人食管癌相关基因编码蛋白质的表达、结构及其功能的研究
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摘要
食管癌的发生发展与多种癌基因、抑癌基因改变有关，但尚未发现与食管癌有高度相关的基因。为了探索新的食管癌相关基因，本室用mRNA差异显示技术与5'-RCAE(Rapid Amplification of cDNA Ends)方法从正常人食管上皮与高癌家族的食管癌组织中分离与克隆出四个基因片段，序列同源比较为未知新基因，并获得了其中一个基因片段的全长cDNA序列，命名为食管癌相关基因-1(Esophageal Cancer Related Gene-l，ECRG-1)。本论文旨在研究ECRG-1基因所编码蛋白质的结构与功能。为此，我们从以下几个方面进行了研究。
1．ECRG-1基因编码蛋白质的结构与功能分析及其三维结构的模建：应用生物信息学(Bioinfomatics)方法分析了ECRG-1基因编码蛋白质的性质与结构，进行蛋白质同源序列比较，预测其功能并模建该蛋白质的三维结构。结果表明ECRG-1基因编码蛋白质是一个跨膜蛋白，分子量为45kD，与丝氨酸蛋白酶同源性较高，推测ECRG-1基因编码蛋白质的功能与细胞形态及其生长有关。以丝氨酸蛋白酶(Serine Protease)晶体结构为模板，利用同源模建原理，从理论上设计了ECRG-1基因编码蛋白质的三维结构。
2．ECRG-1基因编码蛋白质在原核细胞中的表达：应用RT-PCR方法克隆与分离出ECRG-1基因的蛋白编码区序列，经测序与5'-RCAE钓取的序列相符合。以质粒pGEX-4T-1为表达载体，在大肠杆菌中诱
Many tumor suppresser genes and oncogenes are involved in the initiation and development of esophageal cancer (EC). However, no gene directly associated with EC has been found, In recent years, in order to find novel gene related to EC four differential fragments were isolated and cloned by the effective technique of mRNA differential display with comparing the differential gene expression between normal esophageal epithelia and EC. The four fragments were found no remarkable similarity to the known sequence in homology analysis with the public data of GenBank. These four fragments may represent novel genes. The full-length of one sequence was obtained by the technique of 5'-RCAE (Rapid Amplification of cDNA Ends) . The sequence was assigned as esophageal cancer related gene-1 (ECRG-1). The aim of this thesis is to explore the structure and function of ECRG-1 gene. The results are as follow:
1. The structure and function of the protein encoded by ECRG-1 gene was primarily predicted by bioinfonnatics. The result shows the protein is a transmembrane protein that has remarkable similarity to serine protease. The molecular weight of the protein encoded by ECRG-1 gene was about 45 kD. The function may play an essential role in cell growth and maintenance of cell
1．ECRG-1基因编码蛋白质的结构与功能分析及其三维结构的模建：应用生物信息学(Bioinfomatics)方法分析了ECRG-1基因编码蛋白质的性质与结构，进行蛋白质同源序列比较，预测其功能并模建该蛋白质的三维结构。结果表明ECRG-1基因编码蛋白质是一个跨膜蛋白，分子量为45kD，与丝氨酸蛋白酶同源性较高，推测ECRG-1基因编码蛋白质的功能与细胞形态及其生长有关。以丝氨酸蛋白酶(Serine Protease)晶体结构为模板，利用同源模建原理，从理论上设计了ECRG-1基因编码蛋白质的三维结构。
2．ECRG-1基因编码蛋白质在原核细胞中的表达：应用RT-PCR方法克隆与分离出ECRG-1基因的蛋白编码区序列，经测序与5'-RCAE钓取的序列相符合。以质粒pGEX-4T-1为表达载体，在大肠杆菌中诱
Many tumor suppresser genes and oncogenes are involved in the initiation and development of esophageal cancer (EC). However, no gene directly associated with EC has been found, In recent years, in order to find novel gene related to EC four differential fragments were isolated and cloned by the effective technique of mRNA differential display with comparing the differential gene expression between normal esophageal epithelia and EC. The four fragments were found no remarkable similarity to the known sequence in homology analysis with the public data of GenBank. These four fragments may represent novel genes. The full-length of one sequence was obtained by the technique of 5'-RCAE (Rapid Amplification of cDNA Ends) . The sequence was assigned as esophageal cancer related gene-1 (ECRG-1). The aim of this thesis is to explore the structure and function of ECRG-1 gene. The results are as follow:
