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Considerations for proteolytic labeling–optimization of 18 O incorporation and prohibition of back‐exchange
Author(s) -
Storms Henricus F.,
van der Heijden Robert,
Tjaden Ubbo R.,
van der Greef Jan
Publication year - 2006
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2738
Subject(s) - chemistry , trypsin , chromatography , electropherogram , isoelectric focusing , capillary electrophoresis , proteolytic enzymes , mass spectrometry , electrophoresis , proteolysis , hydrolysis , proteome , urea , kinetics , biochemistry , enzyme , physics , quantum mechanics
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 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 © 2006 John Wiley & Sons, Ltd.