title: Trypsin is the primary mechanism by which the (18)O isotopic label is lost in quantitative proteomic studies.
abstract: Labeling with (18)O is currently one of the most commonly used methods for incorporating a stable isotopic label into samples for comparative proteomic studies. In this approach, isotopic labeling involves the enzymatic digestion, typically performed with trypsin, of a protein population in (18)O-water, which incorporates the stable isotope into the C termini of the newly formed peptides. Although trypsin is often used to facilitate isotopic incorporation after digestion, it is typically overlooked that this same mechanism can lead to isotopic loss even under conditions such as low pH where it is assumed that trypsin is inactive. To examine the role that trypsin plays in isotopic loss, several experiments were performed on the rate of delabeling under conditions relevant to multidimensional proteomic experiments. Results from these studies demonstrate that enzyme-facilitated exchange of (18)O in the peptide with (16)O in the aqueous solvent was the major process by which the label is removed from the peptides, even under conditions of low pH and temperature where trypsin is thought to be inactive. This study brings the rapid, tryptic-facilitated exchange to the attention of laboratories using this scheme to prevent inaccuracies in quantitative labeling due to loss of the isotopic label.
authors.affiliation: Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30302-4712, USA.
authors.forename: Peggi M
authors.initials: PM
authors.lastname: Angel
authors.forename: Ron
authors.initials: R
authors.lastname: Orlando
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000032:analysis*; D011506:Proteins / Q000378:metabolism; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D014357:Trypsin / Q000378:metabolism*; D014867:Water / Q000737:chemistry
chemical_list: D010103:Oxygen Isotopes; D010455:Peptides; D011506:Proteins; D020543:Proteome; D014867:Water; D014357:Trypsin; D010100:Oxygen