1. The structure and function of the protein encoded by ECRG-1 gene was primarily predicted by bioinfonnatics. The result shows the protein is a transmembrane protein that has remarkable similarity to serine protease. The molecular weight of the protein encoded by ECRG-1 gene was about 45 kD. The function may play an essential role in cell growth and maintenance of cell
引文
1 Nation Research Council, Mapping and sequencing the human genome. National Academic Press, 1998
2 Marshall E. Emphasis turns from mapping to large-scale sequencing. Science, 1995, 268: 1270-1271
3 Fraser C M, Gocayne J D, White O, et al. The minimal gene complement of Myoplasma genitalium. Science, 1995, 270: 397-403
4 Fleischmann R D, Adams M D, White O, et al. Whole genome random sequencing and assembly of Haemophilis influenzea Rd. Science, 1995, 269: 496-512
5 Blatmen F B, G Plunkett Ⅲ, Bloch C A, et al. The complete genome sequence ofE. coli K 12. Science, 1997, 277: 1453-1462
6 Goffeau A, Barrel B G, Bussey H, et al. Life with 6000 genes. Science, 1996, 274: 546-567
7 李林，吴家睿，李伯良．蛋白质组学的产生及其重要意义，生命科学，1998．11：49-50
8 Nowak R. Entering the postgenome era. Science, 1995, 270: 368-371
9 Kahn P. From genome to proteome: looking at a cell's protein. Science, 1995, 270: 369-370
10 Mild Y, Swensen J, Shattuck-Eidens D,et al. A Strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994, 266: 66-71
11 李华川，陆士新．食管癌组织中Rb基因突变与表达的研究．中华肿瘤杂志，1993，15：412-41412 Liang Y Y, Estere A, Martel- Planche G, et al. P53 mutations in esophageal tumors from high-incidence areas of China. Int J Cancer, 1995, 61: 611-614
13 李华川，陆士新．食管癌组织中抑癌基因APC、MCC突变的研究．中华肿瘤杂志，1995，17：9-12
14 Maesawa C, Tamura G, Nishizuka S, et al. Inactivation of the CDKN2 gene by homozygous deletion and de Novo methylation is associated with advanced stage esophageal squamous cell carcinoma. Cancer Res, 1996, 56: 3875-3878
15 梁苑苑，陆士新．食管癌癌变过程中c-myc与HER-1癌基因表达的研究．中华肿瘤杂志，1991，13：168-170
16 梁苑苑，郭永军，陆士新，等．食管癌中五种不同基因改变的分析．中华肿瘤杂志，1993，15：408-411
17 郭永军，陆士新，梁苑苑，等．原发性及亚硝胺诱导的食管癌中 int-2基因的扩增．中华肿瘤杂志，1993，15：91-93
18 Jiang W, Kahn SM, Tomita N, et al. Amplification and expression of the human cyclin D gene in esophageal cancer. Cancer Res, 1992, 52: 2980-2983
19 苏涛，刘海玲，陆士新，等．人食管癌相关基因cDNA片段的克隆与初步鉴定．中华肿瘤杂志，1998，20：254-257
20 刘海玲，张登学，陆士新，等5′RACE技术钓取未知基因中的应用．癌症，1999，18：106-112
21 Leytus S P, Loeb K R, Hagen F S, et al. A novel trypsin-like serine protease (hepsin) with a putative transmembrane domain expressed by human liver and hepatoma cells. Biochemistry, 1988, 27: 1067-1074
22 Tsuji A, Torres-Rosado A, Arai T, et al. Hepsin , a cell membrane-associated protease. Characterization, tissue distribution and gene??localization. J. Biol. Chem, 1991, 266: 16948-16953
23 Torres-Rosado A, O'shea K S, Tsuji A, et al. Hepsin, a putative cell-surface serine protease, is required for mammalian cells growth. Proc. Natl. Acad. Sci. U.S.A. 1993, 90: 7181-7187
