Detection of Intracellular Ceramide

Ceramide is an important intermediate in sphingolipid metabolism and is present in all eukaryotic cells. Ceramide consists of an even chain of fatty acids (2 to 28 carbons) linked to a sphingomyelin group through an amide bond. Ceramide is an important structural component of cell membranes, as well as an intracellular second messenger involved in the regulation of cell growth, proliferation, differentiation, senescence and apoptosis.

Synthetic Pathway of Ceramide

There are three major synthetic pathways for ceramide as follows:

(1) The ab initio pathway via palmitoyl coenzyme A and serine, which occurs in the endoplasmic reticulum

(2) The sphingolipid hydrolysis pathway that converts sphingolipids to ceramides, which occurs in the plasma membrane, lysosomes, Golgi apparatus, and mitochondria

(3) The salvage pathway that converts complex sphingolipids into sphingomyelin to generate ceramides via reacylation reactions, occurring in the lysosome and intracellular bodies.

Pathways of ceramide synthesis (Holthuis et al., 2001).

Intracellular Ceramide Assay

Different types of ceramides in cells can exert different or even opposite effects. Determination of various ceramides in biological samples is important to study the biological effect mechanism of ceramides.

The complexity of ceramide and the tediousness of the assay process have long prevented a rapid, effective and large-scale method for qualitative and quantitative determination of ceramides. The analysis of ceramides has been limited to the overall level or a particular ceramide, and the content of several ceramides in tissues or cells is rarely measured simultaneously and categorically. The exploration of multiple reaction monitoring (MRM) mode using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS) allows the simultaneous determination of different types of ceramides in cell extracts.

HPLC-MS provides an analytical tool for the quality evaluation and control of ceramide extracts, as well as technical support for the comprehensive analysis of intracellular ceramide content. Here is the general process:

• Configuration of ceramide stock solution and validation of assay system

Prepare ceramide standard stock solution and set aside. Aspirate each ceramide standard stock solution and dilute it step by step with methanol to prepare the standard curve series solution.

• Cell culture
• Cellular lipid extraction
• Ceramide qualitative analysis

The lipid extracted samples of the cells to be tested were subjected to a full scan of the parent ion mode. The precursor ion range is set at a mass-to-charge ratio of 300 to 1000 based on the relative molecular weight of the ceramide. The relative molecular weight of the compound is determined by the quasi-molecular ions of the obtained compound, and thus the possible types of ceramides are screened. The type of ceramide present in the cells to be tested is finally confirmed by comparing the results with the corresponding ceramide standard mass spectra.

• Ceramide quantitative analysis

Cellular lipid samples to be tested are extracted and ceramide is measured under the same chromatographic conditions, and the peak areas are recorded. The recovery of the additional internal standard is calculated first and used as the extraction rate of ceramide. Then the recoveries of the internal standards are used to adjust the peak areas of the other ceramides. The adjusted peak areas are then substituted into the respective standard curves and the content of each ceramide is calculated according to the external standard method. Finally, the total protein content (mg/mL) is used for standardization to obtain the content of each ceramide in ug/mg protein in the cells to be tested.

Creative Proteomics offers a platform for the ceramides quantitative analysis using HPLC-ESI-MS in multiple reaction detection mode with the advantages of high sensitivity, reproducibility, rapidity and accuracy, and without the need for sample derivatization.

Reference

1. Holthuis, J. C., Pomorski, T., Raggers, R. J., Sprong, H., & Van Meer, G. (2001). The organizing potential of sphingolipids in intracellular membrane transport. Physiological reviews, 81(4), 1689-1723.