摘 要
在了解到生物體內可以發揮大部份功能的重要物質是蛋白質後，蛋白質的研究與應用就成為後基因體時代發展的重點，蛋白質體學則成為一項重要的研究方法。大腸桿菌是最常被用來表現重組蛋白質的宿主細胞，其蛋白質的資料庫也持續更新而相當接近。
本研究是利用蛋白體學來分析宿主菌大腸桿菌(Escherichia coli BL21)以及含重組基因的大腸桿菌(Escherichia coli BL21含重組過的pGEX質體)有無誘導重組基因表現蛋白體的不同。同時也使用硝酸銀和二胺銀兩種銀染方法來偵測蛋白質; 研究發現二胺銀的方法可以偵測出較多的蛋白質，即靈敏度較高，但相較於二胺銀的方法，需要較久的操作時間和較大的藥品消耗量，膠片上的直紋和橫紋也比較多，會影響分析。在增加蛋白質回溶時間到5分鐘後，硝酸銀法可以偵測出的蛋白質點並不會比使用二胺銀法的方法少，為低分子量範圍(小於20K Da)的蛋白質點還是以二胺銀法能染出較多的點。所以綜合而言使用硝酸銀法有較多的優點。
在蛋白質鑑定的部份，已用ESI-MS/MS(Q-STAR)鑑定出膠片上的兩個蛋白質，gapA (glyceraldehyde 3-phosphatedehydrogenase，MW 35.2K Da , pI 6.58 )與dnaK (Chaperon Hsp70，MW 69K Da , pI 4.83)。其中gapA具有守護者蛋白質(houesekeeping protein)的特性，可以做為正規化因子，以更準確的比較蛋白質表現量的變化。
而重組菌(未誘導和誘導)所欲表現的目標蛋白質GST-nanA-Arg5在粗抽液的二維膠體電泳圖上並未發現，即使是將蛋白質分析的總量增加到500 μg也看不到。但是在經過純化的蛋白質溶液其二維膠體電泳及使用西方轉漬的結果都顯示有大量的目標蛋白質存在，西方轉漬的結果也顯示含GST的蛋白質不只一個。粗抽液中目標蛋白質為何沒出現以及西方轉漬所呈現的結果則需要更進一步的探討。

Abstract
The study and application of cellular proteins is the main field for research in the post-genome era proteomics provides a powerful tool for understanding the roles of proteins in an organism. Escherichia coli is most frequently as a host cell for the expression of recombinant proteins. Databases related to Escherichia coli updating and approaching to be complete.
This study is to analyze the differences of the proteomes of the host cell Escherichia coli BL21, induced and non-induced recombinant Escherichia coli BL21 (including a recombinant plasmid). Alternatively, we used silver nitrate and silver diammine protocols to detect proteins and finding on the two-dimensional electrophoresis (2-DE) gels. We could detect more proteins by using silver diammine protocol. However, in contrast to silver nitrate protocol, the silver diammine protocol was time consuming and costly. Also, vertical and horizontal streaks were more observable and subject to interfere software analysis. The silver nitrate was thus superior. As the protein redissolving time is increasing to five minutes, the number of spots appeared on the gel was not less than that by using the method of silver diammine.
On 2-DE gels, we have identified two protein spots: gapA (glyceraldehyde 3-phosphatedehydrogenase，MW 35.2K Da , pI 6.58 ) and dnaK (Chaperon Hsp70，MW 69K Da , pI 4.83), by using LC-MS/MS. Protein gapA is one of the housekeeping proteins and can serve as the standard for the normalization of proteins for quantitative analysis.
We did not find the target protein: GST-nanA-Arg5 on both the 2-DE gels of proteins from induced and non-induced recombinant Escherichia coli BL21 cells, even when the amounts of proteins loaded to gels was increased to 500 μg. However, the presence of the target protein on the 2-DE gel of the purified protein was obvious, confirmed by western blotting. The result from western blotting implied that there were several proteins associated with GST tag. We need to investigate more detail on why the target protein in the original extracted solution did not show up on the 2-DE gel and to carry out the experiments of western blotting.

