摘要
同许多昆虫一样,家蚕雌性附腺由分泌部和贮存部组成,分泌部分泌的胶状蛋白贮存于贮存部中。胶状粘液蛋白主要作用是将产出的卵粘附于外界的物质上,以保证昆虫在有大风大雨的自然环境中也能正常继代。从形态学和解剖学上的研究发现,家蚕雌性附腺是逐步发育的,但非常有趣的是雌性附腺在羽化前的2～3天开始突然大量分泌粘液蛋白,并在贮存部中积蓄,导致贮存部迅速的膨大。而家蚕欧洲种的一些品种产下的卵为天然散卵,这些品种的雌体不能正常分泌胶状粘性物质,经研究确认为遗传性状,命名为Ng突变。家蚕雌性附腺是一个特殊器官,合成和分泌特异蛋白质在短时间内完成,对雌性附腺的研究有助于搞清楚细胞、组织或器官发育分化的调节机制。
蛋白质组学是在蛋白的水平大规模研究基因表达的产物,能够直接的研究蛋白质的表达水平和相关蛋白的活性状态。家蚕的雌性附腺无论是在形态上还是在功能上在发育过程上都发生了显著的变化。因此,本研究利用双向电泳和质谱相结合的蛋白质组学的研究方法对不同发育阶段的雌性附腺分泌部表达的蛋白进行比较研究,以进一步研究这个复杂的生物学过程。
1.探讨了家蚕不同组织包括雌性附腺、丝腺、中肠和血液等的总蛋白提取和样品制备方法,优化不同组织来源蛋白的一向等电聚焦电泳(固相胶条)和二向SDS电泳的实验条件。由于组织的特异性,对不同的组织采用不同的蛋白提取方法和电泳条件。研究结果表明,对雌性附腺、中肠和丝腺用不同的裂解缓冲液提取蛋白和用不同的电泳参数进行双向电泳分离,能获得较好的蛋白质提取率、较高的分辨率(>1000蛋白点)、良好的重复性。
2.为了获得理想的家蚕雌性附腺分泌部组织在不同发育阶段的蛋白质表达图谱,对不同发育阶段的正常和Ng突变体的雌性附腺的分泌组织蛋白质样品制备和电泳进行了多次重复实验,确立了实验条件,取得良好的实验结果,建立了蛋白质参考图谱。利用分析软件(Image master)可以检测到800以上个蛋白点,而且不同发育时期的蛋白分布图基本相似,蛋白点主要分部在分子量30kD到70kD和等电点pH4～8的范围内。进一步对凝胶进行对比和软件匹配以及表达量的分析,发现有11个蛋白点的表达量在不同的发育时期表达量差异达到1.5
The colleterial gland of the silkworm, Bombyx mori, is referred to the female accessory system. The gland developed from the imaginal disc located in the ninth abdominal segment. Morphological and cytological observations have shown that the colleterial gland is differentiated into two parts, a tubular secretory region and a reservoir lobe. During certain developing stage, glue-like substance will be secreted by the secretory region of the gland and stored in the reservoir lobe. In silkworm, like some other insects, the glue like proteins are released to the surface o f eggs when egg-laying, then the glue-like substance will be hardened to fix the eggs on the surface of plants or other substrata, such that they aren't easily detached even in a strong wind or a heavy rain. The colleterial gland of silkworm grows gradually until day 8 after pupal ecdysis, then enlarged markedly due to the accumulation of glue-like substances before moth emergence. The Ng (a gene symbol for No-glue mutation located on chromosome linkage 12-21.8) mutant was one of the more than 400 mutations recorded in silkworm gene bank. The female moth of Ng mutant silkworm secreted only little glue-like substance and laid loose eggs naturally. The observation of anatomy showed that the typical shape of the colleterial gland had already formed in 7 or 8 days after pupal ecdysis in certain silkworm variety. However, the most interesting phenomenon is that the relatively small secretory region of colleterial gland can secrete a large amount of glue-like substance only in two or three days in silkworm. Interestingly, the female moth of Ng mutant secreted only little glue-like substance. Whereas some achievements in this field had been acquired, the molecular mechanisms of the secretion in a large quantity of glue-like proteins during a short period and the Ng mutant formation were still not understood well. Proteomics is a large-scale study of the gene expression at the protein level, which
ultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins. Key elements of classical proteomics are the separation of proteins in a sample using immobilized pH gradient two-dimensional gel electrophoresis (2-DE) and their subsequent identification by biological mass spectrometry. The purpose of choosing colleterial gland in the present research material w as n ot o nly t hat t he c olleterial g land m orphologically enlarged m arkedly and secreted glue-like substance in one or two days, and that its mechanisms had not been clear. During this development, different functional genes might be activated and some s pecial p roteins i nvolved w ith c olleterial g land d evelopment and s ecretion o f glue-like proteins might be expressed. Moreover, there was a very good experiment material available in our silkworm genebank that was the silkworm strain E981 and its Ng mutant line. Therefore, we investigated the utility of a proteomic approach to improve the understanding of this complex bioprocess.1. In this research, the main methods of the total proteins extraction and preparation of different organs of insect (Bombyx mori) including the colleterial gland, mudgut, silkgland and hemolymph and the conditions of isoelectric focusing and SDS-PAGE were discussed and optimized. The methods and conditions of protein preparation and electrophoresis of different organs were different. The proteins extracted from colleterial gland, midgut and middle silk gland then separated by two-dimensional electrophoresis, the results showed that higher protein extraction rate, resolution (more than 1,000 spots detected by software), better reproducibility and more sample volume were acquired in the present experiment.2. In order to establish the 2D PAGE profiles database during different development stages, all the steps from protein sample preparation to the two-dimensional electrophoresis were operated more than two times, and the conditions were optimized. The image analysis software (Image master) typically detected more than 800 spots on each gel following sliver staining, and the protein spots distribution pattern were nearly same during different stages between normal and Ng mutant
