The phosphorylation site and desmethionyl N-terminus of Drosophila phosrestin I in vivo determined by mass spectrometric analysis of proteins separated by two-dimensional gel electrophoresis

Post-translational modifications of proteins play crucial roles in modulating many cellular processes, In order to understand the physiological roles of post-translational protein modifications it is imperative to determine the nature of the change in chemical structure involved in each protein modification, In our earlier work, we developed a method for the study of protein modification through a streamlined combination of two-dimensional gel electrophoresis, in-gel digestion, high performance liquid chromatography and electrospray ionization tandem quadrupole mass spectrometry, Using this method we determined the in vivo phosphorylation site of phosrestin I to be Ser(366). Since our earlier work described the method only briefly, we will present a full description of the method in this paper, In addition, by using this method, we also show that the N-terminus of phosrestin I is desmethionylated in vivo, These techniques are easily adapted to the study of other proteins and will serve as powerful tools for the study of post-translational protein modifications in general.