Current two‐dimensional electrophoresis technology for proteomics

Proteomics - Tập 4 Số 12 - Trang 3665-3685 - 2004
Angelika Görg1, Walter Weiss, Michael J. Dünn
1Department of Proteomics, Technische Universität München, Freising-Weihenstephan, Germany. [email protected]

Tóm tắt

Abstract

Two‐dimensional gel electrophoresis (2‐DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g.multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2‐DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2‐DE enables the separaration of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post‐translational modifications. This is in contrast to liquid chromatography‐tandem mass spectrometry based methods, which perform analysis on peptides, whereMrand pIinformation is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2‐DE technology with IPGs (Görget al.,Electrophoresis2000,21, 1037–1053), has overcome the former limitations of carrier ampholyte based 2‐DE (O'Farrell,J. Biol. Chem. 1975,250, 4007–4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5–12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow‐overlapping IPGs provide increased resolution (δpI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2‐DE can resolve more than 5000 proteins simultaneously (˜2000 proteins routinely), and detect and quantify < 1 ng of proteinperspot. In this article we describe the current 2‐DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2‐DE with IPGs; protein detection and quantitation; computer assisted analysis of 2‐DE patterns; protein identification and characterization by MS; two‐dimensional protein databases.

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Tài liệu tham khảo

10.1002/elps.1150090913

10.1002/(SICI)1522-2683(20000401)21:6<1037::AID-ELPS1037>3.0.CO;2-V

10.1038/nature01511

10.1002/pro.5560070320

10.1021/ac980320p

10.1038/13690

10.1002/1097-0061(20000630)17:2<81::AID-YEA22>3.0.CO;2-Z

10.1038/10890

10.1038/85686

10.1016/S0165-9936(03)00508-9

10.1016/S0958-1669(99)80004-4

10.1016/S1367-5931(00)00167-8

10.1128/MMBR.66.1.39-63.2002

10.1016/S1367-5931(01)00275-7

10.1016/S1389-0344(01)00095-8

10.1002/1615-9861(200201)2:1<3::AID-PROT3>3.0.CO;2-R

10.1016/S0021-9258(19)41496-8

10.1016/S0021-9258(19)41192-7

10.1007/BF00281458

Görg A., 1985, Am. J. Hum. Genet., 37, 922

10.1002/1522-2683(20000701)21:13<2610::AID-ELPS2610>3.0.CO;2-H

10.1002/1522-2683(200108)22:14<2865::AID-ELPS2865>3.0.CO;2-Y

10.1038/349545a0

10.1002/elps.1150180306

10.1002/elps.1150190850

Görg A., 1999, Methods Mol. Biol., 112, 197

10.1002/(SICI)1522-2683(19990101)20:4/5<712::AID-ELPS712>3.0.CO;2-Y

10.1002/1615-9861(200206)2:6<727::AID-PROT727>3.0.CO;2-2

10.1002/pmic.200300720

Dunn M. J., 1993, Gel Electrophoresis: Proteins, 41

10.1002/(SICI)1522-2683(19990101)20:4/5<660::AID-ELPS660>3.0.CO;2-Q

Rabilloud T., 1999, Methods Mol. Biol., 112, 9

10.1002/pmic.200300471

Dunn M. J., 2001, Proteomics – from protein sequence to function, 43

10.1002/1522-2683(200206)23:11<1745::AID-ELPS1745>3.0.CO;2-A

10.1002/mas.10075

10.1002/elps.1150091106

10.1016/0003-2697(89)90008-0

10.1002/pmic.200300450

10.1002/pmic.200390057

Simpson R. J., 2004, Eur. Pharm. Rev., 1, 25

10.1002/elps.1150190526

10.1002/elps.1150191108

10.1002/(SICI)1522-2683(19990101)20:4/5<705::AID-ELPS705>3.0.CO;2-Q

10.1002/pmic.200390037

10.1002/(SICI)1522-2683(20000401)21:6<1054::AID-ELPS1054>3.0.CO;2-8

10.1006/abio.2000.4514

Thoenes L., 2003, Proteomic Forum 03, 93

10.1021/pr025564b

10.1002/(SICI)1522-2683(19990101)20:4/5<826::AID-ELPS826>3.0.CO;2-A

10.1002/yea.320110702

10.1002/(SICI)1522-2683(19990101)20:4/5<701::AID-ELPS701>3.0.CO;2-5

10.1002/pmic.200300492

10.1002/elps.1150190540

10.1006/abio.1999.4203

10.1002/(SICI)1522-2683(20000401)21:6<1202::AID-ELPS1202>3.0.CO;2-I

10.1126/science.274.5289.998

10.1002/1615-9861(200110)1:10<1200::AID-PROT1200>3.0.CO;2-Q

10.1016/0009-9120(95)02017-9

10.1161/01.RES.0000071748.48338.25

10.1016/S0378-4347(98)00314-4

10.1016/0076-6879(74)31078-6

10.1002/elps.1150180519

10.1002/yea.320110602

10.1006/abio.2000.4868

10.1002/elps.11501301167

