van der Geer P, Hunter T
Molecular Biology and Virology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037.
Electrophoresis. 1994 Mar-Apr;15(3-4):544-54. doi: 10.1002/elps.1150150173.
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.
鉴定蛋白质磷酸化位点对于评估单个位点在通过磷酸化调节特定蛋白质过程中的作用至关重要。在此，我们综述一种我们开发的用于鉴定磷酸化位点的方法，该方法基于用位点特异性蛋白酶消化32P标记的蛋白质，并在薄层纤维素板上对消化产物进行二维分离，第一维采用电泳，随后第二维采用色谱法。这种方法非常灵敏，每分钟仅需几百次32P衰变就能获得可重复的磷酸肽图谱。我们还报告了分析完整磷酸化蛋白和从二维分离中回收的单个磷酸肽的磷酸氨基酸含量的方法，其中对材料进行部分酸水解，水解产物通过一维或二维电泳在薄层纤维素板上分离。最后，我们描述了通过用位点特异性蛋白酶进行二次消化、手动埃德曼降解和免疫沉淀来分析分离的磷酸肽结构的方法，并指出如何将这些信息与肽的二维迁移率结合使用，从蛋白质的已知序列推断磷酸肽的身份。
