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J Biomol Tech. 2007 February; 18(1): 71.
PMCID: PMC2292018

P204-T Targeted Quantitative Protein Analysis in Human Plasma Using High-Resolution Multiple Selected Reaction Monitoring Assays on a Triple Quadruple Mass Spectrometer

Abstract

There is huge need for discovery and validation of novel biomarkers for early diagnoses of various diseases. Usually, a common endpoint for a biomarker discovery experiment is a list of putative marker proteins, and a reasonable next step will be to perform targeted quantitative measurements of these proteins in an expanded patient population to assess their validity as markers. Analytical accuracy and precision are required for unambiguous quantitative analysis of these targeted proteins from complex biological fluids, such as human plasma/serum. Wide dynamic range and high sensitivity are also critical for detecting low-abundance proteins from the complex samples.

One approach for this application is the use of tandem mass spectrometry to monitor a unique peptide (or peptides) from a protein of interest by a selected reaction monitoring (SRM) assay, or by simultaneous analysis of many peptides by a multiple selected reaction monitoring (mSRM) assay. This approach can be extended further to provide absolute quantitation of targeted proteins by incorporation of appropriate stable isotope-labeled peptides as internal standards. While mSRM assays are sensitive for targeted peptides, in a complex matrix, such as human serum, assay selectivity can become a major issue. It is often difficult to differentiate between the targeted peptide signal and matrix background, particularly when quantifying many very low abundance proteins. The unique high-resolution SRM (h-SRM) capability of the TSQ Quantum Ultra can help to significantly overcome this problem and increase mSRM assay specificity.

In this presentation, we demonstrate the TSQ Quantum Ultra mass spectrometer’s unparalleled capability for highly sensitive and accurate multiple protein quantitation from human plasma by using high-resolution multiple reaction assays. Over 300 mSRM transition assays were developed for detecting major proteins and known biomarkers simultaneously from human plasma by using both unit mass resolution and high-resolution on the Q1 quadruple.


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