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The new generation of MALDI TOF/TOF instruments can be used to generate informative identification and relative quantification data using iTRAQ chemistries. This presentation introduces a novel automated iTRAQ relative quantification software package for the AXIMA-TOF2 MALDI TOF/TOF mass spectrometer. The package uses and extends onwards from existing LC-MALDI software. Interfacing of mass spectral data, relative quantification data, and Mascot search results in an interactive, visual correlation providing a highly practical end-user package.
The user interface has multi-level organization. The primary level shows Mascot database search hits along with an average quantification ratio for each protein along with significance scoring. The software correlates the peak intensity/area of the iTRAQ reporter ions to the protein identifications assigned by Mascot using MS/MS spectra in order to relatively quantify the protein components in labeled samples. Further interrogation of the individual peptide ratios may be found at the second visualization level along with the relevant spectra. The user interface allows input of iTRAQ kit–specific software correction factors, allowing duplex through to 8-plexed sample scenarios.
In proof-of-principle experiments combining iTRAQ relative quantification methodology with the AXIMA-TOF2, two equimolar aliquots of a six-protein mixture were derivatized with iTRAQ reagents 114 and 117 respectively. Relative quantification calculations were performed on the reporter ions and highlighted a 1:1 ratio of 114:117 with an error of ±15%, which is entirely consistent with the literature. We have also established the necessity to acquire relative intensity information for more than four peptide pairs in order to reliably quantify the parent protein.