Recent advances in MS instrumentation and progresses in phosphopeptide 
enrichment, in conjunction with more powerful data analysis tools, have 
facilitated unbiased characterization of thousands of site-specific 
phosphorylation events. Combined with stable isotope labeling by amino acids in 
cell culture metabolic labeling, these techniques have made it possible to 
quantitatively evaluate phosphorylation changes in various physiological states 
in stable cell lines. However, quantitative phosphoproteomics in primary cells 
and tissues remains a major technical challenge due to the lack of adequate 
techniques for accurate quantification. Here, we describe an integrated strategy 
allowing for large scale quantitative profiling of phosphopeptides in complex 
biological mixtures. In this technique, the mixture of proteolytic peptides was 
subjected to phosphopeptide enrichment using a titania affinity column, and the 
purified phosphopeptides were subsequently labeled with iTRAQ reagents. After 
further fractionation by strong-cation exchange, the peptides were analyzed by 
LC-MS/MS on an Orbitrap mass spectrometer, which collects CID and high-energy 
collisional dissociation (HCD) spectra sequentially for peptide identification 
and quantitation. We demonstrate that direct phosphopeptide enrichment of 
protein digests by titania affinity chromatography substantially improves the 
efficiency and reproducibility of phosphopeptide proteomic analysis and is 
compatible with downstream iTRAQ labeling. Conditions were optimized for HCD 
normalized collision energy to balance the overall peptide identification and 
quantitation using the relative abundances of iTRAQ reporter ions. Using this 
approach, we were able to identify 3557 distinct phosphopeptides from HeLa cell 
lysates, of which 2709 were also quantified from HCD scans.