High-Resolution Capillary Isoelectric Focusing−Electrospray Ionization Mass Spectrometry for Hemoglobin Variants Analysis

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Abstract
On-line capillary isoelectric focusing (CIEF)−electrospray ionization mass spectrometry (ESIMS) as a two-dimensional separation system is employed for high-resolution analysis of hemoglobin variants A, C, S, and F. The effects of moving ionic boundary inside the CIEF capillary and MS scan rate on the separation resolution and mass detection of hemoglobin variants are investigated. The formation of a moving ionic boundary due to the replacement of background electrolyte counterions with sheath liquid counterions can be minimized by combining cathodic mobilization with a gravity-induced hydrodynamic flow. Hemoglobin variants F and A, with a pI difference of 0.05 pH unit, are almost baseline resolved and identified in CIEF−ESIMS. The concentration detection limit for each hemoglobin variant is in the range of 10-8 M, comparable to that obtained in two-dimensional gel electrophoresis using silver staining. Initial preconcentration during the focusing step and the use of single-ion monitoring scan mode are responsible for improving detection limits.