Lu J, Zenobi R
Department of Chemistry, ETH Zentrum, Universitästrasse 16, Zürich, CH-8092, Switzerland.
Anal Biochem. 1999 May 1;269(2):312-6. doi: 10.1006/abio.1999.4038.
Labeled proteins are extensively used in molecular biology and environmental science. The determination of the composition and label ratio is very important for monitoring the efficiency of their separation and purification. In this paper a novel method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry was developed for this purpose. The results obtained for three commercial labeled proteins showed that they are mixtures of different conjugates. In some cases, the label ratio obtained by UV spectrometry and MALDI mass spectrometry was strikingly different. For fluorescent labels such as fluorescein isothiocyanate, MALDI mass spectrometry determines the number of covalently bound labels, whereas UV absorption yields both bound and adsorbed labels. For biotinylated proteins, label ratios obtained by the 4-hydroxyazabenzene-2'-carboxylic acid (HABA)-avidin method were found to be much smaller those determined by MALDI mass spectrometry. The HABA-avidin method may therefore not be suitable for the determination of biotin label ratios.
标记蛋白在分子生物学和环境科学中有着广泛的应用。确定其组成和标记比例对于监测其分离和纯化效率非常重要。本文为此开发了一种基于基质辅助激光解吸/电离（MALDI）飞行时间质谱的新方法。对三种商业标记蛋白的分析结果表明，它们是不同共轭物的混合物。在某些情况下，通过紫外光谱法和MALDI质谱法获得的标记比例存在显著差异。对于异硫氰酸荧光素等荧光标记物，MALDI质谱法测定共价结合标记物的数量，而紫外吸收则同时给出结合和吸附的标记物。对于生物素化蛋白，发现通过4-羟基氮杂苯-2'-羧酸（HABA）-抗生物素蛋白法获得的标记比例远低于MALDI质谱法测定的比例。因此，HABA-抗生物素蛋白法可能不适用于生物素标记比例的测定。
