Evaluation of the effect of the immunopurification-based procedures on the CZE-UV and CZE-ESI-TOF-MS determination of isoforms of intact alpha-1-acid glycoprotein from human serum

摘要

Differences in a-1-acid glycoprotein (AGP) peptidic and glycan moieties originate several udisoforms, whose modifications have been related to different pathophysiological situa- udtions. Differences in the isoforms of AGP existing in serum of individuals suffering from uddifferent diseases compared to healthy ones could be potentially used as biomarkers. udCZE has been proven to be a useful technique for the analysis of glycoprotein isoforms. udHowever, direct CZE analysis of AGP isoforms in serum samples needs efficient puri- udfication methods that allow the protein analysis. In this work two new and fast methods udto purify AGP from human serum are evaluated in regard to their effect on the deter- udmination of isoforms of the intact glycoprotein by CZE-UV and by a developed CZE-ESI- udTOF-MS method. Both preparation methods, which differ in the pre-treatment of the udsample prior to an anti-AGP immunochromatographic step are shown to be adequate to udanalyze isoforms of intact AGP. Comparison of both purification methods by CZE-UV udand CZE-ESI-TOF-MS indicates that serum AGP purified without acidic precipitation as udpre-treatment is more adequate due to AGP higher yield, which leads to better CZE-Mass udspectra. Both CZE methods show no indication that acidic precipitation influences the udglycosylation (including sialylation) of AGP.