Simplified capillary isoelectric focusing with chemical mobilization for intact protein analysis.

We report a capillary isoelectric focusing system based on a sequential injection method for simplified chemical mobilization. This system was coupled to an ion trap mass spectrometer with an electrokinetically pumped nanoelectrospray interface. The nanoelectrospray emitter employed an acidic sheath electrolyte. To simplify focusing and mobilization, a plug of ammonium hydroxide was first injected into the capillary, followed by a section of mixed sample and ampholyte. During focusing, the NH3H2O section worked as catholyte. As focusing progressed, the NH3H2O section was titrated to lower pH by the acidic sheath electrolyte. Chemical mobilization started automatically once the ammonium hydroxide was consumed by the acidic sheath flow electrolyte, which then acted as the mobilization solution. In this report, the lengths of the NH3H2O section and sample were optimized. With a 1 m long capillary, a relative short plug of the NH3H2O section (3 cm) produced both fast migration and reasonable separation resolution. The simplified capillary isoelectric focusing mass spectrometry system produced base peak intensity relative standard deviation of 8.5% and migration time relative standard deviation <= 0.6% for myoglobin and cytochrome C in triplicate runs.