colleterial gland. Furthermore, most of the protein spots arranged from 30 kD to 70kD and pH 4 to 8. Under the help of the software, it was found that most protein's expression volume in different development days were no significant difference, however, interestingly, the volume variation of 11 protein spots that changed more than 1.5 folds in expression level compared with moth emergence day, and 3 spots (spots 1, 2and 3) only expressed in the last one or two days of pupal stages and in the day of moth emergence and no expression in Ng mutant. All these differential proteins were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Spot No. 1 was unknown protein in the silkworm and its highest identity protein was found in fruit fly. Spots No. 2 and 3 were the same protein, cytosolic actin A3; Spot No. 9 was identified as eukaryotic translation initiation factor 5 A (eIF-5A). Actin is a highly c onserved protein of the contractile system, present in the cytoskeleton of all eucaryotic cells. Actins are involved in a variety of cellular events such as motion, chromosome segregation, transport of macromolecules and endo-and exocytosis. Interestingly, inBombyx m ori silk gland cells, cytoplasmic actin is an abundant component of apical bundles of micro filaments, which participates in exocytosis of silk proteins. The actins only expressed in the two days when the glue-like proteins are secreted markedly. Furthermore, there were no actins expressed in the Ng mutant silkworm that secreted little glue-like substance during the whole development stage. The main content of secretions from both colleterial gland and silk gland are proteins, it indicates that actins are important functional proteins, which might participate or regulate the secretion of colleterial gland, and probably play similar function in silk gland. The eIF-5A is important for the protein translation of eukaryotic cell, and its expression volume was increased gradually during the stage of glue-like protein secretion, therefore, it is important for protein synthesis. Except that, these differential expressed proteins might be related to the Ng mutant form and glue proteins secretion.3. For further study the difference between the normal and Ng mutant colleterial gland at proteomics level, the Fluorescence two-dimensional difference gel electrophoresis
(2-DIGE) coupled with mass spectrometry were used to investigate mutant specific changes in the proteome of the secretory region of colleterial gland (normal and Ng mutant) in the moth emergence day. It was used Cy3 labeled proteins isolated from the normal colleterial gland and Cy5 labeled the proteins from the Ng mutant colleterial gland, and separated on the same 2-D gel along with a Cy2 labeled mixture of both 2 normal and mutant samples as an internal standard. Over 1000 protein spot-features were analyzed in the paired normal and mutant comparison. DeCyder software was used for simultaneous comparison of abundance of abundance changes and detected 20 spots which changed more than two-fold in expression level, and the expressed volume variation of some proteins even got almost 10 times. Using peptide mass fingerprint matrix-assisted laser desorption/ionization-time of flight mass spectrometry and public protein database, it was identified 4 proteins including PI09 protem-Bombyx mori. (spot 5), 50S ribosomal protein L5 (spot 9), Replication initiation protein (spot 13), Actin Al - silkworm (spot 17). According to the function of these differential expressed proteins, it indicated that these differential expressed proteins which connected to the protein synthesis and secretion might be have the relationship with glue-like protein secretion and Ng mutant form.4. The secreted protein mixture was separated by immobilized pH gradient two-dimensional gel electrophoresis. The results showed that more than 70 protein or polypeptide were detected in the gel (24 cm length), and the expressed volume of about 30 spots were abundance. According the distribution of these spots in gel, most of them arranged from pH 4-7, and the molecular weight of most protein and polypeptide more than 45 kD. It was identified 33 spots by mass spectrometry to understand the composition of the complex mixture. Peptide mass fingerprint of those protein spots were obtained and compared in the public database, the results suggested that most of the protein were the family of ribosomal protein occupied 30.4% among the identified proteins including the spot 3(60S ribosomal protein L4) ^ 5(60S ribosomal protein L14) .. 11 (40S ribosomal protein S6K 17(ribosomal protein
S27a) , 18 (60S ribosomal protein L7a) -. 27 (60S ribosomal protein L4) > 32 (60S ribosomal protein L6). Furthermore, two spots, 23 (Heat shock 70 kDa protein cognate 4) and 33 (Heat shock-related 70 kDa protein 2) , were identified as heat shock proteins that might be act as molecular chaperones to regulate protein folding and transfer the glue-like proteins out of special cells. Accepted that, some spots were identified as RNA-binding protein, elongation factor and so on, all the proteins composed the complex protein mixture secreted by colleterial gland ofBombyx mori.
