Selective and sensitive quantification of the cytochrome P450 3A4 protein in human liver homogenates through multiple reaction monitoring mass spectrometry

PROTEOMICS ◽  
2016 ◽  
Vol 16 (21) ◽  
pp. 2827-2837 ◽  
Author(s):  
Anna Cieślak ◽  
Isabelle Kelly ◽  
Jocelyn Trottier ◽  
Mélanie Verreault ◽  
Ewa Wunsch ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Human Liver ◽  
Multiple Reaction Monitoring ◽  
Cytochrome P450 3A4 ◽  
Reaction Monitoring ◽  
Liver Homogenates

Targeted Analysis of Eicosanoids Derived from Cytochrome P450 Pathway by High Resolution Multiple Reaction Monitoring Mass Spectrometry

2021 ◽  
Author(s):  
Tanize Acunha ◽  
Viviani Nardini ◽  
Ana Paula Ferranti Peti ◽  
Morgana Kelly Borges Prado ◽  
Luiz Alberto Beraldo Moraes ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
High Resolution ◽  
Multiple Reaction Monitoring ◽  
Reaction Monitoring ◽  
Targeted Analysis

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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