Yu Tao, Traina Joseph A, Pungor Erno, McCaman Michael
Berlex Biosciences, Richmond, CA 94804, USA.
Anal Biochem. 2006 Dec 1;359(1):54-62. doi: 10.1016/j.ab.2006.08.018. Epub 2006 Sep 5.
Standard SDS-PAGE analysis of a pegylated protein was able to confirm an increase in its molecular size after reaction with an activated polyethylene glycol (PEG) but could do little to identify the extent of pegylation or to support characterization of the consistency of the modified protein. In this article, we demonstrate the utility of the capillary electrophoresis technology (using a microfluidic system) in analyzing the pegylation pattern of a recombinant protein over a range of 1-12 PEGs per polypeptide. Confirmatory data from mass spectrometry analysis of pegylated adducts are also presented. These allowed independent confirmation of the extent of pegylation. This electrophoretic analysis gives a robust, reproducible, and direct characterization of PEG adducts. We found that traditional estimation of PEG adducts by an indirect colorimetric (trinitrobenzene sulfonic acid) reaction, which detects loss of free amino groups, was quite erroneous for the recombinant protein in our study as well as several commercially available pegylated proteins. These results support the use of this capillary electrophoresis device for precise characterization of pegylated proteins.
对聚乙二醇化蛋白质进行标准的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析，能够证实其与活化聚乙二醇（PEG）反应后分子大小增加，但在确定聚乙二醇化程度或支持对修饰后蛋白质一致性的表征方面作用不大。在本文中，我们展示了毛细管电泳技术（使用微流控系统）在分析重组蛋白的聚乙二醇化模式方面的效用，该模式为每个多肽含有1至12个PEG。还给出了对聚乙二醇化加合物进行质谱分析的验证数据。这些数据能够独立确认聚乙二醇化程度。这种电泳分析对PEG加合物给出了可靠、可重复且直接的表征。我们发现，通过间接比色法（三硝基苯磺酸）反应对PEG加合物进行传统估算，该反应检测游离氨基的损失，对于我们研究中的重组蛋白以及几种市售聚乙二醇化蛋白来说是相当错误的。这些结果支持使用这种毛细管电泳装置对聚乙二醇化蛋白进行精确表征。
