin. Coomasie blue-stained protein spots were excised from the gel using a spot picker (Amersham Biosciences). and matched.br/cbsp/pagini- ciais/profound. The match is significant if the score is greater than 56 (Po0. iodoacetic acid modification of cysteine. In cases where there is no significant match (due to not enough mass value .

Gracilaria gracilis (Stackhouse) Steentoft. Forty-two well-defined protein spots were chosen. The isoelectric focusing (IEF) was performed on 7 cm immobilized pH gradients strips. and 5–8 (Fig. digested. and TCA/acetoneprecipitated proteins. or inaccurate mass). Whenever necessary. changii proteome was successfully established using the 7 cm IPG strips of pH range 3–10. The 15 peptides identified matched to proteins of Gracilaria species such as Gracilaria tenuistipitata var. The resulting 2-DE gels showed mainly unfocused proteins. lysis buffer-extracted.8 and (b) the phenol/chloroform method. Two of these protein spots (SSP no. The 2-DE protein profile consisted of mostly protein spots with a molecular mass lower than 66. G. 4–7. The presence of these fibrillar polymers renders protein extraction difficult. probably because these are isomers that migrated close to each other as visualized in the Coomasie blue-stained gel. a best match is reported. Approximately 516 protein spots were detected in the merged image. however.7 mg protein/g of seaweed powder). WONG ET AL.Y. changii protein. The proteins were visualized by silver staining. There were no matches to any G. excised.4 kDa. Based on this observation. and the Cryptophyta (Guillardia theta Hill et Wetherbee). however.8-extracted. The G. changii proteins were extracted using (a) Tris buffer pH 8. MS-Digest (http://prospector. phenol/chloroform extraction (5. Defined protein spots were noted. and the molecular mass and pI of the hit are close to the experimental mass and pI. Acleto et Foldvik (single match). and Gracilariopsis lemaneiformis (Bory de Saint-Vincent) E. the proteins were focused using 17–18 cm IPG strips of pH 4–7 and pH 5–8. Irvine et Farnham (three matches). 2003). The best match must belong to the alga family. The peptides masses were queried against the available public protein databases. . followed by lysis buffer protein extraction (8. Improved protein separation was observed with the narrower pH (i. 2a). the coverage of the protein by the matching peptides must have a minimum of 14% with at least four matched peptides. Several pro- FIG. MS was then performed as an initial attempt to identify the proteins. The remaining protein spots resulted in insignificant matches that would require other means of identification such as mass spectrometry/mass spectrometry (MS/MS) and amino-acid sequencing. A composite gel image was obtained by merging the two pH range gel images through exclusion of overlapping protein spots. Approximately 424 protein spots were detected in the pH 4–7 gels and 186 proteins spots in pH 5–8 gels. Proteins extracted using the phenol/chloroform method. 3301 and 3203) were matched to a R-phycoerythrin a subunit. The applications of these tools in alga research would certainly complement ongoing alga genome projects such as the brown macroalga Ectocarpus siliculosus (Dillwyn) Lyngbye sequencing initiatives by the Marine Genomic Europe Network. Hence. The Tris buffer pH 8.htm) was used to perform in silico digestion to assist peptide matching and to obtain theoretical masses and pH of the identified proteins.32 mg/g). The present study is aimed at developing a model multicellular alga proteome using the red seaweed.ucsf.22 mg/g). and the gels were relatively clean with minimal streaking.116 P. G. The remaining identified proteins matched proteins belonging to other red algae such as the Porphyra pulchra Hollenberg (Bangiales). we report 15 protein spots that yielded significant matches (Table 1). pI 4–7. All four protein extraction methods examined in the present investigation resulted in different quantity and quality of protein yield. were poor (Fig.00 mg/ g). Dawson. Here. RESULTS The red seaweeds have tough cell walls that contain cellulose fibrils embedded in an extensive gelatinous matrix composed of a variety of sulfated galactose polymers (Michel et al. 2. changii genomic sequences in the public databases. for analytical purposes. changii. pH 4–7 and 5–8) IPG strips as compared with the pH 3–10 IPG strips. and subjected to MALDI-ToF MS. using 100 mg protein extract. were very well resolved by 2DE (Fig. and the vertical axis shows protein separation by molecular mass. the phenol/chloroform protein extraction method was used for all the subsequent protein sample preparations. and TCA/acetone precipitation (3. and was mainly distributed in a pH range of 3–7. Cyanidioschyzon merolae strain 10D (Cyanidiales). probably due to the lack of G. DISCUSSION Two-dimensional gel electrophoresis and MS have become important tools for protein studies in many fields. Two-dimensional gels of Gracilaria changii proteins. 2b). 3a–c). -F.e. The 2-DE gels resolution of the Tris buffer pH 8.0/msdigest. liui Zhang et Xia (six matches). Horizontal axis of the gel shows protein separation by IEF point.edu/ ucsfhtml4. Porphyridium aerugineum Geitler (Porphyridiales). with a limited number of resolved protein spots and the presence of vertical and horizontal streakings.8 protein extraction yielded the most protein (24. and the polymers also cause interference during IEF.

