Protein profiling by capillary isoelectric focusing, reversed‐phase liquid chromatography, and mass spectrometry

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Abstract
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 µg 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.

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