Combined Enzymatic and Data Mining Approaches for Comprehensive Phosphoproteome Analyses

Abstract

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 (TiO 2 )-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 TiO 2 -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-␥ for 5 min. TiO 2 enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-␥ exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-␥ signaling events involved in vesicle traf-

Phosphoproteomic Analysis of the Mouse Brain Cytosol Reveals a Predominance of Protein Phosphorylation in Regions of Intrinsic Sequence Disorder

“…Other studies have reported the use of enzymatic dephosphorylation to validate phosphopeptide identifications; however, between 33 and 63% of peptides found after phosphopeptide enrichment were unphosphorylated, and therefore only peptides characterized in the dephosphorylated sample that matched phosphopeptides in the phosphorylated sample were useful (34,35). It is clear that a subset of phosphopeptides were not amenable to detection under our LC-MS/MS conditions in their phosphorylated form.…”

Large-scale analysis of lysine SUMOylation by SUMO remnant immunoaffinity profiling

“…For label-free quantitative proteomics analyses, Orbitrap raw LC-MS data files were transformed into peptide maps using in-house peptide detection and clustering software 43 . Peptide maps belonging to one experiment were clustered and aligned using clustering parameters of D m/z: 0.02 and time tolerance of ± 2 min (wide), ± 0.5 min (narrow).…”

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the BasicC-Terminal Residue

“…Mass spectrometry (MS) has become the technique of choice for the qualitative [2] and quantitative [3–5] study of protein phosphorylation because of its unparalleled versatility and sensitivity. ‘Bottom-up’ phosphoproteomics analysis takes place at the peptide-level and usually requires phosphopeptide enrichment [6–8] because of the generally sub-stoichiometric levels of phosphorylated peptides relative to their non-modified counterparts [9] and their somewhat reduced ionization efficiency [10, 11], both factors that are detrimental in data-dependent analysis (DDA) strategies where the selection and fragmentation of precursor ions is dictated by their relative intensities.…”