Considerations for proteolytic labeling-optimization of O-18 incorporation and prohibition of back-exchange

Proteolytic O-18 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 (H2O)-O-18, Since the former allows for higher sample concentrations and thus more favorable kinetics. It was demonstrated that the inhibitory effect of urea on O-18 incorporation could be compensated by the use of higher sample concentrations. Furthermore, it was shown that heat-deactivation of trypsin prevents O-18/O-16 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 addititives. 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. Copyright (c) 2006 John Wiley & Sons, Ltd.