High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins.
The molecular weight measurement of intact Escherichia coli proteins separated by isoelectric focusing-immobilized pH gradient (IEF-IPG) gels and analyzed by mass spectrometry is presented. Two methods are discussed: (i) electrospray ionization (ESI) mass spectrometry (MS) of extracted proteins, and (ii) matrix-assisted laser desorption/ionization (MALDI)-MS analysis directly from IEF-IPG gels. Both ESI and MALDI methods yield sub-picomole sensitivity and good mass measurement accuracy. The use of an array detector for ESI-MS was essential to discriminate against contaminating background ions and to selectively detect high mass protein ions. MALDI-MS offers high-throughput analysis of one- and potentially two-dimensional (2-D) gels. The "virtual 2-D" gel method with first-dimensional IEF separation and the second dimension as molecular mass determination by MS, is a particularly promising method for protein analysis due to its ultra high sensitivity and correspondence to classical 2-D gels. Further sensitivity enhancements for the MALDI-MS method are provided by post acceleration detection optimized for high mass time-of-flight analysis.
Loo
J A
JA
Chemistry Department, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105, USA. joseph.loo@wl.com
Brown
J
J
Critchley
G
G
Mitchell
C
C
Andrews
P C
PC
Ogorzalek Loo
R R
RR
D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry*; D005782:Gels; D007525:Isoelectric Focusing / Q000379:methods*; D008970:Molecular Weight; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D001426:Bacterial Proteins; D005782:Gels