Protein profiling by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry.
An automated system for intact protein analysis is described that combines capillary isoelectric focusing (CIEF), reversed-phase liquid chromatography (RPLC), and electrospray ionization-mass spectrometry (ESI-MS). Performance is demonstrated with a complex yeast enzyme concentrate. CIEF is performed with a microdialysis membrane-based cathodic cell that permits pI fractions to be sampled and stored for subsequent LC-MS analysis. A total of 50 microg protein is loaded onto the capillary. Ten fractions are stored which span the pI range 3-10. Each fraction is subsequently cleaned on a reversed-phase trap column and then characterized by LC-MS. MaxEnt1 is used to deconvolute the raw mass spectra to obtain the molecular weight (MW) of intact proteins/peptides in the sample. A two-dimensional display of pI vs. MW is illustrated for the 500 most prevalent species as identified by MaxEnt1.
Zhou
Feng
F
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
Johnston
Murray V
MV
D002853:Chromatography, Liquid / Q000379:methods*; D007525:Isoelectric Focusing / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
D011506:Proteins