引文
Aebersold R H, Leavitt J, Saavedra R A et. Al. Eletroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-poly-acrylamide gels for direct sequence analysis. J BiolChem, 1986, 261: 4229～4238
Alain Mangé, Eric Julien, Jean-Claude Prudhomme, Pierre Couble. A strong inhibitory element down-regulates SRE-stimulated transcription of the A3 cytoplasmic actin gene ofBombyx mori. J Mol Biol., 1997, 265: 266～274
Anderson, N. L. and Anderson, N. G. Proteome and Proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998, 19: 1853～1861
Andrew Alban, Stephen Olu David, Lennart Bjorkesten et al. A novel experimental design for comparative two-dimensional gel analysis: Two dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics, 2003, 3: 36～44
Andrew Alban, Stephen Olu David, Lermart Bjorkesten, et al. A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics, 2003, 3: 36～44
Arenkov P, Kukhtin A, Gemmell A et al. Protein microchips: Use for immtmoassay and enzymatic reactions. Anal Bichem, 2000, 278: 123～131
Arisawa N., Fugo H.. Development and protein synthesis of colleterial glands in the female silkworm, Botnbyx mori. Jpn. J. Appl. Ent. Zool., 1990, 34: 227～235
Amott D, O'Connell K L, King K L, et al. An integrated approach to proteome analysis-Identification of proteins associated with cardiac hypertrophy. Analytical Biochemistry, 1998, 258: 1～18
Bairoch A., Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic. Acids Res., 2000, 28: 45～48
Bartlet Jones M., Jeffery W. A., Hansen H. F. et al.. Peptide ladder sequencing by mass-spectrometry using novel, volatile degradation reagent. Rapid commun. Mass Spectrom., 1994, 8: 737～742
Bradford M M. A rapid and sensitive method for the quantitation of microgram qualities of proteins utilizing the principle of dye binding. Analytical Biochemistry, 1976, 72: 248～254
Brush M. Dye hard: protein gel staining products. Scientist, 1998, 12: 16～22
Bulet P., Uttenweiler Joseph S., in: Kamp, R. M., Kyriakidis D., Choli-Papadopoulou, T. Proteome and Protein Analysis, Springer Verlag, Heidelberg, 2000, 157～174
Carr S. A., Annan R. S. Overview ofpeptide and protein analysis by mass spectrometry. In: Current protocols in molecular biology, 1997, Wiley, New York.
Chair B. T., Wang R., Beavis R. C. et al.. Protein ladder sequencing. Science, 1993, 262: 89～92
Chaurand P, luetzenkirchen F, Spengler B. Peptide and protein identification by matrix-assisted laser desorption ionization(MALDI) and MALDI -post-source decay time of flight mass spectrometry. J. Am. Soc. Mass Spectrom, 1999, 10: 91-103
Christiansen J., Houen G. Comparison of different staining methods for polyvinylidene difluoride membranes. Electrophoresis, 1992, 13: 179～183
Christine Eymann, Annette Dreisbach, Dirk Albrecht. et al. A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics, 2004, 4: 0
Couble, P., Blaes, N., Prudhomme, J. C. Actin microfilaments and fibroin secretion in the silk gland cells of Bombyx mori. Exp cell Res., 1984, 151: 322～331
Da'rio Eluan Kalumea, Sylvie Kieffera, Kate Rafna et al. Sequence determination of three cuticular proteins and isoforms from the migratory locust, Locusta migratoria, using a combination of E dman degradation and mass spectrometric techniques. Biochimica et Biophysica Acta, 2003, 1645: 152～163
David B. Friedmen, Salisha Hill, Jeffrey W. Keller et al. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics, 2004, 4: 793～811
David B. Friedman, Salisha Hill, Jeffery W. Keller et al.. Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics, 2004, 4: 793～811
David R. Goodlett, Eugene C. Y. Stable isotopic labeling and mass spectrometry as a means to determine differences in protein expression. Trends in Analytical Chemistry, 2003, 22(5): 282～290
Deng, W., Burland, V., Plunkett, G, et al. Genome sequence of Yersinia pestis KIM. J. Bacteriol. 2002, 184(16): 4601～4611
Dove A. P roteomics: translating genomics into products? Nature Biotechnol., 1999, 17: 233～236
Dunn, M. J. Two-dimensional polyacrylamide gel electrophoresis. In: Advances in electrophoresis. Vol. 1, VCH, Weinheim, 1987, 1～109
Emili A Q, Cageny H Large scale functional analysis using peptide or protein arrays. Nat. Biotechnol, 2000, 18: 393～397
Fenn J B, Mann M, Meng C K, et al. Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989, 246: 64～71
Fernandez J., Gharahdaghi F., Mische S. M. Routine identification of proteins from sodium dodecyl sulfate-polyacrylamide gelelectrophoresis(SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry(MALDI-TOF-MS). Electrophoresis, 1998, 19: 1036～1045
Figeys D, Aebersold. High sensitivity analysis of proteins and peptides by capillary electrophoresis-tandem mass spectrometry: recent developments in technology and applications. Electrophoresis, 1998, 19: 885～892
