Combined Enzymatic and Data Mining Approaches for Comprehensive Phosphoproteome Analyses

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. Comprehensive phosphoproteome analyses present sizable analytical challenges in view of suppression effects of phosphopeptides and the variable quality of MS/MS spectra. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of titanium dioxide (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 a salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity that 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 multiphosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-γ for 5 min. TiO2 enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-γ exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-γ signaling events involved in vesicle trafficking, production of reactive oxygen species, and mRNA translation. 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. Comprehensive phosphoproteome analyses present sizable analytical challenges in view of suppression effects of phosphopeptides and the variable quality of MS/MS spectra. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of titanium dioxide (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 a salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity that 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 multiphosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-γ for 5 min. TiO2 enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-γ exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-γ signaling events involved in vesicle trafficking, production of reactive oxygen species, and mRNA translation. In biological systems, signal transduction pathways are primarily guided through post-translational modifications (PTMs) 1The abbreviations used are: PTM, post-translational modification; AP, alkaline phosphatase; DHB, 2,5-dihydroxybenzoic acid; IFN-γ, interferon-γ; ROS, reactive oxygen species; TiO2, titanium dioxide; FA, formic acid; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; IPI, International Protein Index; RT, retention time; cPLA2, cytosolic phospholipase A2; eIF, eukaryotic translation initiation factor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; mTOR, mammalian target of rapamycin; ACN, acetonitrile; BCA, bicinchoninic acid; i.d., inner diameter. 1The abbreviations used are: PTM, post-translational modification; AP, alkaline phosphatase; DHB, 2,5-dihydroxybenzoic acid; IFN-γ, interferon-γ; ROS, reactive oxygen species; TiO2, titanium dioxide; FA, formic acid; TPCK, l-1-tosylamido-2-phenylethyl chloromethyl ketone; IPI, International Protein Index; RT, retention time; cPLA2, cytosolic phospholipase A2; eIF, eukaryotic translation initiation factor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; mTOR, mammalian target of rapamycin; ACN, acetonitrile; BCA, bicinchoninic acid; i.d., inner diameter. of proteins to transmit information from extracellular stimuli into the cytoplasm and nuclei of cells for changes in cytoskeletal structure, vesicle transport, and gene transcription (1Kaufmann H. Bailey J.E. Fussenegger M. Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis.Proteomics. 2001; 1: 194-199Crossref PubMed Scopus (157) Google Scholar). Protein phosphorylation is the most abundant reversible PTM and a major regulator of protein activity or stability. About one-third of proteins encoded by the human genome are assumed to be phosphorylated during their life cycle (2McLachlin D.T. Chait B.T. Analysis of phosphorylated proteins and peptides by mass spectrometry.Curr. Opin. Chem. 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