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Briefings in Functional Genomics and Proteomics Advance Access published online on June 25, 2008
Briefings in Functional Genomics and Proteomics, doi:10.1093/bfgp/eln032
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© The Author 2008. Published by Oxford University Press. For permissions, please email: journals.permissions@oxfordjournals.org

Proteomics: from hypothesis to quantitative assay on a single platform. Guidelines for developing MRM assays using ion trap mass spectrometers

Bomie Han and Richard E. Higgs

Corresponding author. Bomie Han, Lilly Corporate Center, Drop Code GL54, Indianapolis, IN 46285, USA. Tel: 317-433-5189; Fax: 317-277-0173; E-mail: bomiehan{at}lilly.com

High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.

Keywords: multiple reaction monitoring, assay validation, spiked recovery, HPLC-MS, quantification, proteomics, LTQ


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