20. Multiple Reaction Monitoring Cubed (MRM3): A New Strategy for Clinical Validation of Putative Biomarkers in Crude Serum at Low nanogram/mL Level
Mon 4:54 PM - PosterSplash Track 2
Jean-Philippe Charrier
Tanguy Fortin, bioMerieux, Marcy L'étoile , France
Arnaud Salvador, Université de Lyon, Lyon 1, Lyon, France
Jean-Philippe Charrier, bioMerieux, Marcy L'étoile, France
Christof E. Lenz, Applied Biosystems Germany, Darmstadt, Germany
Florence Bettsworth, bioMerieux, Marcy L'étoile, France
Xavier Lacoux, bioMerieux, Marcy L'étoile, France
Geneviève Choquet-Kastylevsky, bioMerieux, Marcy L'étoile, France
Jérôme Lemoine, Université de Lyon, Lyon 1, Lyon, France
Stable isotope dilution-selected reaction-monitoring mass spectrometry (SID-SRM-MS), or stable isotope dilution-multiple reaction-monitoring mass spectrometry (SID-MRM-MS), carried out in triple quadrupole instruments has emerged as a promising alternative to ELISA for validation of putative protein biomarkers discovered during proteomics projects. However, mass spectrometry-based quantitation of plasma proteins in the low nanogram/mL range currently requires extensive sample fractionation steps which hampers its implementation within clinical laboratories.

In the present work, we describe a new method called Multiple Reaction Monitoring Cubed (MRM3) that significantly improves the specificity and sensitivity of detection of proteotypic peptides in comparison with conventional MRM.

The method consists of : i) Q1 selection at unit resolution of a proteotypic peptide ion ii) Q2 CID activation of the selected proteotypic peptide ion iii) Q3 trapping at unit resolution of the most intense fragment ion that defines an optimal SRM transition iv) activation of this species to generate fragment ions of the second generation, v) mass selective scan of the second generation fragment ions, vi) extraction of specifically defined signature of secondary product ions to reconstruct a chromatogram.

As an illustration, five proteins (TP171, TP435, TP574, Core NS4 and PSA) were spiked into human plasma samples at concentrations ranging from 5 nanograms to 1000 nanograms/mL. MRM3 reconstructed ion chromatograms were used to generate a standard concentration curve for each protein and from this, a limit of detection (LOD) in the low nanogram/mL range (between 5 and 50 ng/ml, depending on the protein) was observed. On average, we estimated a 5 fold improvement of this limit as compared to MRM mode because of the significant reduction in the background interfering species. Furthermore, MRM3 dramatically improved both the linearity of the response and the precision for the weakest concentration points because of the improved fidelity of the peak. The power of MRM3 method is illustrated by the assay of prostate specific antigen (PSA) in non-depleted sera of patients which correlated well with the ELISA tests.

In conclusion, MRM3 quantitation strategy provides much higher confidence towards the specificity of detection of peptides in complex mixtures and allows protein quantification at low nanogram/mL range in crude serum or plasma.