目錄
中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅲ
目錄……………………………………………………………………..Ⅴ
圖目錄…………………………………………………………………...V
表目錄…………………………………………………………………VII
第一章 緒論……………………………………………………………..1
1.1 研究動機與目的…………………………………………....1
1.2 研究原理……………………………………………………4
1.2.1蛋白質定量法…………………………………………4
1.2.2 IEF之研究原理……………………………………….4
1.2.3 SDS-PAGE之研究原理………………………………5
1.2.4 MALDI-TOF質譜研究原理………………………….6
1.3 文獻回顧…………………………………………………...…8
1.3.1 Isoelectric focusing……..………………………….…8
1.3.2 SDS-PAGE………………………………………..…10
1.3.3 銀染…………………………………………………11
1.3.4 二維電泳分析………………………………………13
1.3.5 蛋白膠內酵素水解…………………………………14
1.3.6 MALDI-TOF質譜儀………………………………...15
1.3.7 E.coli 的蛋白體…………………………………..…16
第二章 實驗藥品與設備……………………………………………....18
2.1 實驗藥品……………………………………………….….….18
2.1.1 Escherichia coli 培養以及蛋白質的保存…………...…18
2.1.2蛋白質濃度測定…………………………………………19
2.1.3 IPG strip之rehydration……………………………….…19
2.1.4 SDS-PAGE………………………………………………20
2.1.5銀染………………………………………………………21
2.1.5.1 二胺銀法(silver diammine)…………………………...20
2.1.5.2 硝酸銀法(silver nitrate)…………………….………...22
2.1.6 西方轉漬………………………………………….…….22
2.1.7 蛋白質膠內酵素水解…………………………………..24
2.1.8 peptide extraction………………………………………..24
2.1.9 Matrix製備…………………………………………..…..25
2.2 實驗設備……………………………………………………...26
第三章 實驗步驟與分析方法…………………………………………28
3.1 E. coli BL21的培養及蛋白質保存…………………………28
3.1.1 基因重組菌 E. coli BL21的培養……………………...29
3.1.2 宿主菌 E. coli BL21的培養………………………..….30
3.1.3有誘導之基因重組菌E. coli BL21 的培養……………31
3.2 蛋白質濃度測定……………………………………….….….31
3.2.1 檢量線製作……………………………………..………31
3.2.2 濃度測定…………………………………………….….31
3.3 等電點分離（IEF）………………………………….………32
3.4 SDS-PAGE…………………………………………………….33
3.5 銀染…………………………………………………………...35
3.5.1 硝酸銀法(Silver nitrate)………………………………….35
3.5.2 二胺銀法(silver diammine)………………………………36
3.6 軟體分析(PDquest)…………………………………………...38
3.7 西方轉漬……………………………………………………...39
3.8蛋白質膠內酵素水解…………………………………….……41
3.9 ZipTip純化……………………………………………………42
3.10 MALDI-TOF質譜分析………………………………………43
3.11電灑離子化串聯質譜儀(ESI-MS/MS)分析及蛋白質鑑定…44
第四章 實驗結果與討論………………………………………………45
4.1蛋白質濃度測定……………………………………………….45
4.2不同銀染方法之比較………………………………………….47
4.3 宿主菌以及有無誘導的重組菌之蛋白體的比較……………51
4.4 純化後之蛋白質溶液之二維電泳分析與西方轉漬………….60
4.5 大量蛋白質(加入量為500μg) ………………………………..63
4.6 MALDI-TOF 質譜分析……………………………………...64
4.7 蛋白質鑑定(protein identification)…………………………….67
第五章 結論與建議……………………………………………………68
參考文獻………………………………………………………………..71
附錄.A
附錄.B
圖目錄
圖1.1 蛋白質進行IEF時移動之示意圖(Dr. R A Rastall, 2000)……...5
圖1.2 strip與SDS-PAGE之操作示意圖……………………….………6
圖1.3 MALDI-TOF 質譜儀之原理示意圖…………………………......7
圖4.1 蛋白質檢量線……………………………………………….…..46
圖4.2誘導之重組菌蛋白質的二維電泳膠片以硝酸銀法染色..................................................................................................48
圖4.3 誘導之重組菌蛋白質的二維電泳膠片以二胺銀法染色
……………………………………………………………………49
圖4.4 誘導之重組菌蛋白體二維膠體電泳膠片以硝酸銀法染色……………………………………………………………….50
圖4.5 宿主菌的蛋白體二維電泳膠片以二胺銀法染色……….……..52
圖4.6 重組菌蛋白體之二維電泳膠片以二胺銀法染色……………...53
圖4.7 宿主菌蛋白體之二維電泳膠片以硝酸銀法染色……….……..54
圖4.8 重組菌蛋白體之二維電泳膠片以硝酸銀法染色的結果……...55
圖4.9 加入的蛋白質量為60 μg時，誘導之重組菌蛋白體二維電泳膠片以硝酸銀法染色……………………………………………..58
圖4.10加入的蛋白質量為70 μg時，誘導之重組菌蛋白體二維電泳膠片以硝酸銀法染色……………………………………………..59
圖4.11 純化後之蛋白質溶液的二維電泳分析圖以硝酸銀法染色 (蛋白質含量為(15μg)…………………………………………….60
圖4.12 西方轉漬圖，原始二維膠片來自圖4.13…………………….61
圖4.13 純化後蛋白質溶液之二維電泳膠片使用硝酸銀法，西方轉漬轉印之原始膠片………………………………………………61
圖4.14 樣品加入量為500 μg時，誘導之重組菌的蛋白體二維電泳圖以硝酸銀法染色……………………………………………....63
圖4.15 蛋白質點2之MALDI-TOF質譜圖………………………...65
圖4.16 蛋白質點3之MALDI-TOF質譜圖………………………...65
圖4.17 二維膠片電泳圖中蛋白質點1-4的位置…………………...67
表目錄
表3.1 LB medium………………………………………………………28
表3.2 Low salt washing buffer………………………………………….28
表3.3 Stock buffer………………………………………………………28
