Rapid prefractionation of complex protein lysates with centrifugal membrane adsorber units improves the resolving power of 2D-PAGE-based proteome analysis.
BACKGROUND
Two-dimensional gel electrophoresis (2D-PAGE) has proven over the years to be a reliable and efficient method for separation of hundreds of proteins based on charge and mass. Nevertheless, the complexity of even the simplest proteomes limits the resolving power of 2D-PAGE. This limitation can be partially alleviated by sample prefractionation using a variety of techniques.


RESULTS
Here, we have used Vivapure Ion Exchange centrifugal adsorber units to rapidly prefractionate total fission yeast protein lysate based on protein charge. Three fractions were prepared by stepwise elution with increasing sodium chloride concentrations. Each of the fractions, as well as the total lysate, were analyzed by 2D-PAGE. This simple prefractionation procedure considerably increased the resolving power of 2D-PAGE. Whereas 308 spots could be detected by analysing total protein lysate, 910 spots were observed upon prefractionation. Thorough gel image analysis demonstrated that prefractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate.


CONCLUSIONS
Prefractionation with Vivapure Q spin columns proved to be a simple, fast, reproducible, and cost-effective means of increasing the resolving power of 2D-PAGE using standard laboratory equipment.
Doud
Mary Kathryn
MK
Harvard NIEHS Center Proteomics Facility, Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, USA. mdoud@hsph.harvard.edu
Schmidt
Michael W
MW
Hines
David
D
Naumann
Claudia
C
Kocourek
Andreas
A
Kashani-Poor
Noushin
N
Zeidler
Robert
R
Wolf
Dieter A
DA
D002498:Centrifugation / Q000295:instrumentation; D005591:Chemical Fractionation / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D005656:Fungal Proteins / Q000032:analysis* / Q000302:isolation & purification; D007091:Image Processing, Computer-Assisted / Q000379:methods; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012568:Schizosaccharomyces / Q000737:chemistry
D005656:Fungal Proteins; D020543:Proteome