title: Quantification of protein phosphorylation by liquid chromatography-mass spectrometry.
abstract: The identification and quantification of specific phosphorylation sites within a protein by mass spectrometry has proved challenging when measured from peptides after protein digestion because each peptide has a unique ionization efficiency that alters with modification, such as phosphorylation, and because phosphorylation can alter cleavage by trypsin, shifting peptide distribution. In addition, some phosphorylated peptides generated by tryptic digest are small and hydrophilic and, thus, are not retained well on commonly used C18 columns. We have developed a novel C-terminal peptide (2)H-labeling derivatization strategy and a mass balance approach to quantify phosphorylation. We illustrate the application of our method using electrospray ionization liquid chromatography-mass spectrometry by quantifying phosphorylation of troponin I with protein kinase A and protein kinase C. The method also improves the retention and elution of hydrophilic peptides. The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage. Measurement of phosphorylation is shown to be linear (relative standard error <5%) with a detection limit of <10%.
authors.affiliation: Cell and Molecular Biology Program and Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Previs
authors.forename: Peter
authors.initials: P
authors.lastname: VanBuren
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Begin
authors.forename: Jim O
authors.initials: JO
authors.lastname: Vigoreaux
authors.forename: Martin M
authors.initials: MM
authors.lastname: LeWinter
authors.forename: Dwight E
authors.initials: DE
authors.lastname: Matthews
mesh_terms: D002853:Chromatography, Liquid; D017868:Cyclic AMP-Dependent Protein Kinases; D003903:Deuterium; D013058:Mass Spectrometry / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis*; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D011493:Protein Kinase C; D040901:Proteomics / Q000379:methods; D019210:Troponin I
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D019210:Troponin I; D003903:Deuterium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C