24 Remington S J, Mattews B W. A systematic approach to the comparison of protein structures. J. Mol. Biol, 1980, 140: 77-99
25 Rao S T, Rossman M G. Comparison of super-secondary structures in proteins. J. Mol. Biol, 1973, 76: 241-256
26 Blundell T L, Sibando B L, Steinberg M J E, et al. Knowledge-based prediction of protein structures and the design of novel molecules. Nature, 1987, 326: 347-352
27 Sali A, Blundell T L. Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming. J. Mol. Biol, 1990, 212: 403--428
28 Holm L, Sander C. Database algorithm for generating protein backbone and side-chain co-ordinates from a C_α trace. Application to model building and detection of co-ordinate errors. J. Mol. Biol, 1991, 218: 183-194
29 Warwicer J, Watson H C. Calculation of the electric protential in the active site cleft due to α-helixdiploes. J. Mol. Biol, 1982, 157: 671
30 Van Gunsteren W F, Berendsen H J C. Computer simulation of molecular dynamics: methodology, applications, and perspectives in chemistry. Angew. Chem Int Ed Engl, 1990, 29: 992-1023
31 Kirkpatrick S, Gelatt C D, Vecchi M R et al. Optimization by simulated annealing. Science, 1983, 220: 671-680
32 J．萨姆布鲁克，E．F弗里奇，T．曼尼阿蒂斯．《分子克隆实验指??南》 第二版 科学出版社 北京 1992
33 Mosmann T, et al. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods, 1983, 65: 55
34 鄂征 主编．《组织培养和分子细胞学技术》北京出版社 北京 1995 214-219
35 寿思明．生产重组医用蛋白质的三种途径．生命的化学，1998，18：24-25
36 孙志贤 主编．《现代生物化学理论与研究技术》军事医学科学出社 北京 1995 452-458
37 Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 1988, 67: 31-40.
38 沈珝琲，方福德 主编．《真核基因表达调控》高等教育出版社 北京 1996
39 赵卫红，寿好长，陈家佩．G_2+M期阻滞与肿廇．国外医学肿瘤分册，1999，26：76-78
40 F．奥斯伯，R．布伦特，R．E．金斯顿，等．《精编分子生物学实验指南诊科学出版社 北京 1998
41 胡志远，邢桂春，贺福初．用脂质体法转染COS-7细胞条件的优化．军事医学科学院院刊，1997，21：125-127
42 Lim K, Chae C B. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta- galactosidase. Biotechniques, 1989, 7: 576
43 Weber M, Moller K, Welzeck M. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells. Bioteclmiques, 1995, 19: 9301 Nation Research Council, Mapping and Sequencing the Human Genome, National Academic Press, 1998
2 Murray J C, Buetow K H, Weber J L, et al. A comprehensive human linkage map with centimorgan density. Science, 1994, 265: 2049-2054
3 Adams M D, Kerlavage A R, Fleischmann R D, et al. Initial assessment of human gene diversity and expression patterns based upon 83 million nuleotides ofcDNA sequence Nature, 1995, 377: Suppl 3-297
4 Dib C, Faure S, Fizames c, et al. A comprehensive genetic map of the human genome based on 5,246 microsatellites. Nature, 1996, 380: 152-154
5 Fraser C M, Gocayne J D, White O, et al. The minimal gene complement of Myoplasma genitalium. Science, 1995, 270: 397-403
6 Fleischmann R D, Adams M D, White O, et al. Whole genome random sequencing and assembly of Haemophilis influenzea Rd. Science, 1995, 269: 496-512
7 Blatmen F B, G Plunker Ⅲ, Bloch C A, et al. The complete genome sequence ofE. coli K 12. Science, 1997, 277: 1453-1462
8 Goffeau A, Barrel B G, Bussey H, et al. Life with 6000 genes. Science, 1996, 274: 546-567
