Current methods for phosphoproteome analysis have several limitations. First, 
most methods for phosphopeptide enrichment lack the specificity to truly purify 
phosphopeptides. Second, fragmentation spectra of phosphopeptides, in particular 
those of phosphoserine and phosphothreonine containing peptides, are often 
dominated by the loss of the phosphate group(s) and therefore lack the 
information required to identify the peptide sequence and the site of 
phosphorylation, and third, sequence database search engines and statistical 
models for data validation are not optimized for the specific fragmentation 
properties of phosphorylated peptides. Consequently, phosphoproteomic data are 
characterized by large and unknown rates of false positive and false negative 
phosphorylation sites. Here we present an integrated chemical, mass 
spectrometric and computational strategy to improve the efficiency, specificity 
and confidence in the identification of phosphopeptides and their site(s) of 
phosphorylation. Phosphopeptides were isolated with high specificity through a 
simple derivatization procedure based on phosphoramidate chemistry. 
Identification of phosphopeptides, their site(s) of phosphorylation and the 
corresponding phosphoproteins was achieved by the optimization of the mass 
spectrometric data acquisition procedure, the computational tools for database 
searching and the data post processing. The strategy was applied to the mapping 
of phosphorylation sites of a purified transcription factor, dFOXO and for the 
global analysis of protein phosphorylation of Drosophila melanogaster Kc167 
cells.