Our optimized TiO2 phosphopeptide enrichment methodologies are combined with a state-of-the-art nanoACQUITY™ Ultra Performance or Ultra-high pressure LC (UPLC) system (Waters Corp.) /LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) platform to provide a very robust phosphoprotein profiling and phosphoproteomics analysis. Figure 1 schematically outlines workflows for identification and quantitation of phosphorylated proteins and peptides. The workflow is generally divided into two options: one for TiO2 enrichment of phosphopeptides after the protein(s) of interest are purified (e.g. SDS PAGE) or immunoprecipitated and another for more complex phosphopeptide mixtures which are directly extracted from tissue or whole cell extracts. The first is a more targeted approach to discover numerous sites of phosphorylation on proteins of interest, whereas the latter is directed towards a more global phosphopeptide discovery based approach. For most of these approaches, three different quantitative methods (SILAC, iTRAQ, and label-free quantitation) can be introduced at steps indicated by “Q” to determine changes of phosphorylation between two or more samples. The type of quantitation method to be used will be based on the sample amount, complexity, and the desired outcome for the project. Additional work flows for locating phosphotyrosine sites using anti-phosphotyrosine antibodies with immunoprecipitation can also be utilized.