Identification of oxidized phosphatidylcholines in nontargeted metabolomics

The present research was carried out in the laboratory of Prof. Coral Barbas, at the Center for Metabolomics and Bioanalysis (CEMBIO) of the Faculty of Pharmacy of the CEU-San Pablo University of Madrid, in the ambit of the Erasmus+ Program (coordinator Prof. Federica Pellati) and under the supervision of Prof. Joanna Godzien. Nontargeted metabolomics is a powerful high-throughput analytical approach, capable to unravel the yet-not-comprehended mechanisms of pathologies and discover biomarkers of disease. However, this approach suffers several limitations. Identification process in nontargeted metabolomics is known to be the major bottleneck in the whole procedure. The main reason for this is the great variety of chemicals in the human metabolome, together with the lack of pure standards for chemical structure confirmation and the limited amount of available information from databases. For these reasons, characterization of metabolites appears to be a major priority for the development of metabolomics. The study presented in this work focuses on the characterisation of the oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC). The interest in these products comes from their role in both pathogenesis of a great number of oxidative stress-related diseases. The aims of the present study are: The measurement of the accurate masses of the oxidation products of PAPC by mean of a liquid chromatography-mass spectrometry technique; The research of diagnostic signals for a rapid and reliable identification of oxidation within the phosphocholines based on tandem mass spectrometry; The elucidation of the product fragmentation pattern produced by the collision induced dissociation (CID) tandem mass spectrometry. In order to achieve these goals, in the present study a liquid chromatography-mass spectrometry-hyphenated technique (LC-MS) was employed and an oxPAPC standard (Avanti Polar Lipids, Inc. AL, USA) purchased. The wide range of metabolite coverage of LC, as well as the high sensitivity and accurate mass detection of a Q-TOF (quadrupole-time of flight) mass spectrometer, made this technique the most appropriate one for this study. After making a list from the scientific literature of all the known possible products arising from PAPC oxidation, an MS analysis was set in order to take a fingerprint of the standard mix. This allowed more masses to be added to the list for the tandem mass spectrometry (MS2) experiment. MS and MS2 spectra were inspected with Mass Hunter Qualitative software (Agilent, B.06.00). In conclusion, in this study, accurate masses in both MS negative and positive mode of PAPC's oxidation products were measured. The fragmentation pattern allowed the identification of these products as well. Furthermore, the diagnostic signals indicating the presence of oxidation within PCs were defined. These data are stored in an in-house database accessible to the database-mediator “CEU mass mediator” (CMM). Furthermore, a new functionality will be added to CMM for the identification of oxidized fatty acids from oxidized phosphocholines and semi-automated recognition of oxidation type.