CE-based sample quality assessment prior to 2-D gel electrophoresis: towards the standardization of gel-based proteomics.
2-DE remains one of the most commonly used separation techniques for complex protein mixtures. This article describes a new approach to 2-DE sample assessment using SDS capillary gel electrophoresis (in Beckman Coulter sieving medium) combined with multi-pixel detection. The performance of this platform was investigated using protein samples prepared for 2-DE. The capability to characterize 2-DE sample was tested and the results show that the repeatability of peak migration time and intensity are better than 2% RSD. The system gives good resolution, accurate molecular mass assignment, as well as absolute and relative quantification of proteins. Notably, this study also demonstrates the use of this platform to screen the quality of simple and complex 2-DE samples. Implementation of this technique in the proteomics workflow will not only improve the success rate of 2-DE, but will also enable sample verification before 2-DE and allow the relative quantification of proteins. The validation of differential protein expression is also demonstrated using the combined information of relative molecular mass and relative quantification. It is the first time that a rapid and visual evaluation method is reported for the quality assessment of 2-DE samples.
Techanukul
Tanasit
T
Institute of Biomedical Engineering, Imperial College London, London, UK.
Pereira
Fiona
F
Lipka
Alexandria
A
Suckling
Jason
J
Wood
Steven L
SL
Lewis
Phil
P
Hassard
Stuart
S
Cass
Anthony E G
AE
Nagy
Judit M
JM
D015180:Electrophoresis, Gel, Two-Dimensional; D005782:Gels / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics; D011786:Quality Control
D005782:Gels; D011506:Proteins