High reproducibility of large-gel two-dimensional electrophoresis

被引:44
作者
Challapalli, KK
Zabel, C
Schuchhardt, J
Kaindl, AM
Klose, J
Herzel, H
机构
[1] Humboldt Univ, Inst Theoret Biol, D-10115 Berlin, Germany
[2] Charite Univ Med Berlin, Inst Human Genet, Berlin, Germany
[3] Charite Univ Med Berlin, Microdiscovery GmbH, Berlin, Germany
[4] Charite Univ Med Berlin, Dept Neuropediat, Berlin, Germany
关键词
Huntington's disease; proteomics; reproducibility; two-dimensional gel electrophoresis; variability;
D O I
10.1002/elps.200405979
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Two-dimensional gel electrophoresis (2-DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2-DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER(TM). Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2-DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low-intensity spots. This indicates a high technical reproducibility of the 2-DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two-dimensional (2-D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot-by-spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.
引用
收藏
页码:3040 / 3047
页数:8
相关论文
共 19 条
[1]   INTERLABORATORY REPRODUCIBILITY OF YEAST PROTEIN-PATTERNS ANALYZED BY IMMOBILIZED PH GRADIENT 2-DIMENSIONAL GEL-ELECTROPHORESIS [J].
BLOMBERG, A ;
BLOMBERG, L ;
NORBECK, J ;
FEY, SJ ;
LARSEN, PM ;
LARSEN, M ;
ROEPSTORFF, P ;
DEGAND, H ;
BOUTRY, M ;
POSCH, A ;
GORG, A .
ELECTROPHORESIS, 1995, 16 (10) :1935-1945
[2]   Proteomics in 2002: A year of technical development and wide-ranging applications [J].
Figeys, D .
ANALYTICAL CHEMISTRY, 2003, 75 (12) :2891-2905
[3]   Extracting information from cDNA arrays [J].
Herzel, H ;
Beule, D ;
Kielbasa, S ;
Korbel, J ;
Sers, C ;
Malik, A ;
Eickhoff, H ;
Lehrach, H ;
Schuchhardt, J .
CHAOS, 2001, 11 (01) :98-107
[4]  
Klose J, 1999, METH MOL B, V112, P147
[5]   Genetic analysis of the mouse brain proteome [J].
Klose, J ;
Nock, C ;
Herrmann, M ;
Stühler, K ;
Marcus, K ;
Blüggel, M ;
Krause, E ;
Schalkwyk, LC ;
Rastan, S ;
Brown, SDM ;
Büssow, K ;
Himmelbauer, H ;
Lehrach, H .
NATURE GENETICS, 2002, 30 (04) :385-393
[6]  
Klose J, 1999, METH MOL B, V112, P67
[7]   2-DIMENSIONAL ELECTROPHORESIS OF PROTEINS - AN UPDATED PROTOCOL AND IMPLICATIONS FOR A FUNCTIONAL-ANALYSIS OF THE GENOME [J].
KLOSE, J ;
KOBALZ, U .
ELECTROPHORESIS, 1995, 16 (06) :1034-1059
[8]   Reproducibility of polypeptide spot positions in two-dimensional gels run using carrier ampholytes in the isoelectric focusing dimension [J].
Lopez, MF ;
Patton, WF .
ELECTROPHORESIS, 1997, 18 (3-4) :338-343
[9]  
Mahon P, 2001, ELECTROPHORESIS, V22, P2075, DOI 10.1002/1522-2683(200106)22:10<2075::AID-ELPS2075>3.0.CO
[10]  
2-C