We investigated the effect of pH and the additive monoethanolamine on 18O-atom incorporation into the C-terminal carboxy group of peptide fragments during tryptic digestion in 18O-labeled water. Although amidase activity was sufficient for digestion at pH 6-11, the second 18O-atom incorporation at the carboxy oxygen site was inhibited at pH 11 or above. The addition of at least 50 mM monoethanolamine into the reaction mixture also inhibited the carboxy oxgen exchange without reduction in amidase activity. Therefore, tryptic digestion for 18O-single labeling should be performed in 50 mM phosphate buffer (pH 11) containing 50 mM monoethanolamine. The production ratios of 18O-single labeled peptides were over 85%, and these results were independent of amino acid sequence. We also investigated the linearity of the 18O-single labeled to unlabeled ratio (18O1/18O 0). The use of y ions for calculation of the 18O 1/18O0 ratio gave a better correlation between the observed and theoretical 18O1/18O 0 ratios in the range of 0.1 to 10 than did the use of precursor ions. In the analysis of a pseudobiomarker spiked into human serum, the present 18O-single labeling method was found to be robust because it was not affected by incomplete LC separation. The present 18O-single labeling method represents a useful tool for quantitative proteomics using nanoLC-ESI-MS/MS.
- O-single labeling
- quantitative proteomics