Multiple Reaction Monitoring (MRM)

Abstract

We previously reported on a method for the quantitative analysis of the 45 most abundant plasma proteins, based on liquid chromatography MRM/mass spectrometry, with quantitation accuracy provided by the use of stable isotopically labeled standard peptides. In this paper, we describe the use of this MRM method for the discovery of biomarkers characteristic of the presence or absence of coronary artery disease. Quantitative analysis of peptides specific for 44 of these abundant proteins was performed on plasma from a group of 38 patients classified as coronary artery disease-positive or coronary artery disease-negative. Statistical analysis of the results resulted in the discovery of a panel of 5 proteins with discriminative concentrations between patients with and without coronary artery disease, with a P value of <0.05. An initial evaluation of the analytic pipeline by cross-validation resulted in an estimated sensitivity and specificity of 74% for the test samples. This demonstrates that MRM-mass spectrometry-based quantitation offers a potential methodology for both biomarker discovery and biomarker validation. Similar to other discovery methodologies (eg, isobaric tags for relative and absolute quantitation), MRM-mass spectrometry provides the sensitivity and accuracy needed in the discovery phase, but, unlike the others, MRM-mass spectrometry offers the high throughput needed for clinical validation. Thus, this advantage eliminates the need for the development and validation of different discovery and clinical validation methods. ### Mass Spectrometry Mass spectrometry (MS) is an analytical chemistry technique based on the determination of the mass (actually, the mass-to-charge ratio) of an analyte ion. This analyte can be fragmented inside the mass spectrometer, which is under vacuum, to give charged product ions. This process is known as collision-induced dissociation . The masses of the fragment ions can then be determined in a second stage of MS, giving rise to the terms MS/MS or tandem mass spectrometry (see Figure 1a). Because …