Reversible protein phosphorylation is a central cellular regulatory mechanism in 
modulating protein activity and propagating signals within cellular pathways and 
networks. Development of more effective methods for the simultaneous 
identification of phosphorylation sites and quantification of temporal changes 
in protein phosphorylation could provide important insights into molecular 
signaling mechanisms in various cellular processes. Here we present an 
integrated quantitative phosphoproteomics approach and its application for 
comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) 
gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O 
labeling plus (16)O/ (18)O-methanol esterification for quantitation, a 
macro-immobilized metal-ion affinity chromatography trap for phosphopeptide 
enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by 
neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear 
ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were 
identified and quantified including receptors, kinases, proteins associated with 
small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were 
also identified as differentially expressed followed by LPA stimulation, and we 
have shown evidence of pseudopodia subcellular localization of one of these 
candidate proteins. These results demonstrate the efficiency of this 
quantitative phosphoproteomics approach and its application for rapid discovery 
of phosphorylation events associated with LPA gradient sensing and cell 
chemotaxis.