Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10825973 | Methods | 2013 | 28 Pages |
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 â¼2700 phosphoproteins. Nearly 7400 phosphorylation events from â¼2300 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.
Keywords
CREBPMSFCRTC2CIDAGCAcOHTFAHEPESFDRIMACXICDTTACN(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)2-(N-morpholino) ethanesulfonic acidLC–MS/MSm/zS/NAcetonitrilescxTrifluoroacetic acidAcetic acidFormic acidstrong cation exchangecollision induced dissociationdithiothreitolSILACMass spectrometryPhosphorylationPhenylmethanesulfonyl fluorideMeSfalse discovery ratemass to charge ratioSignal-to-noise ratiostable isotope labeling by amino acids in cell cultureQuantitative proteomicsLiverliquid chromatography–tandem mass spectrometryimmobilized metal affinity chromatographyextracted ion chromatogramautomatic gain control
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Authors
Joshua T. Wilson-Grady, Wilhelm Haas, Steven P. Gygi,