Mass Spectrometry in High-Throughput Clinical Biomarker Assays: Multiple Reaction Monitoring

2012 ◽  
pp. 117-137 ◽  
Author(s):  
Carol E. Parker ◽  
Dominik Domanski ◽  
Andrew J. Percy ◽  
Andrew G. Chambers ◽  
Alexander G. Camenzind ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Clinical Biomarker

scholarly journals MitoPlex: A Targeted Multiple Reaction Monitoring Assay for Quantification of a Curated Set of Mitochondrial Proteins

2019 ◽  
Author(s):  
Aleksandr B. Stotland ◽  
Weston Spivia ◽  
Amanda Orosco ◽  
Allen M. Andres ◽  
Roberta A. Gottlieb ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Skeletal Muscle ◽  
High Throughput ◽  
Metabolic Flux ◽  
Multiple Reaction Monitoring ◽  
Mitochondrial Protein ◽  
Carbon Chain ◽  
Reaction Monitoring ◽  
In Vivo Models ◽  
Central Carbon

SummaryMitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed ‘MitoPlex’. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (218 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, ‘MitoPlex plus metabolites’ indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.

scholarly journals Calcineurin Activity Assay Measurement by Liquid Chromatography–Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode

2014 ◽  
Vol 60 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Lynn Carr ◽  
Anne-Laure Gagez ◽  
Marie Essig ◽  
François-Ludovic Sauvage ◽  
Pierre Marquet ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Mononuclear Cells ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Activity Assay ◽  
Peripheral Blood Mononuclear ◽  
Blood Concentrations ◽  
Transplant Patients

Abstract BACKGROUND Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical. We developed a new method amenable to routine use. METHODS Using liquid chromatography–multiple reaction monitoring mass spectrometry (LC-MRM-MS), we quantified CN activity by measuring the dephosphorylation of a synthetic phosphopeptide substrate. A stable isotope analog of the product peptide served as internal standard, and a novel inhibitor cocktail minimized dephosphorylation by other major serine/threonine phosphatases. The assay was used to determine CN activity in peripheral blood mononuclear cells (PBMCs) isolated from 20 CNI-treated kidney transplant patients and 9 healthy volunteers. RESULTS Linearity was observed from 0.16 to 2.5 μmol/L of product peptide, with accuracy in the 15% tolerance range. Intraassay and interassay recoveries were 100.6 (9.6) and 100 (7.5), respectively. Michaelis–Menten kinetics for purified CN were Km = 10.7 (1.6) μmol/L, Vmax = 2.8 (0.3) μmol/min · mg, and for Jurkat lysate, Km = 182.2 (118.0) μmol/L, Vmax = 0.013 (0.006) μmol/min · mg. PBMC CN activity was successfully measured in a single tube with an inhibitor cocktail. CONCLUSIONS Because LC-MRM-MS is commonly used in routine clinical dosage of drugs, this CN activity assay could be applied, with parallel blood drug concentration monitoring, to a large panel of patients to reevaluate the validity of PBMC CN activity monitoring.

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