磷酸肽
色谱法
埃德曼退化
化学
水解
纤维素
电泳
蛋白酵素
生物化学
肽
肽序列
酶
基因
作者
Peter van der Geer,Tony Hunter
标识
DOI:10.1002/elps.1150150173
摘要
Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the analysis of the phosphoamino acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.
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