Protocol for the measurement of secreted acetylcholine in the lumen of mouse intestine using LC-MS

Abstract

Acetylcholine (ACh) is a small-molecule neurotransmitter that regulates a wide range of neuronal and non-neuronal physiological functions. The non-neuronal roles of ACh are highly tissue specific, requiring precise measurements of its concentration in situ. Here, we present a protocol for measuring secreted ACh in the lumen of the mouse intestine. We describe the procedures for sample collection, preparation, and metabolite purification. Finally, we detail the use of liquid chromatography-mass spectrometry (LC-MS) to quantify ACh levels. For complete details on the use and execution of this protocol, please refer to Ndjim et al.¹.

Keywords: Immunology; Metabolomics; Neuroscience.

Graphical abstract

Figure 1

Workflow of the ACh sample preparation and analysis

Figure 2

Calibration curve for acetylcholine quantification Plotted are mean peak areas resulting from 4 replicate injections of acetylcholine solutions in the concentration range between 0 nM to 2000 nM. Error bars indicate standard deviation of means.

Figure 3

Acetylcholine detection using MRM transitions Quantifier (left) and qualifiers (right) Acetylcholine peaks in 50 nM standards (A), 1000 nM standards (B) and in samples (C). Occasionally, contaminants can be observed in qualifiers and/or quantifier transitions, especially at low concentrations. See panels (A and C).