An Improved Two-Dimensional HPLC Method for Endogenous 2'-Deoxy-ADPR and 2'-Deoxy-NAD

Area of Science:

• Biochemistry
• Immunology
• Cell Biology

Background:

• Transient receptor potential melastatin 2 (TRPM2) channels are crucial in immune cells.
• 2 -deoxy-adenosine diphosphoribose (2d-ADPR) acts as a potent activator of TRPM2 channels.
• CD38 enzyme synthesizes 2d-ADPR from 2 -deoxy-nicotinamide adenine dinucleotide (2d-NAD).

Purpose of the Study:

• To develop and validate a method for quantifying endogenous 2d-ADPR and 2d-NAD.
• To establish the cell number required for reliable measurement of these metabolites.

Main Methods:

• Development of a two-dimensional High-Performance Liquid Chromatography (2D-HPLC) method.
• Quantification of 2d-ADPR and 2d-NAD in Jurkat T cells.

Main Results:

• The 2D-HPLC method successfully quantified endogenous 2d-ADPR and 2d-NAD.
• A sample size of 25 million Jurkat T cells was determined to be sufficient for accurate metabolite quantification.

Conclusions:

• A robust 2D-HPLC method is now available for measuring key TRPM2 channel regulators.
• This method enables further research into the roles of 2d-ADPR and 2d-NAD in immune cell function.