Figeys D, Gygi S P, Mckinnon G. et al. An integrated microfluidics-tandem mass spectrometry system for automated protein analysis. Anal Chem, 1998, 70: 3728～3734
Fleischmann R D et al. Whole-genome random sequencing and assembly of Haemophilus influenzae. Science, 1995, 269: 496～512
Francesca Levi-Schaffer, Vladislav Temkin, Hans-Uwe Simon et al. Proteomic analysis of human eosinophil activation mediated by mast cells, granulocyte macrophage colony stimulating factor and tumor necrosis factor alpha. Proteomics, 2002, 2: 1616～1626
Frank Stadler, Dinah Hales. Highly-resolving two-dimensional electrophoresis for the study of insect proteins. Proteomics, 2002, 2: 1347～1353
Gefeng Lia, b, R. William Currie, Imtiaz S. Ali. Insulin potentiates expression of myocardial heat shock protein 70. European Journal of Cardio-thoracic Surgery, 2004, 26: 281-288
Gert Van den Bergh, Lutgarde Arckens. Fluorescent two-dimensional difference gel electrophoresis unveils the potential of gel-based proteomics. Current opinion in Biotechnology, 2004, 15: 38～43
Gharahdaghi F., Weinberg C. R., Meaghor D. A. et al. Mass spectrometric of protein from Silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis, 1999, 20: 601-605
Gharahdaghi F., Weinberg C. R., Meaghor D. A. et al. Mass spectrometric of protein from Silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity. Electrophoresis, 1999, 20: 601～605
Gorg A., Obemaier C, Boguth G et al., The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000, 21(6): 1037～1053
Gorg A., Obermaier C., Boguth G et al. Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins. Electrophoresis, 1997, 18: 328～337
Gorg A., Postel W., Gunther S., et al. The current state of two-dimensional electrophoresis with immobilized pH gradient. Electrophoresis, 1988, 9(9): 531～546
Grinblat, Y., Brown, N. H. and Kafatos, F. C. Isolation and characterization of the Drosophila translational elongation factor 2 gene. Nucleic Acids Res., 1989, 17(18): 7303～7314
Guedes S M, Vitorino R, Tomer K, et al. Drosophila melanogaster larval hemolymph protein mapping. Biochem Biophys Res Commun, 2003, 312(3): 545～554
Guido Tirone, Rita M. P. Development of Female Accessory Gland of Chrysomya Putoria(Widemann)(Diptera: Calliphoridae) During Oogenesis. Int. lnsect Morphl. and Embryol., 1997, 26: 1～7
Gygi S P, Rist B, Aebersold R. Measuring gene expression by quantitative proteome analysis. Curt Opin Biotech, 2000, 11: 396～401
Gygi S P, Rist B, Cerber S A et al. Quantitative analysis of complex protein mixtures using isotopecoded affinity tags. Natural Biotechnol, 1999, 17: 994～999
Heine G. Zucht H, Schuhmann M, et al. High-resolution peptide mapping of cerebrospinal fluid a novel concept for diagnosis and research in central nervous systems diseases. J Chromator Analyt Technol Life Sci, 2002, 782: 353-361
Henzel W. J., Billeci T. M., Stults J. T. et al. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in proteins sequence databases. Proc. Natl. Acad. Sci. USA, 1993, 90: 5011～5015
Henzel W. J., Billeci T. M., Stults J. T., et al.. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence database. Proc. Natl. Acad. Sci. USA, 1993, 90: 5011～5015
Herbert B. Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis, 1999, 20: 660～663
Herbert B., Molly M. P., Gooley A. A. et al. Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as a reducing agent. Electrophoresis, 1998, 19: 845～851
Hewick R M, Hunkapiller M W, Hood L E, Dreyer W J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem, 1981, 256: 7990～7997
Hirano H. The present status of proteome analysis. Chem. Biol., 1998, 36(12): 771～777
Hiroshi Fujii, Yutaka Kawaguchi. Changes of protein and nucleic acid synthesis in the developing colleterial glands of the silkworm, Bombyx mori. Sci. Bull. Agr., Kyushu Univ., 1994, 49: 61～66
Iborra, F., Buhler, J. M. Protein subunit mapping. A sensitive high-resolution method. Anal. Biochem., 1976, 74: 503～511
James P., Quadroni M., Carafoli E. et al. Protein identification by mass profile fingerprinting. Biochem. Biophys. Res. Commun., 1993, 195: 58～64
James P., Quadroni M., Carafoli E. et al.. Protein identification by mass profile fingerprinting. Biochem. Biiophys. Res. Commun., 1993, 195: 58～64
Jin YX, Chen YY, Xu MK, et al. Studies on middle silkgland proteins of cocoon sex-limited silkworm(Bombyx mori L.) using two-dimensional polyacrylamide gel electrophoresis. J. Biosci. 2004, 29(1): 45～49
John C. Wheeler, Vanessa King, John Tower, et al. Sequence requirements for upregulated expression of Drosophila hsp70 transgenes during aging. Neurobiology of Aging, 1999, 20: 545～553
Joseph Tambunan, Pei Kan Chang, Hong Li, et al. Molecular cloning of a cDNA encoding a silkworm protein that contains the conserved BH regions of Bc1-2 family proteins. Gene, 1998, 212: 287～293
Julien Bouley, Christophe Chambon, Brigitte Picard. Mapping of bovine skeletal muscle proteins using two-dimensional gel electrophoresis and mass spectrometry. Proteomics. 2004, 4: 1811～1824
