Characterization of proteins by sequential isoelectric focusing on immobilized pH gradients and electrospray mass spectrometry.

The coupling of isoelectric focusing on immobilized pH gradients (IPG) with electrospray-mass spectrometry (ES-MS) was applied to the characterization of proteins according to two different and important properties, such as net surface charge and molecular mass. From a technical point of view, these methods are complementary, since ES-MS requires ion-free samples as usually supplied by isoelectric focusing on IPGs. This report describes the experiments carried out on model proteins to demonstrate the feasibility of the sequential application of these two techniques for the characterization of proteins. A minimum of 5 micrograms protein was needed for good signal by mass spectrometry. The following proteins were studied: myoglobin, truncated interleukin-6-mutein, recombinant cytochrome c551 and insulin-like growth factor I. Extraction from the IPG matrix was carried out in 70% acetonitrile/30% water/0.05% trifluoroacetic acid either by passive diffusion or by centrifugation through a 0.22 micron Amicon membrane, with protein recoveries of 80-85%.