confirmed that the phenol/ chloroform protein extraction method that involved protein precipitation by isopropanol and protein resolubilization using urea and detergents is reliable and suitable for MS analysis.9% of wet weight (Norziah and Chio 2000). The sequential steps in the phenol/chloroform protein extraction method probably removed most of the interfering compounds such as pigments. allophycocyanin. 3. 5-bisphosphate carboxylase/oxygenase (RUBISCO). Solubilization of the resulting protein pellets with urea in combination with detergents further improved the quality of the protein extract. Hydrophobic proteins such as the membrane proteins could be lost during extraction owing to their inherent solubility.8 containing urea. (b) pI 4–7. Even though protein yield is much lower than those extracted with Tris and lysis buffer and also considering that seaweed protein content is approximately 6. Successful identifications of the photosynthetic pigments characteristics of the Rhodophyta. nonetheless.PROTEOME OF GRACILARIA CHANGII 117 FIG. and phycocyanin. the enzyme that . Ribulose-1. Despite these potential shortcomings the extraction protocol yielded a reasonable number of seaweed proteins that were highly resolved by the 2-DE. These spots represented perhaps the high abundance of soluble G. six nuclear-encoded proteins and one from a plasmid. the precipitation steps using isopropanol could have excluded most of the alcohol- soluble proteins. Protein spots with identifications are marked by arrow and standard spots numbers (SSPs). Computational gel analysis revealed approximately 500 proteins spots. changii proteins. The availability of this plastid genome and an additional few nuclear-encoded red algal genes in the public databases. The proteins were visualized with silver stain. and tributylphosphine. identification of the remaining protein spots was hampered. (d) 18 cm/17 cm IPG strips of pI 4–7 and (e) pI 5–8. In the current study. One hundred micrograms and 500 mg of proteins were resolved using 7 cm immobilized pH gradients (IPG) strips of (a) pI 3–10. and it was found that the phenol/chloroform extraction method gave the most reliable and consistent results. enabled successful matching of the G. phycoerythrin. 3. An adequate number of proteins was resolved by 2-DE. and the proteins could be used for MS. changii peptides to seven chloroplast-encoded proteins.[(3-cholamidopropy/dimethyammonia]-1-propanesulfonate. The high molecular mass proteins may also be lost during IEF steps as these proteins would have difficulty migrating into the IEF strips. and salt that usually interfere with IEF. as only one complete chloroplast genome of a Gracilarialean alga. polysaccharides. (c) pI 5–8. at least 15 well-resolved G. Although a total of over 500 protein spots were resolved in the pH range 4–8. 2004). The proteins were extracted using phenol/chloroform and solubilized in Tris buffer pH 8. tein extraction methods were evaluated. Gracilaria tenuistipitata. however. changii protein spots were successfully identified from 2-DE gels by peptide mass fingerprinting. Likewise. Two-dimensional gels of Gracilaria changii proteins. is presently available in the public data base (Hagopien et al. it was counterbalanced by a relatively clean protein sample. nucleic acids.

