Quantitative Analytical Techniques of Proteins Based on Two-Dimensional Gel Electrophoresis

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1 PROGRESS IN CHEMISTRY Vol. 18 No. 4 Apr., ( ; ; ) 2DE,, 2DE,, 2DE ;, (DIGE) ;, 2DE : O ; Q51 : A : X(2006) Quantitative Analytical Techniques of Proteins Based on Two-Dimensional Gel Electrophoresis Long Xiaohui 1 Mo Zhihong 2 Zhang Yaozhou (1. College of Life Science, Zhejiang University, Hangzhou , China ; 2. College of Chemistry and Engineering, Chongqing University, Chongqing , China ; 3. College of Life Science, Zhejiang University of Science and Technology, Hangzhou , China) Abstract Quantitative analytical techniques of proteins based on two2dimensional gel electrophoresis,depending on the comparing of altered levels of proteins between different samples to obtain quantitative changes, are informative for comprehesive understanding functions and biological mechanism of proteins in cells. Conventional methods in which protein samples are separated on individual gels are introduced, and shortcomings of these methods that limit their application in general 2DE analysis are discussed. More approachs including differential in2gel electrophoresis (DIGE), differential exposure approach, and stable isotope approach are stated. The latter methods permitting separation on a single gel containing mixed samples, have superior power in quantitative analysis compared to conventional methods. Various quantitative protocols, advantages and limitations of these protocols as well as further development of quantitative techniques based on 2DE are discussed. Key words two2dimensional gel electrophoresis ; quantitative techniques ; DIGE ; isotope2coded method [1,2 ],,,, ( two2 dimensional gel electrophoresis, 2DE) ( mass : , : (No ) 3 3 e2mail com

2 ] spectrometry,ms),2de [3, 2DE ( ) [6,7 ], ( SYPRO Ruby) ( 3 H 35 S), [14 ] [15 ] MS, ngΠ Julie [16 ] ( G2250), 35 S G2250, G2250 Cawood [17 ] [8,9 ],, 4 (Asp Ala Leu Gly) G2250, [10 ] 2DE, mrna,, [18 ], 2 [11 ], 20 50ngΠ, ; [19 ] 4, 0. 1ngΠ, ( PI), ( M r ),, 2DE, ( isoelectric focusing, IEF) SDS2, PI M r, 2DE ( in vivo), Leu [20 ] 32 S2Met [21 ] [12,13 ], X,, (, 2DE ), (multiphoton detection,mpd) [22 ], 7 8 ( X 3 ) MS 1 2DE 2DE SYPRO Ruby [23 ], (post2electrophoresis) 2DE, 1 2ngΠ [24 ] SYPRO Ruby 2,, ; MS ;

3 476 18,, 2 ( differential in2gel electrophoresis, DIGE) 2,, [25 ] DIGE,, 2 1 Cy3 Cy5, Fig. 1 Chemical structure of Cy3 and Cy5 dyes 211 Lys DIGE Cy2 [31 ] Cy2 DIGE, ( pre2, Cy3Π5 [26,27 electrophoresis) ] N2,Cy2 Cy3, Cy5, Lys DIGE (Lys) 2 ( Lys DIGE ),, 3 % 5 %, 2DE 2DE,, (Cy3Π5 540Π620 nm 590Π680, nm ), Zhou [32 ] 1 500,, ;, M r 2 (, ) Cy3Π5 ( 434Π464Da) : ( ), 1 ( SYPRO Ruby CBB ) M r, MS ; Lys, Cys DIGE, PI 2. 2 Cys DIGE Kolkman [28 ] DIGE DIGE, Zhou [32 ] Saccharomyces cerevisiae Cys DIGE, DIGE Cy3Π5 ( ; ) Cys ( Lys) [29 ] [30 ],, Cys DIGE, ( 011ngΠ 1ngΠ )

