BMC Plant Biol. 2010 Nov 18;10:252.

MRM Methods for High Precision Shift Measurements in H/DX-MS

Conventional methods for the measurement of deuterium exchange data suffer limitations arising from extensive deuteration protocols, MS domain data collection and short chromatographic run times. Together, these limitations create complex spectra with reduced dynamic range and the potential for significant spectral overlap. For applications of mass shift perturbation detection, we present an improved method for targeting sequence coverage, based upon multiple reaction monitoring (MRM) on a triple quadrupole platform. Two classes of MRM detection are described and validated, each requiring only two transitions per peptide. These methods were developed through simulations of MRM sampling of isotopic distributions, and validated by shift measurements for a range of peptides. An MRM method involving resolved offset transmission windows in Q1 and a fixed window in Q3 provides a generic and sensitive approach. Shift measurement precision by MRM is demonstrated to match that of conventional high resolution determinations (~5% RSD) and exceed the dynamic range by over two orders of magnitude in concentration. Optimized MRM methods were applied to a set of peptides selected to discriminate between two different classes of antimitotic drugs binding to a/b-tubulin. The precision of the MRM methods are comparable with higher resolution conventional methods, and additionally simplify data analysis. A set of guidelines is presented to facilitate rapid development of MRM shift assays for any peptides arising from H/DX workflows.

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