title: Considerations for proteolytic labeling-optimization of 18O incorporation and prohibition of back-exchange.
abstract: Proteolytic (18)O labeling is a very powerful tool for differential analysis applied to proteome studies. However, it is a relatively new technique and the optimization of the labeling process still needs some attention. We found that the two-step post-proteolytic labeling should be favored over the conventional digestion of proteins in H(2) (18)O, since the former allows for higher sample concentrations and thus more favorable kinetics. It was demonstrated that the inhibitory effect of urea on (18)O incorporation could be compensated by the use of higher sample concentrations. Furthermore, it was shown that heat-deactivation of trypsin prevents (18)O/(16)O back-exchange. In addition, no non-specific hydrolysis of the peptides could be observed as a result of the heating. Heat inactivation of trypsin opens the way for the use of capillary electrophoresis as a separation technique in proteolytic labeling studies, as it abolishes the need for use of detrimental additives. Analysis of a labeled protein digest by capillary isoelectric focusing/mass spectrometry showed the applicability of the method. No back-exchange was observed across the entire electropherogram.
authors.affiliation: Division of Analytical Biosciences, Leiden/Amsterdam Center for Drug Research, P.O. Box 9502, 2300 RA Leiden, The Netherlands. harriestorms@zonnet.nl
authors.forename: Henricus F
authors.initials: HF
authors.lastname: Storms
authors.forename: Robert
authors.initials: R
authors.lastname: van der Heijden
authors.forename: Ubbo R
authors.initials: UR
authors.lastname: Tjaden
authors.forename: Jan
authors.initials: J
authors.lastname: van der Greef
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D010103:Oxygen Isotopes / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*
chemical_list: D010103:Oxygen Isotopes; D020543:Proteome