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 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.
Johnson
Kenneth L
KL
Department of Biochemistry and Molecular Biology, W. M. Keck FT-ICR Mass Spectrometry Laboratory, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.
Muddiman
David C
DC
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry*; D012709:Serum Albumin / Q000032:analysis / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014168:Transferrin / Q000032:analysis / Q000737:chemistry
D001059:Apoproteins; D010103:Oxygen Isotopes; D010455:Peptides; D020543:Proteome; D012709:Serum Albumin; D014168:Transferrin; C028284:apotransferrin