CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

Abstract

Alpha-1-acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample-consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8-3.8) in a capillary. Intraday RSD values < or = 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70-22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.