9 Wilson R, Ainscough R, Anderson K, et al. 2.2 mb of contiguous nucleotide sequence from chromosome Ⅲ of C. Elegans. Nature, 1994, 368: 32-38
10 Kahn P, From genome to proteome: looldng at a cell's proteins. Science, 1995, 270: 369-37011 Swinbanks D. Government backs proteome proposal. Nature, 1995, 378: 653
12 Wilkins M R, Sanchez J C, Gooley A A, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotech & Genetic Engineering Rev, 1995, 13: 19-50
13 Bin L, Zwilling R, Pallin V, et al. Two dimensional gel electrophoresis of Gaenorhabdilis elegans homogenates and identification of protein spots by mirosequencing. Electrophoresis, 1997, 18: 557-562
14 Gerreiro N, Djordievic M A, Rolfe B G, et al. New Rhizobium leguminosarum flavonoid induced proteins revealed by proteome analysis of differentially displayad proteins. Mol Plant Mirobe Interact, 1997, 10: 506-516
15 Wasinger V C, Humphery S I, Bjellqist B. Proteomic "contig" of Ochrobactrum anthropi, application of extensive pH gradients. Electrophoresis, 1997, 18: 1373-1383
16 Van Bogelen R A, Neidhardt F C, Olson E R, et al. Escherichia coli proteome analysis using the gene-protein database. Electrophoresis, 1997, 18: 1243-1251
17 Yan J X, Williams K L, Hochstrasser O F, et al. The Dictyostelium discoidenm proteome the SWISS-2D PAGE database of the multicellular aggregate (Slug). Elecphoresis, 1997, 18: 491-49
18 Cells J E, Rasmussen H H, Vanderckhove J, et al. Human 2-D PAGE databases for proteome analysis in health and disease: http://biobose,dik/egi-bin/celis. FEBS Lett, 1996, 398: 129-134
19 Payne W E, Garrels J I. Yeast protein database (YPD): a database for the complete proteome of saccharomyces cerevisiae. Nucliec Acids Res, 1997, 25: 57-62
20 Hodges P E, Payne W E, Garrels J I, et al. The yeast protein database??(YPD): a curated proteome database for Saccharomyces cerevisiae. Nucliec Acids Res, 1998, 26: 68-72
21 Humphery S I, etal. J Prot Chem, 1997, 16: 537
22 Wilkins M R, Sanchez J C, Williams K L, et al. Current challenges and future applications for protein maps and post-translational in proteome projects. Electrophoresis, 1996, 17: 830-
2 Marshall E. Emphasis turns from mapping to large-scale sequencing. Science, 1995, 268: 1270-1271
3 Fraser C M, Gocayne J D, White O, et al. The minimal gene complement of Myoplasma genitalium. Science, 1995, 270: 397-403
4 Fleischmann R D, Adams M D, White O, et al. Whole genome random sequencing and assembly of Haemophilis influenzea Rd. Science, 1995, 269: 496-512
5 Blatmen F B, G Plunkett Ⅲ, Bloch C A, et al. The complete genome sequence ofE. coli K 12. Science, 1997, 277: 1453-1462
6 Goffeau A, Barrel B G, Bussey H, et al. Life with 6000 genes. Science, 1996, 274: 546-567
7 李林，吴家睿，李伯良．蛋白质组学的产生及其重要意义，生命科学，1998．11：49-50
8 Nowak R. Entering the postgenome era. Science, 1995, 270: 368-371
9 Kahn P. From genome to proteome: looking at a cell's protein. Science, 1995, 270: 369-370
10 Mild Y, Swensen J, Shattuck-Eidens D,et al. A Strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994, 266: 66-71
