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The analysis of phosphopeptides from complex protein mixtures remains a challenging procedure. APAF uses an Applied Biosystems 4000 Q-Trap hybrid quadrupole linear ion-trap instrument. This instrument is well suited to the analysis of post-translational modifications as diagnostic fragment ions can be detected by precursor ion scanning. In this scan type, Q1 is scanned across a mass range and ions are fragmented in Q2. Q3 is set to transmit only the mass of the diagnostic fragment, (-79 m/z in the case of a phosphopeptide). In a follow-up experiment, precursor ions are selected from fragmentation and product ions scanned out of the ion-trap to provide MS/MS sequence information. Often the site of phosphorylation can be determined from this spectra.
iTRAQ™ reagents are isobaric amine-specific reagents that allow relative quantitation of up to four different samples in a single MS/MS experiment. iTRAQ™ reagents eliminate the need of conducting separate MS experiments to compare expression changes. A further advantage is that quantitative values are obtained for all identified proteins from each of the four mixed analytes - meaning there are no "gaps" in data tables. iTRAQ™ experiments can usually conducted via LC/MS/MS, with the preferred approach utilizing a 2D-nanoLC/MS/MS system and Q-STAR mass spectrometer. For more technical information regarding iTRAQ™ visit (www.appliedbiosystems.com).
N-linked oligosaccharides can be released from 2D gel or PVDF spots. The released oligosaccharides are analysed by LC ESI MS/MS. The glycoprotein can also be identified after the release of the oligosaccharide using MALDI MS or nanoLC ESI MS/MS technique.
This technique can be used to analysis targeted peptides and proteins. The accurate mass and the MS/MS of the parent peptide ions will be acquired. Analysis from the MS/MS data would produce the sequence of the peptides.
For further details or advice on this APAF service please contact us at ms@proteome.org.au. | |
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