Table 1.
Advantages and disadvantages of methods used for the quantification of adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) or for the measurement of phosphodiesterases (PDE) activity.
| Method | AMP | cAMP | PDE Enzyme Activity | Advantages | Disadvantages | Ref. |
|---|---|---|---|---|---|---|
| HPLC-MS/MS | + | + | HPLC is associated with speed, efficiency and accuracy; it can be automated and requires minimal training | can be costly, requires a large amount of expensive organics, has low sensitivity for certain compounds | [28] | |
| CD Spectroscopy | + | pre-steady and steady-state kinetics; real-time detection | micromolar detection limit | [29] | ||
| qPCR | + | dynamic range of quantification, high sensitivity and precision | not ideal for multiplexing, setting up requires high technical skills, high equipment cost, intra- and inter-assay variation, RNA lability, DNA contamination | [30] | ||
| IHC | + |
Fluorescent detection: multiplexing, good target co-localization, high dynamic range Chromogenic detection: sensitivity and long-lasting signal |
Fluorescent detection: low sensitivity, susceptible to photobleaching Chromogenic detection: narrow dynamic range |
[30] | ||
| ELISA immunoassays | + | + | accuracy, high sensitivity, specificity | laborious assay procedure | [31] | |
| RIA | + | implicity and high sensitivity | a scintillation counter is required | [32] | ||
| AlphaScreen assay | + | high sensitivity, rapid and simple, great dynamic range | requires special instrument capable to read A-counts | [33] | ||
| Transcreener assay | + | a single set of detection reagents can be used for all of the enzymes in a family and all acceptors substrates | no need for fluorescently labelled substrates, no signal background, expensive kit | [34] | ||
| IMAP-FP | + | an antibody-independent system | may interfere with other negatively charged reactants | [35] | ||
| IMAP-TR-FRET | + | ease of detection for kinase inhibitors | may interfere with ATP and other negatively charged compounds in high concentrations | [35] |
HPLC-MS: high performance liquid chromatography-mass spectrometry; CD: circular dichroism; IHC: immunohistochemistry; qPCR: quantitative polymerase chain reaction; ELISA: enzyme-linked immunosorbent assay; RIA: radioimmunoassay; IMAP/FP: IMAP (Immobilized Metal Ion Affinity Particle) assay in fluorescence polarization detection mode; IMAP/TR-FRET: IMAP assay/time-resolved fluorescence energy transfer.