Abstract
3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~106 cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1–500 ng/mL cell extract.
Highlights
3′, 5′ – Cyclic adenosine monophosphate is a ubiquitous second messenger that is involved in many cellular functions and bio logical processes [1], such as memory consolidation [2], immune function [3], and metabolism [4]. cAMP is synthesized in the cell by the enzyme adenylyl cyclase (AC) as a response to the binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs) [5]
The method allows for fast determination of cAMP in samples containing ~ 106 cells
The isocratic RPLC-MS/MS method here described for determina tion of cAMP in HT-29 epithelial cells showed a good sensitivity, se lectivity and repeatability
Summary
3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and bio logical processes [1], such as memory consolidation [2], immune function [3], and metabolism [4]. cAMP is synthesized in the cell by the enzyme adenylyl cyclase (AC) as a response to the binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs) [5]. As a more selective alternative, LC-MS/MS methods have been employed [13,14,15,16,17]. We here explored the use of an novel RPLC column which was com patible with a 100% aqueous mobile phase (often suited for highly polar analytes). The method is suited for extracts from ~106 cells per well using the well plate format. This approach provided satisfactory per formance (fast, selective and repeatable quantifications). The method was used to investigate the requirements for retrograde transport of CT to the Golgi apparatus, the ER and the release of its enzymatic moiety to the cytosol, where it increases the levels of cAMP
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