Virtual 2-D gel electrophoresis: visualization and analysis of the E. coli proteome by mass spectrometry.
Mass spectrometric surface analysis of isoelectric focusing gels provides an ultrasensitive approach to proteome analysis. This "virtual 2-D gel" approach, in which mass spectrometry is substituted for the size-based separation of SDS-PAGE, provides advantages in mass resolution and accuracy over classical 2-D gels and can be readily automated. Protein identities can be postulated from molecular mass (+/-0.1-0.2% for proteins of <50 kDa in size) and pI (+/-0.3 pH unit) and confirmed by MALDI in-source decay of the intact protein (providing sequence spanning up to 43 residues) or by peptide mass mapping following gel-wide chemical cleavage. Additionally, posttranslational modifications such as fatty acid acylation can be detected by the mass-resolved heterogeneity of component hydrocarbon chains. Sensitivity was evaluated by comparing the number of proteins detected by this method to equivalently loaded silver-stained 2-D gels. In the 5.7-6.0 pH range, E. coli is predicted to contain 435 proteins; virtual 2-D gels found 250 proteins ranging from >2 to <120 kDa in size present at levels to tens of femtomoles, as compared to the 100 proteins found by silver-staining 2-D gels. Extrapolating this result to the total theoretical proteome suggests that this technology is capable of detecting over 2500 E. coli proteins.
Loo
R R
RR
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Cavalcoli
J D
JD
VanBogelen
R A
RA
Mitchell
C
C
Loo
J A
JA
Moldover
B
B
Andrews
P C
PC
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D020543:Proteome / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome