Weiss M, Manneberg M, Juranville J F, Lahm H W, Fountoulakis M
F. Hoffmann-La Roche Ltd., Pharmaceutical Research-Gene Technologies, Basel, Switzerland.
J Chromatogr A. 1998 Feb 6;795(2):263-75. doi: 10.1016/s0021-9673(97)00983-7.
Fast and reproducible separation and determination of amino acids serves the economical and reliable characterization and quantification of peptides and proteins as well as the identification of proteins by amino acid composition analysis on a large-scale. A prerequisite of a successful compositional analysis is a complete hydrolysis of the peptides and proteins and a quantitative recovery of the residues in the hydrolyzate. We investigated the effect of different acid-hydrolysis methods on the compositional analysis of known proteins in solution and after blotting onto polyvinylidene difluoride membranes and worked out the conditions for the processing of large numbers of samples. The reliability of each method was studied by introducing the analysis data into the AACompIdent software and deducing the protein identification scores. All acid-hydrolysis methods delivered reliable analysis data. The most accurate data were provided by conventional, thermal hydrolysis of proteins in solution in the presence of methanesulfonic acid, closely followed by hydrolysis with hydrochloric acid and microwave radiation-dependent hydrolysis with hydrochloric or methanesulfonic acid, respectively. For blotted proteins, conventional hydrolysis delivered more accurate analysis data in comparison with the microwave radiation-induced hydrolysis. The extraction of the residues from the membrane hydrolyzate was a critical step for unambiguous protein identification. Microwave radiation-induced hydrolysis was responsible for a higher degree of racemization of the residues.
快速且可重复地分离和测定氨基酸有助于对肽和蛋白质进行经济可靠的表征和定量，以及通过大规模氨基酸组成分析来鉴定蛋白质。成功进行组成分析的一个先决条件是肽和蛋白质的完全水解以及水解产物中残基的定量回收。我们研究了不同酸水解方法对溶液中已知蛋白质以及印迹到聚偏二氟乙烯膜上后的蛋白质组成分析的影响，并制定了大量样品处理的条件。通过将分析数据导入AACompIdent软件并推导蛋白质鉴定分数来研究每种方法的可靠性。所有酸水解方法都提供了可靠的分析数据。最准确的数据是通过在甲磺酸存在下对溶液中的蛋白质进行常规热水解获得的，紧随其后的是分别用盐酸水解以及依赖微波辐射的盐酸或甲磺酸水解。对于印迹蛋白质，与微波辐射诱导的水解相比，常规水解提供了更准确的分析数据。从膜水解产物中提取残基是明确鉴定蛋白质的关键步骤。微波辐射诱导的水解导致残基更高程度的消旋化。
