Robust iTRAQ peptide quantification on an LTQ-Orbitrap mass spectrometer
and its application to chemical proteomics
Bernhard Kuester1
1Technical University Munich
Isobaric stable isotope tagging reagents enable multiplexed quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. Until recently, the poor recovery of low mass fragments observed in tandem mass spectra acquired on ion trap mass spectrometers precluded the use of these reagents on this widely available instrument platform. The Pulsed Q Dissociation technique (PQD) allows negotiating this limitation but suffers from poor fragmentation efficiency. In this talk we show that by carefully optimizing instrument parameters, low m/z fragment ion intensities can be generated that enable accurate peptide quantification at the 100 amol level. In addition, side by side comparison of PQD on an LTQ-Orbitrap with CID on a quadrupole TOF instrument using complex protein digests shows that while precision of quantification of 10-15% can be achieved by both approaches, PQD quantifies twice as many proteins. PQD on an LTQ-Orbitrap is also more sensitive than ‘higher energy collision induced dissociation’ (HCD) on the same instrument but at the cost of precision. To demonstrate the significant analytical potential of iTRAQ quantification using PQD on an LTQ-Orbitrap, we applied this strategy to measure the kinase interaction profile of the small molecule drugs Imatinib, Bosutinib and Dasatinib in K562 cells. We show that IC50 values of binding can be obtained for hundreds of proteins in a single experiment.
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