Stable isotope labeling method targeting terminal tyrosine for relative peptide quantitation using mass spectrometry

Many neuropeptides lack suitable amino acid residues for modification by existing selective isotope labeling methods and use in relative quantitation by mass spectrometry. To address this issue, a new stable isotope labeling method that targets tyrosine residues by coupling with light cysteine (d0) or heavy cysteine (d2) in the presence of tyrosinase was developed. Optimal derivatization conditions for 1 μM leucine-enkephalin were achieved when 10 mM cysteine and 200 U/ml tyrosinase at pH 6.8 to 7.2 were used for a 60-min incubation period at room temperature. Under these conditions, leucine-enkephalin present at concentrations as low as 125 nM was successfully labeled. When comparisons between the lightly labeled (d0) and heavily labeled (d2) forms were made, a discrepancy between the actual concentration ratio and the raw peak intensity ratio was observed; this is due to the overlap of an isotopic peak of the d0 with the monoisotopic peak of d2. Fortunately, this discrepancy can be corrected by one of two simple computational approaches described. The quantitative labeling of this method to neuropeptides with the terminal tyrosine was confirmed and provides an alternative when other selective isotope-coded affinity tagging methods are not suitable.