Tyrosine Phosphorylation Site Identification by MALDI-MS
This protocol was adapted from “Proteomic Methods for Phosphorylation Site Mapping,” Chapter 9, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.INTRODUCTION
In this protocol, radiolabeled proteins are separated using SDS-PAGE and digested in-gel. The phosphopeptides are subjected to two-dimensional LC and identified by MALDI-MS and MALDI-PSD. The identification of eight tyrosine phosphorylation sites in the human Gab-1 protein is provided as an example of the method’s utility. The expression and phosphorylation of human Gab-1 with the insulin receptor kinase in vitro are summarized as an example of this approach to identifying phosphorylated tyrosine residues.
