|Matthew T. Olson, Department of Pathology, The Johns Hopkins Hospital |
Alfred L. Yergey, OSD, NICHD, NIH
Paul S. Blank, PPB, NICHD, NIH
Aaron Catlin, OSD, NICHD, NIH
Hui Zhang, Division of Clinical Chemistry, Department of Pathology, The Johns Hopkins Hospital
Peter S. Backlund, OSD, NICHD, NIH
Jonathan A. Epstein, OSD, NICHD, NIH
|INTRODUCTION: Isotope ratios have a long history of use in mass spectrometry (MS) quantification experiments in both research and clinical settings. In methods involving MS scans prior to fragmentation, the isotope cluster provides convoluted but precise information about the distribution of elements and their isotopes in the analyte. This abstract explores how such information may be beneficial in classifying - prior to fragmentation - the probability that a peptide is modified.|
METHODS: Several methods for rapid and accurate calculation of the isotope cluster have been developed. When the isotope cluster for peptides with methionine oxidation were measured by an ABI 4800 MALDI TOF-TOF Analyzer was compared against the predicted clusters for other peptides at the same mass, the perturbations of methionine oxidation could be appreciated by the use of a distance histogram between the measured and theoretical clusters. Additionally, as a predictor of how well this method may perform with other posttranslational modifications (PTMs), a database of all tryptic peptides from every known human protein was used. All of the possible amino acid combinations (AACs) for 1,000 randomly selected monoisotopic masses were retrieved from the peptide composition table. For each of these, a set of commonly encountered PTMs were applied, and the peptide elemental compositions (PECs) at the resulting masses were calculated to determine if any modified peptides were isobaric with unmodified peptides at the predicted mass. The amount of overlap was compared. An abundance of isobaric unmodified PECs at the same mass as modified peptides decreases the usefulness of the isotope cluster to classify the probability of peptide modification. For PTMs that generate elemental compositions that share little or no similarity to the PECs of unmodified peptides at the same mass, the likelihood of the isotope cluster detecting a difference increases, and this will be exploited.
PRELIMINARY DATA: The measured isotope clusters of several peptides with methionine oxidation were noted to have distinct differences from the predicted isotope clusters. None of the randomly sampled phosphorylated peptides were isobaric with unmodified peptides at the same mass indicating that the isotope cluster should be able to distinguish the difference between phosphorylated and non-phosphorylated peptides. Posttranslational modifications with smaller elemental perturbation, including methionine oxidation, have some theoretical overlap with unmodified peptides although this has not been seen on our limited measurements so for. The disparity between this prediction and the measured observations is not eminently clear now.