Protein phosphorylation is a central cell signaling event that underlies a broad spectrum of key physiological processes. Advances in affinity chromatography and mass spectrometry are now providing the ability to identify and quantitate thousands of phosphorylation sites simultaneously. In spite of these remarkable advances, comprehensive phosphoproteome analyses still present sizable analytical challenges in view of suppression effects affecting positive ion detection of phosphopeptides and the variable quality of MS/MS spectra limiting sequence assignment and identification of modification sites. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of TiO2-enriched cell extracts. The correlation of retention times of more than 750 phospho- and dephosphopeptide pairs from J774 macrophage cell extracts indicated that removal of the phosphate groups can impart a gain or a loss in hydrophobicity that is partly explained by the formation of salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity which facilitated peptide sequencing. More importantly, alkaline phosphatase digestion enhanced the overall population of putative phosphopeptides from TiO2-enriched cell extracts providing a unique approach to profile multi-phosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-for 5 min. TiO2 enrichment enabled the identification of 1144 phosphopeptides from 433 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-gamma signaling events involved in vesicle trafficking, production of reactive oxygen species and mRNA translation.