表3.4 lysis buffer………………………………………………………..29
表3.5 IEF之program…………………………………………………...33
表3.6配製12.5﹪Separating gel之配方…………………………….....35
表3.7西方轉漬所需buffer………………………………………….…39
表3.8 ZipTip所需溶液…………………………………………...…….42

參考文獻
Andrej S., Matthias W., Ole V., and Matthias M., “Mass Spectrometric Sequencing of Proteins from Silver Stain Polyacrylamide Gels”, Analytiacl Chemistry, 68, 850-858 (1996)
Angelika G., Christian O., Gunther B., Alois H., Burghardt S., Robert W., and Walter W., “The current state of two-dimensional electrophoresis with immobilized pH gradients”, Electrophoresis, 21, 1037-1053 (2000)
Baglioni P., Bini L., Liberatori S., Pallini V., and Marri L., ”Proteome analysis of Escherichia coli W3110 expressing an heterologous sigma factor”, Proteomics, 3, 1060-1065, (2003)
Ben H., Marina G., Mahmoud H., Erna O., John M., Sanne P., and Pier G. R., “Reduction and alkylation of proteins in preparation of two-dimensional map analysis Why, when, and how?”, Elecrophoresis, 22, 2046-2057 (2001)
Bjellqvist B., Hughes G. J., Pasquali C., Paquent N., Ravier F., Sanchez J. C., Frutiger S., and Hochstrasser D., “The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences”, Electrophoresis, 14(10), 1023-1031 (1993)
Bjellqvist B., Pasquali C., Ravier F., Sanchez J. C., and Hochstrasser D., “A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale”, Electrophoresis, 14(12), 1357-1365 (1993)
Bjellqvist B., Sanchez J. C., Pasquali C., Ravier F., Paquent N., Frutiger S., Hughes G. J., and Hochstrasser D., “Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins”, Electrophoresis, 14(12), 1375-1378 (1993)
Choe L. H., and Lee K. H., “A comparison of three commercially available isoelectric focusing units for proteome analysis: The Multiphor, the IPGphor and the Protean IEF cell”, Electrophoresis, 21, 993-1000 (2000)
Christine H., Jean-Charles S., Luisa T., Amos B., Denis F. H., and Ron D. A., “Current status of the SWISS-2DPAGE database”, Nucleic Acids Research, 26, 1 332-333 (1998)
Christine H., J.-C. Sanchez, Luisa T., Amos B., and Ron D.A., “The SWISS-2DPAGE database what has changed during the last year”, Nucleic Acids Research, 27, 1, 289-291 (1999)
Christine H., J.-C. Sanchez, Luisa T., Amos B., and Ron D.A., “The 1999 SWISS-2DPAGE database update”, Nucleic Acids Research, 28, 1, 286-288 (2000)
Christian P., Severine F., Marc R. W., Graham J. H., Ron D. A., Amos B., Dominique S., Jean-Charles S., and Denis F. H., “Two-dimensional gel electrophoresis of Escherichia coli homogenates: The Escherichia coli SWISS-2DPAGE database”, Electrophoresis, 17, 547-555 (1996)
Christian S., Stephanie L., Zaoming P., Xin-Ping L., Johannes S., and Peter J., “Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry”, Electrophoresis, 19, 918-927 (1998)
David A., Kathy L. O., Kathleen L. K., and John T. S., “An Integrated Approach to Proteome Analysis Identification of Proteins Associated”, Analytical Biochemistry, 258, 1-18 (1998)
Elisabet G., Kajsa T., Thomas L., Pia D., Karl-Anders K., and Carol L. N., “Identification of proteins from Escherichia coli using two-dimensional semi-preparative electrophoresis and mass spectrometry”, Rapid Communications in Mass Spectrometry, 15, 428-432 (2001)
Ejvind M., Thomas N. K., Henrik V., and Angelika G., “Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorptionionization-time of flight analysis”, Proteomics, 1, 1359-1363 (2001)