11 李华川，陆士新．食管癌组织中Rb基因突变与表达的研究．中华肿瘤杂志，1993，15：412-41412 Liang Y Y, Estere A, Martel- Planche G, et al. P53 mutations in esophageal tumors from high-incidence areas of China. Int J Cancer, 1995, 61: 611-614
13 李华川，陆士新．食管癌组织中抑癌基因APC、MCC突变的研究．中华肿瘤杂志，1995，17：9-12
14 Maesawa C, Tamura G, Nishizuka S, et al. Inactivation of the CDKN2 gene by homozygous deletion and de Novo methylation is associated with advanced stage esophageal squamous cell carcinoma. Cancer Res, 1996, 56: 3875-3878
15 梁苑苑，陆士新．食管癌癌变过程中c-myc与HER-1癌基因表达的研究．中华肿瘤杂志，1991，13：168-170
16 梁苑苑，郭永军，陆士新，等．食管癌中五种不同基因改变的分析．中华肿瘤杂志，1993，15：408-411
17 郭永军，陆士新，梁苑苑，等．原发性及亚硝胺诱导的食管癌中 int-2基因的扩增．中华肿瘤杂志，1993，15：91-93
18 Jiang W, Kahn SM, Tomita N, et al. Amplification and expression of the human cyclin D gene in esophageal cancer. Cancer Res, 1992, 52: 2980-2983
19 苏涛，刘海玲，陆士新，等．人食管癌相关基因cDNA片段的克隆与初步鉴定．中华肿瘤杂志，1998，20：254-257
20 刘海玲，张登学，陆士新，等5′RACE技术钓取未知基因中的应用．癌症，1999，18：106-112
21 Leytus S P, Loeb K R, Hagen F S, et al. A novel trypsin-like serine protease (hepsin) with a putative transmembrane domain expressed by human liver and hepatoma cells. Biochemistry, 1988, 27: 1067-1074
22 Tsuji A, Torres-Rosado A, Arai T, et al. Hepsin , a cell membrane-associated protease. Characterization, tissue distribution and gene??localization. J. Biol. Chem, 1991, 266: 16948-16953
23 Torres-Rosado A, O'shea K S, Tsuji A, et al. Hepsin, a putative cell-surface serine protease, is required for mammalian cells growth. Proc. Natl. Acad. Sci. U.S.A. 1993, 90: 7181-7187
24 Remington S J, Mattews B W. A systematic approach to the comparison of protein structures. J. Mol. Biol, 1980, 140: 77-99
25 Rao S T, Rossman M G. Comparison of super-secondary structures in proteins. J. Mol. Biol, 1973, 76: 241-256
26 Blundell T L, Sibando B L, Steinberg M J E, et al. Knowledge-based prediction of protein structures and the design of novel molecules. Nature, 1987, 326: 347-352
27 Sali A, Blundell T L. Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming. J. Mol. Biol, 1990, 212: 403--428
28 Holm L, Sander C. Database algorithm for generating protein backbone and side-chain co-ordinates from a C_α trace. Application to model building and detection of co-ordinate errors. J. Mol. Biol, 1991, 218: 183-194
29 Warwicer J, Watson H C. Calculation of the electric protential in the active site cleft due to α-helixdiploes. J. Mol. Biol, 1982, 157: 671
30 Van Gunsteren W F, Berendsen H J C. Computer simulation of molecular dynamics: methodology, applications, and perspectives in chemistry. Angew. Chem Int Ed Engl, 1990, 29: 992-1023
31 Kirkpatrick S, Gelatt C D, Vecchi M R et al. Optimization by simulated annealing. Science, 1983, 220: 671-680
32 J．萨姆布鲁克，E．F弗里奇，T．曼尼阿蒂斯．《分子克隆实验指??南》 第二版 科学出版社 北京 1992
33 Mosmann T, et al. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods, 1983, 65: 55
34 鄂征 主编．《组织培养和分子细胞学技术》北京出版社 北京 1995 214-219
35 寿思明．生产重组医用蛋白质的三种途径．生命的化学，1998，18：24-25
36 孙志贤 主编．《现代生物化学理论与研究技术》军事医学科学出社 北京 1995 452-458
37 Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 1988, 67: 31-40.