Jun Sakai, Hironori Ishikawa, Shinichi Kojima et al, Proteomic analysis of rat heart ischemia and ischemia-reperfusion using fluorescence two-dimensional difference gel fluorescence gel electrophoresis. Proteomics, 2003, 3: 1318～1324
Jum X. Yan, Angelica T. Devenish, Robin Wait et al. Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli. Proteomics, 2002, 2: 1682～1698
Kamijo Z., Kawaguchi H., Fujii B. et al.. Instability of messenger and ribosomal RNA in a glue protein mutant of Bombyx mori.. Dev. Growth. Differ., 1978, 20: 283～289
Karas M, Hillenkarnp F. Laster desorption ionization of proteins with molecular mass exceeding 10000 dalt. Anal Chem, 1988, 60: 2299～2301
Katsuiko Yoshida, Masao Nagata. Adhesive strength of the glue substances in the colleterial glands of the silkmoth. Bombyx mori. J. Seric. Sci. Jpn., 1997, 66(6): 453～456
Kaufmann R., Kirsch D., Spengler B. Sequencing of peptides in a time of flight mass spectrometer: evaluation of postsource decay following matrix-assisted laser desorption ionization(MALDI). Int. J. Mass Spectrom. Ion Process, 1999, 131: 355～385
Kazuei Mita, Masahiro, Kasahara, Shin Sasaki et al. The genome sequence of Silkworm, Bombyx mori.. DNA Reaearch , 2004, 11: 27～35
Kelley, R. L. Initial organization of the Drosophila dorsoventral axis depends on an RNA-binding protein encoded by the squid gene. Genes Dev., 1993, 7(6): 948～960
Klein D. J., Moore P. B., Steitz T. A. The roles of ribosomal proteins in the structure assembly, and evolution of the large ribosomal subunit. Journal of molecular Biology, 2004, 340(1): 141～177
Klose J, Kobalz U. Two-dimensional gel electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis, 1995, 16: 1034～1059
Klose J., Kobalz U. Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis, 1995, 16: 1034～1059
Klose, K. Protein mapping by combined isoelecttric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik, 1975, 26: 231～243
Kouyanou, S., Santos, C., Koliaraki, V., Ballesta, P. G. Protein BmP0 from the silkworm Bombyx mori can be assembled and is functional in the Saccharomyces cerevisiae ribosomal stalk in the absence of the acidic P1 and P2 proteins. Gene, 2003, 314: 173～179
Laemmli, U. K. Cleavageof structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680～685.
Li Jiang, Tan Chen, Xiang Qiu et al. Search for differentially expressed proteins involved in the treatment of human nasopharyngeal carcinoma cells with NGX6 using two-dimensional electrophoresis and mass spectrometry. Prog. Biochem. Biophys. 2001, 28(4): 573～577
Louris J. N., Brodbelt J. S., Cooks R. G. et al. Ion isolation and sequential stages of mass spectrometry in a quadropole ion trap mass spectrometer. Int. J. Mass Spectrom. Ion Process, 1990, 96: 117～137
Lu Hong-sheng .The sericultural science in China(Shanghai: Scientific and Technical publishers), 1991
MacBeath G, Schreiber S L. Printing proteins as micoarrays for high-through put function determination. Science, 2000, 278: 1760～1763
Mann M., Hojrup P., Roepstorff P. Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol. Mass Spectrom. 1993, 22: 338～345
Mann M., Hojrup P., Roepstorff P.. Use of mass spectrometric molecular weight information to identify proteins in sequence database. Biol. Mass Spectrom, 1993, 22: 338～345
Marie-Claude Lang, Christophe Cansier, Paola Rizza et al. Comparative study of the expression of cellular proteins in uninfected and HIV infected U937 cells using two dimensional SDS polyacrylamide gel electrophoresis. Chemico-Biological Interactions. 1997, 103: 179～186
Matsudaira P. Sequence analysis from picomole quantifies of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 1987, 262: 10035～10038
Matthew Bogyo, James H H. Proteomics and genomics. Current Opinion in Chemical Biology. 2003, 7: 2～4
Michael E. Grossmann, Benjamin J. Madden, Fan Gao, et al. Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo. Experimental Cell Research, 2004, 297: 108～117
Mita K., Morimyo M., Okano K., et al.. The construction of an EST database for Bombyx mori and its application. Proc Natl Acad Sci U S A. 2003, 100(24): 14121～14126
Molloy M. P., Herbert B., Walsh B. J. et al. Extraction of membrane proteins by differential solubilization for separation using two-dimensional electrophoresis. Electrophoresis, 1998, 19: 837～844
Motoko Yago, Toshimasa Mitamura, Shinji Abe et. al.. Adhesive strength of gule-like substances from the colleterial glands of Antheraea yamamai and Rhodinia fugax. Int wild silkworth & silk, 2001, 6: 11～15
Myers E W., et al. A whole-genome assembly of Drosophila. Science, 2000, 287: 868～877
Nakamura K, Inokuchi T. Amino acid composition and synthesis of glutious substance in the silkworm, Bombyx mori. J. Sericult. Sci Japan, 1977, 46(2): 120～e124.