4 4 477, 25 % 2DE 2DE [36 ] ;, [33 ] Shaw [34 ] 80 %, (Lys ) [34 ],, Cys MS [35 ], 2DE 2DE, 2,, M r (Cy3ΠCys) (Cy3ΠLys) Da Kondo [35 ] 2DE [34, ] 1 500,, % 20 % DIGE ( in vivo ) 14 C2 ( 14 C2Leu) 3 H2Leu, Cys Lys 2DE, [36 ] 2DE IEF PVDF, [37, Cys, ] X, Cys Π, He2Ne 2DE, ;,, 5 %, 5 ; Bernhardt [38 ], 35 S2 ( 35 S2Met),, 35 S2Met 4 2 2DE : (A) Cy3, (B) [34 ], (C) Fig. 2 2DE images of liver homogenate : (A) Cy3 minimal dye label, (B) Cy3 saturation dye label and (C) silver stain [34 ], DIGE,, MS 2DE,, MS [39 ], 4. 1 [ 2 H 0 ]Π[ 2 H 3 ]2 [ 2 H 0 ]Π[ 2 H 3 ]2 2DE

5 [40 ] ( isotope2coded affinity tag, ICAT) [41 ] ICAT 3 : (1) ; (2), ICAT 8 ( [ 2 H 8 ]2ICAT), ( [ 2 H 0 ]2 ICAT) ; (3),,,, Cys MS(MSΠMS) MS ;MSΠMS Cys, ICAT 2DE,, 2DE ;, ICAT 2DE ;, 2DE, ICAT 3 (A) [ 2DE,, 2 H 0 ]Π[ 2 H 3 ]2 [ 2 H 0 ]Π[ 2 H 3 ]2 [ 2 H 0 ]Π[ 2 [42 ] MALDI2MS, (B) (A) H 8 ]2ICAT, ICAT : Π ( [ 2 H 0 ]Π[ 2 H 3 ]2 transferrin taken from rat sera alkylated with [ 2 H 0 ]2 and ) [ 2 H 3 ]2acrylamide, (B) is short intervals taken from (A) [42 ] 2DE, MS [ 2 H 0 ]Π[ 2 H 3 ] [ 2 H 0 ]Π[ 2 H 4 ]2, 3 MS ( ) 85 %, ( mπz) 3 ( Lys 2 ) 100 %, 100 % ( Cys ) 3 [ 2 H 0 ]Π [ 2 H 4 ]22 42 (vinylpyridines,vps) (MALDI2TOF2MS) ( [ 2 H 0 ]Π [ 2 H 3 ]2 ),A,B A Fig. 3 (A) MALDI mass spectrum of an in situ digest of [ 2 H 0 ]Π[ 2 H 3 ]2 [44 2DE ] VPs, B ( A, A 3 ) mπz 3, ( ) [42 ] [44 ],, [ 2 H 0 ]Π[ 2 H 3 ]2, Cys ( Lys 2 [43 ] IEF PI ) (, ) ;,VPs 100 % Cys 80 % 85 % [44 ] ;VPs Cys Lys ; 2 %SDS 15min, VPs 2DE 100 % [43 ], SDS IEF VPs 4,,, Π ([ 2 H 0 ]Π[ 2 H 4 ])

6 4 479 sample A reduction alkylation with [ 2 H 0 ]2VPs sample B dialysis 2D-gel analysis spot excision digestion reduction alkylation with [ 2 H 4 ]2VPs analysis of quantifying and indentification by MS and MSΠMS 4 [ 2 H 0 ]Π[ 2 H 4 ]2VPs 2DE Fig. 4 Scheme for differential labeling of two samples with [ 2 H 0 ]Π[ 2 H 4 ]2VPs (alkylation of Cys residues) 100 %( 2 ),, ;,, PDQuest [48,49 ] [50 ] VPs 2DE, Choe [53 ] MS 100 % 2, 4 Cys, 3 95 % ( coefficient of ( in vitro) 2DE variation,cv) 0152, 30 %, [45 ] [54, ] : 5 2DE ImageMaster ; Phoretix 2D Evolution 2DE ; ProgenesisWorkstation CV [55 ], 2DE CCD 2DE, [46, ] :, ; ; ( differentially regulated ; proteins,drp), [56 ; ] 2DE 2DE ;MS, ( SwissProt,, PSORT TrEMBL, NCBlnr ), [57 ], Melanie PDQuest Phoretix Progenesis 6 [47 ] ( ), DIGE, Decyder (5,6) Melanie23 Z3 [51,52 ],,,, 2DE,, 4 5 ;,

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