10.1002/elps.11501401126

10.1002/elps.1150190539

10.1083/jcb.93.1.97

10.1002/1522-2683(20001001)21:16<3517::AID-ELPS3517>3.0.CO;2-H

10.1016/S0021-9673(98)00929-7

Hochstrasser A. C., 1991, Appl. Theor. Electrophor., 1, 333

10.1016/0003-2697(88)90472-1

10.1002/1615-9861(200209)2:9<1254::AID-PROT1254>3.0.CO;2-A

10.1002/1522-2683(200011)21:17<3639::AID-ELPS3639>3.0.CO;2-V

10.1002/1615-9861(200201)2:1<58::AID-PROT58>3.0.CO;2-G

10.1002/1522-2683(200105)22:9<1603::AID-ELPS1603>3.0.CO;2-I

10.1002/1615-9861(200212)2:12<1652::AID-PROT1652>3.0.CO;2-3

Macko V., 1969, Hoppe‐Seylers Z. Physiol. Chem., 350, 917

10.1111/j.1749-6632.1973.tb47527.x

10.1111/j.1749-6632.1973.tb47518.x

10.1016/0092-8674(77)90176-3

10.1016/0165-022X(82)90013-6

10.1002/elps.11501501182

10.1002/elps.11501601320

10.1002/elps.11501401212

Görg A., 2004, Purifying Proteins for Proteomics, 391

Islam R., 1998, Sci. Tools, 3, 14

10.1002/elps.1150181133

10.1002/elps.11501401209

10.1002/elps.200390163

Righetti P. G., 1990, Immobilized pH Gradients: Theory and Methodology, 53

10.1002/elps.1150110207

10.1002/elps.1150121205

10.1016/0165-022X(80)90008-1

10.1007/978-3-642-57105-3_4

10.1016/0003-2697(91)90475-9

10.1002/elps.11501501223

10.1002/elps.1150180305

10.1002/elps.11501601183

10.1002/elps.1150120910

10.1073/pnas.160270797

10.1002/elps.1150110310

Klose J., 1999, Methods Mol. Biol., 112, 147

10.1002/elps.1150180807

10.1002/elps.1150090906

10.1002/1615-9861(200211)2:11<1630::AID-PROT1630>3.0.CO;2-N

10.1002/pmic.200300589

10.1002/(SICI)1522-2683(20000401)21:6<1104::AID-ELPS1104>3.0.CO;2-C

10.1002/elps.1150190847

10.1002/elps.1150181413

10.1002/elps.1150180124

10.1074/mcp.M300070-MCP200

10.1002/elps.1150080207

10.1002/1522-2683(20000801)21:14<3035::AID-ELPS3035>3.0.CO;2-F

Zhou S., 2004, Proteomics, 4

Görg A., 1999, Methods Mol. Biol., 112, 235

10.1016/0003-2697(78)90230-0

10.1038/227680a0

10.1016/0003-2697(91)90350-3

10.1016/0003-2697(87)90587-2

10.1002/elps.1150191046

10.1002/(SICI)1522-2683(20000401)21:6<1123::AID-ELPS1123>3.0.CO;2-E

10.1016/S1570-0232(02)00043-0

10.1021/ac002709u

10.1007/978-3-642-57105-3_5

10.1002/elps.1150090603

10.1002/elps.200305844

Adams L. D., 1990, Appl. Theor. Electrophor., 1, 279

Fernandez‐Patron C., 1992, Biotechniques, 12, 564

10.1002/(SICI)1522-2683(19990101)20:4/5<732::AID-ELPS732>3.0.CO;2-Q

10.1126/science.6162199

10.1016/0003-2697(80)90470-4

10.1016/0003-2697(86)90222-8

10.1002/1615-9861(200107)1:7<835::AID-PROT835>3.0.CO;2-2

10.1021/ac950914h

Mortz E., 2001, Proteomics, 1, 359, 10.1002/1615-9861(200111)1:11<1359::AID-PROT1359>3.0.CO;2-Q

Link A. J., 1999, Methods Mol. Biol., 112, 285

Patterson S. D., 1993, Biotechniques, 15, 1076

10.1080/07391102.1999.10508356

Drukier A. K., 2000, From Genome to Proteome. 4th Siena Meeting, 123

10.1002/1098-2787(2001)20:1<1::AID-MAS1001>3.0.CO;2-J

10.1002/elps.1150090709

10.1006/abio.1993.1082

10.1002/1615-9861(200205)2:5<486::AID-PROT486>3.0.CO;2-X

10.1002/1615-9861(200104)1:5<699::AID-PROT699>3.0.CO;2-C

10.1002/pmic.200300587

10.1016/S1367-5931(02)00009-1

10.2174/1389557043487330

10.1038/nbt0303-255

10.1016/S0021-9673(98)00115-0

Packer N. H., 1999, Methods Mol. Biol., 112, 341

10.1002/1615-9861(200102)1:2<223::AID-PROT223>3.0.CO;2-B

10.1002/pmic.200300494

10.1002/1615-9861(200102)1:2<194::AID-PROT194>3.0.CO;2-K

10.1002/pmic.200300434

10.1002/elps.200390069

10.1002/pmic.200390006

10.1002/pmic.200300459

10.1002/pmic.200300439

10.1016/j.copbio.2003.12.001

10.1111/j.1432-1033.1983.tb07286.x

10.1002/1615-9861(200203)2:3<229::AID-PROT229>3.0.CO;2-K

10.1002/(SICI)1522-2683(19990801)20:11<2225::AID-ELPS2225>3.0.CO;2-8

10.1016/S0021-9258(18)83728-0

Dunn M. J., 1992, Microcomputers in Biochemistry: A Practical Approach, 215, 10.1093/oso/9780199632534.003.0007

10.1002/pmic.200300731

10.1021/ac991363o

10.1002/(SICI)1522-2683(19990101)20:4/5<664::AID-ELPS664>3.0.CO;2-A

Westermeier R., 2004, Protein Purification, 3rd Edition

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Proteomics

Tập 4 Số 12

3665-3685

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