Nazrul Islam, M. Lonsdale, N. M. Upadhyaya et al. Protein extraction from mature rice leaves for two-dimensional gel electrophoresis and its application in proteome analysis. Proteomics, 2004, 4: 1903～1908
Neuhoff V, Stamm R, Eibl H. Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels. Electrophoresis, 1985, 6: 427～448
Nousiainen M, Rafn K, Skou L, et al. Characterization of Exoskeletal Proteins from the American Lobster, Homarus amerieanus. Biochem Physiol, 1998, 119: 189～199.
Oda Y, Huang K, Cross F R, et al. Accurate quantitation of protein expression and site-specific phosphorylation. Pro Natl Acid Sci USA, 1999, 96: 6591～6596
Park MH, Cooper HL, Folk JE. Identification of hypusine, all un. usual amillo acid, in a protein from human lymphocytes and of spermidine as its biosynthetic preettmor. Proc NailAcad Sci USA. 1981, 78(5): 2869～2873
Pennington S. R., Dunn M. J. Proteomics: from protein sequence to function. BIOS Scientific Publishers Limited, 2002, 144～145
Pennington SR, Wilkins MR, Hochstrasser DF, et al. Proteome analysis: from protein characterization to biological function. Trends Cell Biol., 1997, 7: 168～173
Rabilloud T, Adessi C, Lunardi J, et al. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilised pH gradients. Electrophoresis, 1997, 18: 307～316
Rabilloud T H, Valette C and Lawrence J J. Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis, 1994, 15: 1522～1558
Rabilloud T H, Valette C, Lawrence J J. Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis, 1994, 15: 1522～1558
Rabilloud T. A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis, 1992, 13: 429～439
Rabilloud T. Use of thiourea to increase the solubility of membrane proteins in twodimensional electrophoresis. Electrophoresis, 1998, 19: 758～760
Rabiloud T. H, Adessi C., Giraudel A. et al. Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradient. Electrophoresis, 1997, 18: 307～316
Ramu S. Saravanan, Jocelyn K. C. Rose. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics, 2004, 4: 2522～2532
Rashmi Sharma, Setsuko Komatsu, Hiroaki Noda. Proteomic analysis of brown planthopper: application to the study of carbamate toxicity. Insect Biochemistry and Molecular Biology, 2004, 34: 425～432
Rashmi Sharma, Setsuko Komatsu, Hiroaki Noda. Proteomic analysis of brown planthopper: application to the study of earbamate toxicity. Insect Biochemistry and Molecular Biology, 2004, 34: 425～432
Rebecca J. McNall a, Michael J. Adang. Identification of novel Bacillus thuringiensis CrylAc binding proteins in Manduca sexta midgut through proteomic analysis. Insect Biochemistry and Molecular Biology, 2003, 33: 999～010
Remacha M, Jimenez-Diaz A, Santos C., et al. Proteins P1, P2, and P0, components of the eukaryotic ribosome stalk. New structural and functional aspects. Biochem Cell Biol., 1995, 73: 959～968
Robert Rybczynski, Lawrence I. Gilbert. Prothoracicotropic hormone-regulated expression of a hsp 70 cognate protein in the insect prothoracic gland. Molecular and Cellular Endocrinology, 1995, 115: 73～85
Robert T., Joanne S., Brian M. et al. Validation and development of fluorescence two-dimensional differential gel eleetrophoresis proteomics technology. Proteomics, 2001, 1: 377～396
Robert Tonge, Joanne Shaw, Brian Middleton, et al. Validation and development of fluorescscence two-dimensional gel electrophoresis proteomics technology. Proteomics, 2001, 1: 377～396
Rosenfeld J., Capdevielle J., Guillemot JC. et al. In-gel digestion of proteins for internal sequence analysis after one-or two-dimensional gel electrophoresis. Anal. Bichem., 1992, 203, 173～179
