Mass spectrometric imaging of immobilized pH gradient gels and creation of "virtual" two-dimensional gels.
We have developed a matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) based technique for the detection of intact proteins directly from immobilized pH gradient gels (IPGs). The use of this technique to visualize proteins from IPGs was explored in this study. Whole cell Escherichia coli extracts of various loadings were separated on IPGs. These IPGs were processed to remove contaminants and to achieve matrix/analyte cocrystallization on the surface of the gel. Mass spectra were acquired by scanning the surface of the gel and were assimilated into a "virtual" two dimensional (2-D) gel. This virtual 2-D gel is analogous to a "classical" 2-D gel, except that the molecular weight information is acquired by mass spectrometry rather than by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This mass spectrometry (MS) based technology exemplifies a number of desirable characteristics, some of which are not attainable with classical two-dimensional electrophoresis (2-DE). These include high sensitivity, high reproducibility, and an inherently higher resolution and mass accuracy than 2-D gels. Furthermore, there is a difference in selectivity exhibited between virtual 2-D gels and classical 2-D gels, as a number of proteins are visible in the virtual gel image that are not present in the stained gels and vice versa. In this report, virtual 2-D gels will be compared to classical 2-D gels to illustrate these features.
Walker
A K
AK
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Rymar
G
G
Andrews
P C
PC
D001426:Bacterial Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014584:User-Computer Interface
D001426:Bacterial Proteins