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J Biomol Tech. 2010 September; 21(3 Suppl): S34–S35.
PMCID: PMC2918007

MRM3 Quantitation for Highest Selectivity of Proteins in Complex Matrices

Abstract

RP-120

The use of Multiple Reaction Monitoring (MRM) combined with stable isotope labeled peptides for the quantification of proteins has been actively explored over the last few years and shows great promise for protein and biomarker research. MRM can significantly reduce interferences, providing a dramatic increase in selectivity, a very low baseline, excellent limits of quantitation, and very good linearity. In complex matrices or in higher throughput analysis where sample preparation is reduced or compressed, interferences are not always eliminated. Recently, we have been exploring a novel quantitative approach called MRM3, that combines the hybrid features of the QTRAP® 5500 system (triple quadrupole linear ion trap). In MRM3 analysis, analyte precursor ions are selected in the Q1 quadrupole, fragmented in the Q2 collision cell, and product ions are collected in the linear ion trap (LIT). A suitable product ion is isolated and fragmented in a second step using resonance excitation. Second-generation product ions are collected and scanned out of the LIT to the detector. This scan is performed in a looped fashion across an LC run and the area under the peak can be used for quantitation in the same way that MRM is used, however with dramatically increased specificity. This technique has been applied to a number of biological samples.When quantifying proteins in plasma, a 3-5x better lower limit of quantitation (LOQ) was obtained using MRM3 analysis over MRM through removal of interferences, with final detection limits of ~10 ng/mL. The standard concentration curves were linear over the 3 orders of dynamic range tested. MRM3 is an effective strategy for quantitation of analytes when high background or interferences make standard MRM quantitation difficult.


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