Roux, A. E, Nguyen, V. T., Squire, J. A., et al. A heat shock gene at 14q22: mapping and expression. Hum. MoL Genet.. 1994, 3(10): 1819～1822
Sanchez J C, Rouge V, Pisteur M, Ravier F et al.. Hochstrasser D F. Improved and simplified in-gel sample application using rewelling of dry immobilized pH gradients. Electrophoresis, 1997, 18: 324～327
Scheele, G. A. Two-dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. J. Biol. Chem., 1975, 250: 5375～5385
Shevchenko A., Wilm M., Vorm O. et al. Mass spectrometric sequencing of proteins from sliver-stained polyacrylamide gels. Anal. Chem., 1996, 68: 850～858
Shevchenko A., Wilm M., Vorm O. Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Anal. Chem., 1996, 68: 850～858
Shiba T, Mizote H, Kaneko T. Hypusine. A new amino acid Occurring in bovine brain. Isolation and structural determination. Bio. china Biophys Acta, 1971, 244(3): 523～531
Smith D. F., Faber L. E., Toft D. O.. Purifications of nactivated progesterone receptor and identifications of novel receptor-associated proteins. J. Biol. Chem., 1990, 265: 3396
Smithies O., Poulik M. D. Two-dimensional electrophoresis of serum proteins. Nature, 1956, 177: 1033
Smithies O., Poulik M. D. Two-dimensional electrophoresis of serum proteins. Nature, 1956, 177: 1033
Soren Naaby-Hansen, Michael D. Waterfield, Rainer Cramer. Proteomics-post-genomic cartography to understand gene function. Trends in Pharmacological Sciences, 2001, 22: 376～384
Soren Naaby-Hansen, Michael D. Waterfield, Rainer Cramer. Proteomics-post-genomic cartography to understand gene function. Trends in Pharmacological Sciences, 2001, 22: 376～384
Soren Nabby-Hansen, Michael D W, Rainer C. Proteomics-post-genomic cartography to understand gene function. TRENDS in Pharmacological Sciences, 2001, 22: 376～383
Suzuki K., Hirata S. Genetic studies on the adhesive character of the silkworm eggs. Bull. Seric. Exp. Stn., 1924, 6: 301～318
Suzuki K., Tsujita M. Cytohistological study on the mucous glands of female moths of the silkworm, Bombyx mori. J. Seric. Sci. Jpn., 1937, 8: 89～107
Tonge R, Shaw J, Middleton B, et al. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics, 2001, 1: 377-396
Tonge, R., Shaw, J., Middleton, B., Rowlinson, R. et al. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics, 2001, 1, 377～396
Unlu, M., Morgan, M. E., Minden, J. S. Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis, 1997, 18, 2071～2077
Urquhart B L, Atsalos T E, Humphery-Smith I, et al. "Proteomic contigs" of Mycobactedum tuberculosis and mycobacterium bovis(BCG) using novel immobilized pH gradient. Electrophoresis, 1997, 18: 1384～1392
Van Oers M. M., Van Marwijk M., Kwa M. S., et al. Cloning and analysis of cDNAs encoding the hypusine-containing protein eIF5A of two lepidopteran insect species. Insect Mol. Biol., 1999, 8(4), 531～538
Ventel J. C., Adams M. D., Myers E. W., et al. the sequence of human genome. Science, 2001, 291(5507): 1304～1351
Ventel J. C., Adams M. D., Myers E. W., et al. the sequence of human genome. Science, 2001, 291(5507): 1304～1351
Vierstraete E, Verleyen P, Baggerman G, et al A proteomic approach for the analysis of instantly released wound and immune proteins in Drosophila melanogaster hemolymph. Proc Natl Acad Sci USA. 2004, 101(2): 470～475
Von Eggeling, F., Gawriljuk, A., Fiedler, W., Ernst, G. et al. Fluorescent dual colour 2D-protein gel electrophoresis for rapid detection of differences in protein pattern with standard image analysis soft-ware. Int. J. Mol. Med., 2001, 8, 373～377
Weerawan Amornsak, Tomoka Noda, Okitsugu Yamashita. Accumulation of glue proteins in the developing colleterial glands of the silkworm, Bombyx mori. J. Seric. Sci. Jpn., 1992, 61(2): 123～130