however.34 43.5 4. R.80 6. the peptide sequences could be obtained by a post-source decay (PSD) technique as an extension of the MALDI-ToF MS. liui] Solute carrier protein [Gracilaria gracilis] Hypothetical protein [Porphyra pulchra] c-phycocyanin a subunit [Gracilaria tenuistipitata var. 3d.33 34. Taddei & L. as the peptide masses obtained matched only to RUBISCO proteins. There is no doubt on the identity of the enzyme. SSP No.95 17.32 50.88 9.08 9.56 4.43 20. The nuclear-encoded serine acetyltransferase was matched to that of the ultra small unicellular red alga. b facilitates the primary CO2 fixation step in photosynthesis. liui] Allophycocyanin a subunit [Gracilaria tenuistipitata var. In order to obtain a precise match to G.40 42. The respective SSP no.7 6. Other metabolic enzymes that were identified include a-1.6 4.5 4. liui] a-1.6 10.47 28.44 22. isozyme 5 (Fragment) [Gracilariopsis lemaneiforis] Aminolevulinic acid dehydratase [Gracilaria gracilis] Ribulose biphosphate carboxylase large chain [Porphyridium aerugineum] g-tubulin [Guillardia theta] C-type cytochrome biogenesis protein [Gracilaria tenuistipitata var. had a better peptide mass match to that of Porphyridium aerugineum even though the G.3 4. e Number of peptides that matched the protein sequence. liui] Serine acetyltransferase [Cyanidioschyzon merolae] 73 63 17 25 23 27 18 25 30 14 18 21 16 20 17 8 7 5 6 8 8 4 6 5 8 5 7 6 6 5 17. The mass resolving power of the instrument is also crucial in producing high-resolution mass peaks. changii enzyme sequence is available in the database.01 54. g Calculated pI of the protein.8 6. the observed masses and theoretical masses should have minimal differences. which is plastid encoded and is used to infer taxonomic relationships in several groups of red algae (Freshwater et al.5 5.62 23.20 4. -F. aminolevulinic acid dehydratase.2 5.50 42. and serine acetyltransferase.69 65.00 17. was successfully identified from the G.10 8. In addition. Gracilaria changii proteins identified by MALDI-ToF mass spectrometry. correspond to those marked in Fig. WONG ET AL. changii proteome.81 5. Even though these gene sequences were not presently available for G. Wetherbee.00 22.26 9. changii.95 17.95 5. Miscleavages and incomplete tryptic digestion can result in peptides with a different observed mass as compared with the theoretical mass.70 36. Cyanidioschyzon merolae P. The enzyme identified in this study.1 4.2 5.50 16.49 50657774 50657774 51209969 4325275 30409180 51209989 4325249 11466615 51210030 5689734 13560094 730477 5901583 51209870 6594273 Standard spot numbers. however. liui] Putative NAD-myo-inositol dehydrogenase [Gracilaria gracilis] LysR-like transcriptional regulator [Cyanidioschyzon merolae] ABC transporter subunit [Gracilaria tenuistipitata var.2 4. theta D. This would involve generation of fragment ions.7 5.2 5.30 14. of peptides matchede Theoretical molecular massf (kDa) NCBI accession no.2 5. This is probably because peptide mass fingerprinting identification relies on high mass accuracy and enzyme specificity.01 14. Molecular mass of the protein spot estimated from the 2-DE gel image analysis.30 44. post-translational modifications can result in a shift in molecular mass. a Experimental pIc Protein name Theoretical pIg 3301 3203 7101 6201 2603 5501 1102 7001 2001 2605 4501 9604R 9601R 8702R 8401 a 24.40 15.3 4. Varano.70 40.25 6.4-glucan lyase. Hill & R. changii RUBISCO.41 48. liui] R-phycoerythrin a subunit [Gracilaria tenuistipitata var.60 34.4-glucan lyase. A. c pI of the protein spot estimated from the 2-DE gel image analysis.89 23.20 55.9 4. The peptide sequence in turn can be used to derive nucleotide sequences to generate primers for PCR amplification of the genes for further . f Calculated molecular mass of the protein.75 4. or alternatively use of the higher capability and accuracy mass spectrometer for MS/MS analysis (Spengler 1997). De Luca.9 8.2 R-phycoerythrin a subunit [Gracilaria tenuistipitata var.118 P. TABLE 1. while g tubulin was matched to that of G. d Percentage of the protein sequence covered by the matching peptides.51 33.70 36. R. 1994). Experimental molecular massb (kDa) Coveraged (%) No.7 6.

S. S.. Groot. S. Haynes. 1999).. Wei. 06-02-02-003 BTK/ER/016). Kuroiwa. Groot... J. The matching may improve as other red algal g tubulin sequences become available. Malaysian J. 1999.. Yoshida. K. 2004.. The total proteins identified from the present study. H. Cyanidioschzon merolae. Sano.. Vandekerckhove. is not a mismatch due to sequence similarity. D. H. & Gross. J. theta to those of Galdieria sulphuraria (Keeling et al. Freshwater. 2002... A multicellular alga like G.).. Griffithsia japonica Okamura. Gracilaria species (Gracilariales. J. The match to G. N. C. The present study describes the first successful protein extraction method applicable for 2-DE and MS analysis of proteins from red alga. van der Heijden.PROTEOME OF GRACILARIA CHANGII 119 studies. M. Asakawa. O. Proc. Komatsu.. P. Hays. Chateau-Joubert.. Bailey. Washburn. Ann.. Miyatake. C. N.. 968 FIIU/C968 (En). A. Thevenot. & Kuroiwa. Negroni. Vandekerckhove. 137–68. T. & Chase. Schieltz. T.. S. S. In Cooksey. K. P.. 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