title: Quantitative comparison of the fasted and re-fed mouse liver phosphoproteomes using lower pH reductive dimethylation.
abstract: Phosphorylation is a common but crucial protein posttranslational modification occurring in virtually all known species. A successful technique for identifying phosphorylation sites is via liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition to identification, the introduction of stable isotopes allows for LC-MS based quantification of thousands of phosphorylation sites. Historically, stable isotope labeling by amino acids in cell culture (SILAC) has been the preferred method for introducing stable isotopes for quantification. SILAC is not well suited, however, for quantitative proteomics in larger animals. The introduction of stable isotope instead by reductive dimethylation is an alternative for performing quantitative proteomics in animal tissues. Here we present an improved reductive dimethylation protocol and demonstrate the application of this method in the analysis of the fasted vs. re-fed mouse liver phosphoproteome. In our analysis, greater than 8500 sites were identified from ∼2700 phosphoproteins. Nearly 7400 phosphorylation events from ∼2300 phosphoproteins were reliably quantified. Using a 2-fold change as a cutoff, 390 phosphorylation sites were found to change between fasted and re-fed mice, many of which may have interesting biological interpretations.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Wilson-Grady
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Haas
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D003903:Deuterium; D004032:Diet; D005215:Fasting; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D008099:Liver / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008745:Methylation; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D010750:Phosphoproteins; D003903:Deuterium