38 沈珝琲，方福德 主编．《真核基因表达调控》高等教育出版社 北京 1996
39 赵卫红，寿好长，陈家佩．G_2+M期阻滞与肿廇．国外医学肿瘤分册，1999，26：76-78
40 F．奥斯伯，R．布伦特，R．E．金斯顿，等．《精编分子生物学实验指南诊科学出版社 北京 1998
41 胡志远，邢桂春，贺福初．用脂质体法转染COS-7细胞条件的优化．军事医学科学院院刊，1997，21：125-127
42 Lim K, Chae C B. A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta- galactosidase. Biotechniques, 1989, 7: 576
43 Weber M, Moller K, Welzeck M. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells. Bioteclmiques, 1995, 19: 9301 Nation Research Council, Mapping and Sequencing the Human Genome, National Academic Press, 1998
2 Murray J C, Buetow K H, Weber J L, et al. A comprehensive human linkage map with centimorgan density. Science, 1994, 265: 2049-2054
3 Adams M D, Kerlavage A R, Fleischmann R D, et al. Initial assessment of human gene diversity and expression patterns based upon 83 million nuleotides ofcDNA sequence Nature, 1995, 377: Suppl 3-297
4 Dib C, Faure S, Fizames c, et al. A comprehensive genetic map of the human genome based on 5,246 microsatellites. Nature, 1996, 380: 152-154
5 Fraser C M, Gocayne J D, White O, et al. The minimal gene complement of Myoplasma genitalium. Science, 1995, 270: 397-403
6 Fleischmann R D, Adams M D, White O, et al. Whole genome random sequencing and assembly of Haemophilis influenzea Rd. Science, 1995, 269: 496-512
7 Blatmen F B, G Plunker Ⅲ, Bloch C A, et al. The complete genome sequence ofE. coli K 12. Science, 1997, 277: 1453-1462
8 Goffeau A, Barrel B G, Bussey H, et al. Life with 6000 genes. Science, 1996, 274: 546-567
9 Wilson R, Ainscough R, Anderson K, et al. 2.2 mb of contiguous nucleotide sequence from chromosome Ⅲ of C. Elegans. Nature, 1994, 368: 32-38
10 Kahn P, From genome to proteome: looldng at a cell's proteins. Science, 1995, 270: 369-37011 Swinbanks D. Government backs proteome proposal. Nature, 1995, 378: 653
12 Wilkins M R, Sanchez J C, Gooley A A, et al. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotech & Genetic Engineering Rev, 1995, 13: 19-50
13 Bin L, Zwilling R, Pallin V, et al. Two dimensional gel electrophoresis of Gaenorhabdilis elegans homogenates and identification of protein spots by mirosequencing. Electrophoresis, 1997, 18: 557-562
14 Gerreiro N, Djordievic M A, Rolfe B G, et al. New Rhizobium leguminosarum flavonoid induced proteins revealed by proteome analysis of differentially displayad proteins. Mol Plant Mirobe Interact, 1997, 10: 506-516
15 Wasinger V C, Humphery S I, Bjellqist B. Proteomic "contig" of Ochrobactrum anthropi, application of extensive pH gradients. Electrophoresis, 1997, 18: 1373-1383
16 Van Bogelen R A, Neidhardt F C, Olson E R, et al. Escherichia coli proteome analysis using the gene-protein database. Electrophoresis, 1997, 18: 1243-1251
17 Yan J X, Williams K L, Hochstrasser O F, et al. The Dictyostelium discoidenm proteome the SWISS-2D PAGE database of the multicellular aggregate (Slug). Elecphoresis, 1997, 18: 491-49
18 Cells J E, Rasmussen H H, Vanderckhove J, et al. Human 2-D PAGE databases for proteome analysis in health and disease: http://biobose,dik/egi-bin/celis. FEBS Lett, 1996, 398: 129-134
19 Payne W E, Garrels J I. Yeast protein database (YPD): a database for the complete proteome of saccharomyces cerevisiae. Nucliec Acids Res, 1997, 25: 57-62
20 Hodges P E, Payne W E, Garrels J I, et al. The yeast protein database??(YPD): a curated proteome database for Saccharomyces cerevisiae. Nucliec Acids Res, 1998, 26: 68-72
21 Humphery S I, etal. J Prot Chem, 1997, 16: 537
22 Wilkins M R, Sanchez J C, Williams K L, et al. Current challenges and future applications for protein maps and post-translational in proteome projects. Electrophoresis, 1996, 17: 830-