Farzin G., Catherine R. W., Denise A. M., Brain S. I., and Sheenah M. M., “Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity”, Electrophreis, 20, 601-605 (1999)
Farzin G., Catherine R.W., Denise A. M., Brian S. I., and Sheenah M. M., “Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: A method for the removal of silver ions to enhance sensitivity”, Electrophoresis, 20, 601-605 (1999)
Fatma M., Michel-Yves M., Alain G., Jean-Yves L., Abdelkader B., and Francois B.,” Cold adaptation of Escherichia coli: microbiological and proteomic approaches”, International Journal of Food Microbiology, 89, 171— 184 (2003)
Fountoulakis M. and Gasser R., “Proteomic analysis of the cell envelope fraction of Escherichia coli”, Amino Acids, 24, 19-41 (2003)
Frederic T.M., Ophe.l.D., Abdelkader N., Brigitte M., and Jean-Claude L., “A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode”, MS Microbiology Letters, 215, 7-14 (2002)
Georgia P., Greg G. Q., and David H. S., “On the mechanism of background silver staining during sodium dodecyl sulphate-polyacrylamide gel electrophoresis”, Electrophoresis, 20, 2039-2045 (1999)
Gorg A., Postel W., Domscheit A., and Gunther S., “Two-dimensional electrophoresis with immobilized pH gradient of leaf proteins from barley (Hordeum vulgare): Method, reproducibility and genetic aspects”, Electrophoresis, 9, 681-692 (1988)
Gorg A., Postel W., and Gunther S., “The current state of two-dimensional electrophoresis with immobilized pH gradients”, Electrophoresis, 9, 531-546 (1988)
Gorg A., Postel W., Gunther S., Weser J., Strahler J. R., Hanash S. M., Somerlot L., and Kuick R., “Approach to stationary two-dimensional pattern: Influence of focusing time and Immobiline/carrier ampholytes concentrations”, Electrophoresis, 9, 37-46 (1988)
Hatzimanikatis V., Choe L. H., and Lee K. H., “Proteomics: theoretical and experimental considerations”, Biotechnology Progress, 15, 312-318 (1999)
Hiroyuki K., Takeshi N., and Yoshiya O., “Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorptionionization time-of-flight mass spectrometry”, Rapid Communications in Mass Spectrometry, 15, 1416-1421 (2001)
Hochstrasser D. F., Harrington M. G., Hochstrasser A. C., Miller M., and Merril C. R., “Methods for increasing the resolution of two-dimensional protein electrophoresis”, Analytical Biochemistry, 173, 424-435 (1988)
Hochstrasser D. F., Patchornik A., and Merril C. R., “Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining”, Analytical Biochemistry, 173, 412-423 (1988)
Holland R. D., Duffy C. R., Rafii F., Sutherland J. B., Heinze T. M., Holder C. L., Voorhees K. J., and Lay Jr J. O., “Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells”, Analytical Chemistry, 71, 3226-3230 (1999)
Jacques M. P. D., Jennifer A. B., Alenxander S., Yvan W., Nadia W., Salvo P., and Garry L. C., “Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins”, Proteomics, 3, 1418-1424 (2003)
Lauren M. S., Dawn M. S., Brian S., D. M. Barnhart., and Joan L. S., “ProteoSilve pH dependent expression of periplasmic proteins in E. coli”, Journal of Bacteriology, Aug, 4246-4258 (2002)
Jun J. Y., Robin W., Tom B., Rachel A. H., Jules A. W., Colin H. W., and Michael J. D., “A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorptionionization and electrospray ionization- mass spectrometry”, Electrophoresis, 21, 3666-3672 (2000)