Wildgruber R., Harder A., Obemater C. Towards high resolution: two-dimensional electrophoresis of saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients. Electrophoresis, 2000, 21(13): 2610～2616
Wilkins M. R., Gasteiger E., Bairoch. A. et. al. Protein identification and analysis tools in the ExPASy sever. Methods Mol. Biol., 1999, 112: 531～552
Wilkins M. R., Lindskog I., Gasteiger E. et al. Detailed peptide characterization using PEPTIDEMASS-a World-Wide-Web-accessible tool. Electrophoresis, 1997, 18: 403～408
Wilkins MR, Sanchez JC, Gooley AA et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnology, 1995, 13: 19～50
Wilkins MR, Sanchez JC, Gooley AA et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Genet. Engin. Rev., 1996, 13: 19～50
Williams, K. L. Genomes and proteomes: towards a multidimensional view of biology. Electrophoresis, 1999, 20: 678～688
Wilm M S, Mann M. Analytical properties of the nanoelectrospray ion-source. Anal Chem, 1996, 68: 1～8
Wilm M S, Mann M. Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? Int. J. Mass Spectrom. Ion Processes, 1994, 136: 167～180
Wilm M., Shevchenko A., Houthaeve T. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature, 1996, 379: 466～469
Yates J. R., Speicher S., Griffin P. R. et al.. Peptide mass maps: a highly informative approach to protein identification. Anal Biochem., 1993, 214: 397～408
Yates J. R., Eng J. K., McCormack A. L. Min genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide database. Anal Chem., 1995, 67: 3202～3210
Yi Hu, Gang Wang, Grace Y. J., et al. Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis. Electrophoresis, 2003, 24(9): 1458～1470
Yongqiang Wang, Pingbo Zhang, Hiroshi Fujii et al. Proteomic studies of lipopolysaccharide-induced polypeptides in the silkworm, Bombyx mori. Biosci. Biotechnol. Biochem., 2004, 68(8): 1821～1823
Zenta Kajiura, Takesi Ohshima, Masao Nakagaki et al.. Female accessory glands(colleterial glands) of the wild silkrnoth and their secretory proteins. J. Ser. Sci. Jpn., 1997, 66(6): 413～419
Zhong B. X., Weng H. B., Fang W. H.. Preparation of protein samples for gel electrophoresis by sequential extraction. Journal of zhe jiang university science, 2002. 5: 606～610
Zhou G, Decamp D. et al. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer specific protein markers. Mol Cell Proteomics, 2002, 1(2:): 117～124
陈平,谢锦云,梁宋平.双向凝胶电泳银染蛋白质点的肽质谱指纹图分析.生物化学与生物物理学报,2000,32(4):387～391
成海平,钱小红.蛋白质组研究的技术体系及其进展.生物纪学与生物物理进展.2000,27(6):584～588
程道军,夏庆友,周泽扬等.家蚕cDNA文库构建及大规模EST测序.蚕业科学2003,29(4):335～339
程志祥,熊俊,倪雪峰等.大鼠胰腺蛋白质组研究中双向电泳技术的初步建立.南京医科大学学报(自然科学版).2003,23(2):106～115
郭尧君.SDS电泳技术的实验考虑及最新进展.生物化学与生物物理进展.1991,18(1):32～37
贾宇峰,林秋霞,郭尧君等.蛋白质双向电泳图像分析.生物化学与生物物理进展.2001,28(2):246～250
凌明圣,徐明波,姚志建.分子伴侣HSP70研究进展.国外医学分子生物学分 册,1993,15(5):227～230
明镇寰.生物化学与分子生物学新概念(第一版).杭州:浙江大学出版社.1999,6～9
沈同,王镜岩.生物化学(第二版).高等教育出版社,1991,397～400
王大东.蛋白质与蛋白质组学.国外医学:分子生物学分册.2000,22(3):118～121
王京兰,万晶宏,罗凌等.蛋白质组研究中肽质量指纹谱鉴定方法的建立及应用.生物化学与生物物理学报,2000,32(4):373～378
向仲怀.国际鳞翅目昆虫基因组筑波会议与中国家蚕基因组.蚕业科学,2002,28(4):271～272
向仲怀.中国家蚕基因组与21世纪丝绸之路.蚕业科学,2003,29:321～322
徐豫松,徐俊良,川崎秀树.家蚕脂肪体合成蛋白质变化的研究.蚕业科学,2000,26(4):239～243
许克新,王云川.双向电泳及质谱分析与蛋白质组研究.第四军医大学学报,2002,23(22):2017～2022
颜海燕.家蚕胚胎高温干燥敏感期的蛋白质研究.浙江大学硕士学位论文:16～56
颜新培,钟伯雄,徐孟奎等.家蚕五龄期后部丝腺蛋白质构成与茧层量的关系.蚕业科学,2003,29(4):344～348
颜新培.家蚕卵黄和胚胎蛋白质组及其催青期变动的研究.浙江大学博士学位论文:1～3
詹显全,关勇军,李萃等.人肺腺癌细胞和正常细胞HBE的蛋白质组差异分析.生物化学与生物物理学报,2002,34(1):50～56
钟伯雄,颜海燕,沈飞英等..家蚕蛋白质双向电泳的样品制备方法.蚕业科学,2003,29(4):427～432
钟伯雄.家蚕胚胎发育时期的蛋白质变化及构造分析.遗传学报,1999,26(6):627～633
钟伯雄.五龄家蚕若干组织器官蛋白质数据库构建.遗传学报,2001,28(3):217～224
