A method for calculating 16O/18O peptide ion ratios for the relative quantification of proteomes

A method is described for the identification and relative quantification of proteomes using accurate mass tags (AMT) generated by nLC-dual ESI-FT-ICR-MS on a 7T instrument in conjunction with stable isotope labeling using O-16/O-18 ratios. AMTs were used for putative peptide identification, followed by confirmation of peptide identity by tandem mass spectrometry. For a combined set of 58 tryptic peptides from bovine serum albumin (BSA) and human transferrin, a mean mass measurement accuracy of 1.9 ppm +/- 0.94 ppm (CIMg99%) was obtained. This subset of tryptic peptides was used to measure O-16/O-18 ratios of 0.36 +/- 0.09 (CIM99%) for BSA (mu = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (mu = 1.0) using a method for calculating O-16/O-18 ratios from overlapping isotopic multiplets arising from mixtures of O-16, O-18(1), and O-18(2) labeled C-termini. The model amino acid averagine was used to calculate a representative molecular formula for estimating and subtracting the contributions of naturally occurring isotopes solely as a function of peptide molecular weight. The method was tested against simulated composite O-16/O-18 spectra where peptide molecular weight, O-16/O-18 ratio, O-18(1)/O-18(2) ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the O-16/O-18 ratios were less than three, even for peptides where the number of sulfur atoms was over- or under-estimated. These data demonstrate that for biomarker discovery, it is advantageous to label the proteome representing the disease state with 18 0; and the method is not sensitive to variations in O-18(1)/O-18(2) ratio. This approach allows a comprehensive differentiation of expression levels and tentative identification via AMTs, followed by targeted analysis of over- and under-expressed peptides using tandem mass spectrometry, for applications such as the discovery of disease biomarkers. (C) 2004 American Society for Mass Spectrometry.