Improved comparative proteome analysis based on two-dimensional gel electrophoresis.
The purpose of this study was to test the extent to which differences in spot intensity can be reliably recognized between two groups of two-dimensional electrophoresis gels (pH 4-7, visualized with ruthenium fluorescent stain) each loaded with different amounts of protein from rat brain (power analysis). Initial experiments yielded only unsatisfactory results: 546 spots were matched from two groups of 6 gels each loaded with 200 microg and 250 microg protein, respectively. Only 72 spots were higher (p<0.05), while 58 spots were significantly lower in the 250-microg group. The construction of new apparatuses that allowed the simultaneous processing of 24 gels throughout all steps between rehydration and staining procedure considerably lowered the between-gel variation. This resulted in the detection of significant differences in spot intensities in 77-90% of all matched spots on gel groups with a 25% difference in protein load. This applied both when protein from 24 biological replicates was loaded onto two groups of 12 gels and when two pooled tissue samples were each loaded onto 6 gels. At a difference of 50% in protein load, more than 90% of all spots differed significantly between two experimental groups.
Eravci
Murat
M
Department of Radiology and Nuclear Medicine (Radiochemistry), Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.|A+M Proteome Science, Berlin, Germany.
Fuxius
Sandra
S
Department of Radiology and Nuclear Medicine (Radiochemistry), Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.|A+M Proteome Science, Berlin, Germany.
Broedel
Oliver
O
A+M Proteome Science, Berlin, Germany.
Weist
Stephanie
S
A+M Proteome Science, Berlin, Germany.
Eravci
Selda
S
A+M Proteome Science, Berlin, Germany.
Mansmann
Ulrich
U
Department of Medical Informatics, Biometry and Epidemiology, University of Munich, Germany.
Schluter
Hartmut
H
Department of Internal Medicine IV, Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.
Tiemann
Joachim
J
Department of Internal Medicine IV, Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.
Baumgartner
Andreas
A
Department of Radiology and Nuclear Medicine (Radiochemistry), Charité Universitätsmedizin, Campus Benjamin Franklin, Berlin, Germany.|A+M Proteome Science, Berlin, Germany.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D008297:Male; D020543:Proteome / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012984:Software
D020543:Proteome