Jun X. Y., Angelica T. D., Robin W., Tim S., Steve L., and Sue F., “Fluorecence two-dimensional difference gel electrophoresis and mass spectrometry based proteomics analysis of Escherichia coli “, Proteomics, 2, 1682-1698 (2002)
Kabir Md. M., and Shimizu K., “Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production”, Applied Microbiology and Biotechnoogy, 62,244-255 (2003)
Karin D., Gorji M., Eberhard D., Gerald S., Franz C., Monika C.-P., and Karl B., “Monitoring of protein profiles for optimization of recombinant fermentation processes using public domain databases”, Electrophoresis, 24, 303-310 (2003)
Kathleen M. C., Julie C. N., John C. J., and David A., “Similarity of the Escherichia coli proteome upon completion of different biopharmaceutical fermentation processes”, Proteomics, 1, 1133-1148 (2001)
L. Peng, and K. Shimizu, “Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement”, Applied Microbiology and Biotechnology, 61, 163-178 (2003)
Lella H. C., Wilfred C., and Kelvin H. L., “Proteome analysis of factor for inversions timulation (Fis) overproduction in E. coli”, Electrophoresis, 20, 798-805 (1999)
Mark P. M., Ben R. H., Martin B., and Thierry R., “Proteomic analysis of the Escherichia coli outer membrane”, European Journal of Biochemistry, 267, 2871-2881 (2000)
Martine C. and Brian T. C., “A Robust, Detergent-Friendly Method for Mass Spectrometric Analysis of Integral Membrane Proteins”, Analytical Chemistry, 72, 5655-5658 (2000)
Mee-Jung H., Sang S. Y., and Sang Y. L., “Proteomics of recombinant E. coli XL1-Blue”, Journal of Bacteriology, ?, 301-308 (2001)
Merril C. R., Goldman D., and Van Keuren M. L., “Simplified silver protein detection and image enhancement methods in polyacrylamide gels”, Electrophoresis, 3, 17-23 (1982)
Merril C. R., Switzer R. C., and Van Keuren M. L., “Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain”, Proceedings of the National Academy of Sciences of the United States of America, 76, 4335-4339 (1979)
Nandakumar M. P., Jie S., Babu R., and Mark R. M., “Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via NaOH for two-dimensional electrophoresis”, Journal of Proteome Research, 2, 89-93 (2003)
Oakley B. R., Kirsch D. R., and Morris N. R., ”A simplified utrasensitive silver stain for detecting proteins in polyacrylamide gels”, Analytical Biochemistry, 105, 361-363 (1980)
O’Farrell P. H., “High resolution two-dimension electrophoresis of proteins”, Journal of Biological Chemistry, 250, 4007-4021 (1975)
P. S. Lee and K. H. Lee , ”Escherichia coli─A Model System That Benefits From and Contributes to the Evolution of Proteomics”, Biotechnology and Bioengineering, 84, 7, 801-814 (2003)
Rabilloud T., ”Mechanisms fo protein silver staining in polyacrylamide gels: A 10-year synthesis”, Electrophoresis, 11, 785-794 (1990)
Rabilloud T., ”A comparison between low background silver diammine and silver nitrate protein stains”, Electrophresis, 13, 429-439 (1992)
Paola B., Luca B., and Sabrina L., “Proteome analysis of Escherichia coli W3110 expressing a heterologous sigma factor”, Proteomics, 3, 1060-1065 (2003)
Pferdeort V. A., Wood T. K., and Reardon K. F., “Proteomic changes in Escherichia coli TG1 after metabolic engineering for enhanced tricholoethene biodegradation”, Proteomics, 3, 1066-1069, (2003)
Phillip C., “Review Characterisation of bacterial proteomes by two-dimensional electrophoresis”, Analytica Chimica Acta, 372, 121-145 (1998)
