title: Quantitative protein analysis using enzymatic [¹⁸O]water labeling.
abstract: This unit describes the stepwise procedure for differential oxygen isotope labeling of peptides and mass spectrometric quantification of relative protein levels in comparative proteomic experiments. The [¹⁸O] labeling of peptides happens at the peptide C-terminus and is achieved via the enzymatic oxygen exchange of tryptic peptides via catalysis of immobilized trypsin. Experimental considerations in effective incorporation and stabilization of the oxygen labels are discussed. Methods for mass spectrometric quantification of peptides with differential [¹⁶O] and [¹⁸O] isotopes are presented.
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut.
authors.forename: Mary Joan
authors.initials: MJ
authors.lastname: Castillo
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland.
authors.forename: Kristy J
authors.initials: KJ
authors.lastname: Reynolds
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut.
authors.forename: Alexander
authors.initials: A
authors.lastname: Gomes
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland.
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
authors.affiliation: Department of Chemistry, University of Connecticut, Storrs, Connecticut.
authors.forename: Xudong
authors.initials: X
authors.lastname: Yao
mesh_terms: D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010103:Oxygen Isotopes / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D014867:Water / Q000737:chemistry*
chemical_list: D010103:Oxygen Isotopes; D010455:Peptides; D014867:Water
keywords: 18O-labeling; back-exchange; enzymatic oxygen labeling; immobilized trypsin; quantitative proteomics