Open AccessA method for calculating 16o/18o peptide ion ratios for the relative quantification of proteomes
Author(s) -
Kenneth L. Johnson,
David C. Muddiman
Publication year - 2004
Publication title -
journal of the american society for mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.961
H-Index - 127
eISSN - 1879-1123
pISSN - 1044-0305
DOI - 10.1016/j.jasms.2003.11.016
Subject(s) - chemistry , peptide , mass spectrometry , isotope , chromatography , proteome , tandem mass spectrometry , stable isotope ratio , analytical chemistry (journal) , biochemistry , physics , quantum mechanics
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 16O/18O 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 (CIM99%) was obtained. This subset of tryptic peptides was used to measure 16O/18O ratios of 0.36 +/- 0.09 (CIM99%) for BSA (micro = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (micro = 1.0) using a method for calculating 16O/18O ratios from overlapping isotopic multiplets arising from mixtures of 16O, 18O1, and 18O2 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 16O/18O spectra where peptide molecular weight, 16O/18O ratio, 18O1/18O2 ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the 16O/18O 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 18O; and the method is not sensitive to variations in 18O1/18O2 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.