Range of protein detection by selected/multiple reaction monitoring mass spectrometry in an unfractionated human cell culture lysate

PROTEOMICS ◽  
2012 ◽  
Vol 12 (8) ◽  
pp. 1185-1193 ◽  
Author(s):  
H. Alexander Ebhardt ◽  
Eduard Sabidó ◽  
Ruth Hüttenhain ◽  
Ben Collins ◽  
Ruedi Aebersold
Keyword(s):  
Mass Spectrometry ◽  
Cell Culture ◽  
Human Cell ◽  
Multiple Reaction Monitoring ◽  
Protein Detection ◽  
Reaction Monitoring ◽  
Human Cell Culture ◽  
Cell Culture Lysate

scholarly journals The importance of TNAP/Tnap for dental development in human cell culture and in zebrafish

Bone Reports ◽  
2021 ◽  
Vol 14 ◽  
pp. 101006
Author(s):  
Stephanie Graser ◽  
Sofia Paulus ◽  
Elisa Riekert ◽  
Franz Jakob ◽  
Eva Klopocki ◽  
...  
Keyword(s):  
Cell Culture ◽  
Human Cell ◽  
Dental Development ◽  
Human Cell Culture

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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