Production of ¹⁸O-single labeled peptide fragments during trypsin digestion of proteins for quantitative proteomics using nanoLC-ESI-MS/MS

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.