Rosslyn M. B., Conor O’Byrne, Ian R. B., and Phillip C., “Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns”, Proteomics, 3, 764-766 (2003)
Reiko M., Hikari T., Tsutomu F., Mika K., Noriko S., and Kimie M., “In situ alkylation with acrylamide for identification of cysteinyl residues in proteins during one- and two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis”, Proteomics, 2, 1672-1681 (2002)
Robert H., Brenda L. G., Kenneth M. S., and Diane C. D., “Application of zwitterionic detergents to the solubilization of integral membrane proteins for two-dimensional gel electrophoresis and mass spectrometry”, Proteomics, 2, 1479-1488 (2002)
Seow T. K., Korke R., Liang R. C. M. Y., Ong S. E., Ou K., Wong K., Hu W. S., and Chung M. C. M., “Proteomic investigation of metabolic shift in mammalian cell culture”, Biotechnology Progress, 17, 1137-1144 (2001)
Seow T. K., Ong S. E., Liang R. C. M. Y., Ren E. C., Chan L., Ou K., and Chung M. C. M., “Two-dimensional electrophoresis map of the human hepatocellular carcinoma cell line, HCC-M, and identification of the separated proteins by mass spectrometry”, Electrophoresis, 21, 1787-1813 (2000)
Sheng Z. Colleen K.V.P. and Jack D. H., ”Automated chip-based nanoelectrospray-mass spectrometry for rapid identification of proteins separated by two-dimensional gel electrophoresis”, Ectrophoresis , 24, 3620-3632 (2003)
Sophie R., Sylvie L., Mireille C., Alain V. D., Emmanuelle L.-W., and Thierry R., “About the mechanism of interference of silver staining with peptide mass spectrometry”, Proteomics, 4, 909-916 (2004 )
Sung H. Y., Han M.-J., Sang Y. L., Ki J. J., and Yoo J.-S., “Combined transcriptome and proteome analysis of Escherichia coli during high cell density culture”, Biotechnolgy and Bioengineering, 81, 753-767 (2003)
Sylvie L., Veronique S., and Thierry R., “Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis”, Proteomics, 3, 249-253 (2003)
Takesbi Y., Reiko M., Hikari T., Hiroto K., and Kimie M., “Improvement of automatic in-gel digestion by in situ alkylation of proteins”, Journal of Biomolecular Techniques, 14, 191-196 (2003)
Thierry R., “A comparison between low background silver diammine and silver nitrate protein stains”, Electrophoresis, 13, 429-439 (1992)
Tonella T., Hoogland C., Binz P.-A., and Appel R. D., ”New perspectives in the Escherichia coli proteome investigation”, Proteomics, 1, 409-423 (2001)
Tremoulet F., Duche O., Namene A., and Martinie B., “A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode”, FEMS Microbiology Letters, 215, 7-14 (2002)
Valerie A. P., Thomas K. W., and Kenneth F. R. “Proteomic changes in Escherichia coli TG1 after metabolic engineering for enhanced trichloroethene biodegradation”, Proteomics, 3, 1066-1069 (2003)
Valerie C. W., and Ian H.-S., “small gene-products in E. coli K12”, FEMS Microbiology Letters, 169, 375-382 (1998)
Vassily H., Leila H. C., and Kelvin H. L., “Proteomics: Theoretical and Experimental Considerations”, Biotechnology Progress, 15, 312-318 (1999)
Yan J. X., Wait R., Berkelman T., Harry R. A., Westbrook J. A., Wheeler C. H., and Dunn M. J., “A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry”, Electrophoresis, 21, 3666-3672 (2000)
Zhang N. and Li L.,”Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two-layer sample preparation”, Rapid Communications in Mass Spectrometry, 18, 889-896 (2004)