Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation.
Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.
Richardson
Matthew R
MR
Department of Cellular & Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN, USA.
Liu
Sean
S
Ringham
Heather N
HN
Chan
Victor
V
Witzmann
Frank A
FA
D015180:Electrophoresis, Gel, Two-Dimensional; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000302:isolation & purification; D007525:Isoelectric Focusing; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029968:Escherichia coli Proteins; D020543:Proteome