Zhang Chenhua, Clarke William, Hage David S
Department of Chemistry, University of Nebraska, Lincoln, NE, USA.
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Methods Mol Biol. 2019;1972:41-56. doi: 10.1007/978-1-4939-9213-3_4.
Human alpha-acid glycoprotein (AGP) is an acute phase glycoprotein that has a heterogeneous glycosylation pattern. This pattern can change in certain diseases, which has resulted in interest in using AGP glycoforms as potential biomarkers for these diseases. This report describes a method that uses capillary electrophoresis to characterize and analyze AGP glycoforms both in purified samples of AGP and in human serum. This method uses static and dynamic coatings of poly(ethylene oxide) that are applied to a silica capillary for separation of AGP glycoforms in the reversed-polarity mode of CE and in the presence of negligible electroosmotic flow. Electrophoretic injection is performed onto such capillaries by using a stationary stacking interface between the sample and running buffer. In addition, acidic precipitation and desalting are used to allow for the isolation and the analysis of AGP from only 65 μL of serum. Up to eleven AGP glycoform bands can be reproducibly separated by this method, with the difference in migration time between neighboring bands being 12- to almost 60-fold larger than the standard deviation for the migration time of any given band. A limit of detection down to about 2 nM per glycoform band can be obtained by this method for AGP in serum based on absorbance detection and without the need for further sample modification or labeling.
人α-酸性糖蛋白（AGP）是一种具有异质性糖基化模式的急性期糖蛋白。这种模式在某些疾病中会发生变化，这使得人们对将AGP糖型用作这些疾病的潜在生物标志物产生了兴趣。本报告描述了一种使用毛细管电泳来表征和分析纯化的AGP样品以及人血清中AGP糖型的方法。该方法使用聚环氧乙烷的静态和动态涂层，将其应用于硅胶毛细管，以便在CE的反相模式下且在电渗流可忽略不计的情况下分离AGP糖型。通过在样品和运行缓冲液之间使用固定堆积界面，在这种毛细管上进行电泳进样。此外，采用酸性沉淀和脱盐方法，仅从65μL血清中即可分离和分析AGP。用这种方法可重复分离出多达11条AGP糖型条带，相邻条带之间的迁移时间差异比任何给定条带迁移时间的标准偏差大12至近60倍。基于吸光度检测，该方法对血清中的AGP每糖型条带的检测限可低至约2 nM，且无需对样品进行进一步处理或标记。
