Wounding stress causes rapid increase in concentration of the naturally occurring 2',3'-isomers of cyclic guanosine- and cyclic adenosine monophosphate (cGMP and cAMP) in plant tissues.

3',5'-Cyclic guanosine monophosphate (cGMP) and 3',5'-cyclic adenosine monophosphate (cAMP) are well reported second messenger molecules involved in cellular signal transduction, in physiological functions such as neurotransmission in animals and in the modulation of cell growth and differentiation. In plants, 3',5'-cyclic nucleotides have been implicated in the regulation of ion homeostasis, hormone and stress responses. The behavior of the 2',3'-cyclic nucleotide variants is also known in animal tissue but no quantitative information is available about 2',3'-cAMP and 2',3'-cGMP in plant material. A recently developed HILIC-SPE/LC-MS/MS method for the analysis of cyclic nucleotides in blood and animal tissue was therefore adapted to measure 2',3'-cAMP and 2',3'-cGMP concentrations in plant material. Cyclic nucleotide concentrations were measured in Arabidopsis thaliana (Col-0) leaves before and after the application of wounding stress. A significant (∼5-fold) up-regulation of 2',3'-cAMP and 2',3'-cGMP was measured in Arabidopsis leaves compared to the control samples. The results indicate a thus far unreported strong correlation between plant stress and both 2',3'-cAMP and 2',3'-cGMP levels in plant material, and may open new avenues towards understanding the role of cyclic nucleotides in plants.

Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).