New tools based on mass spectrometry approaches for the identification of nitrated complex lipids

Field: Chemistry (Physical Sciences)

Over the last years, the interest in products resulting from endogenous nitration reactions has increased, namely in nitro-fatty acids (NO2-FAs) that are considered as important signaling molecules. Although NO2-FA were detected in vivo as free species or adducted to proteins, esterified forms were only recently reported as nitrated phospholipid (NO2-PLs) and nitrated triacylglycerides (NO2-FA-TAG), together with the occurrence of lipoxidation adducts of NO2-PLs. The free, esterified and nucleophilic-adducted NO2-FA, generated in in vitro mimetic systems and in vivo, were detected using mass spectrometry (MS)-based approaches, coupled or not to liquid chromatography, with the identification of their typical fragmentation pattern and characteristic reporter ions under tandem MS (MS/MS) conditions. However, recent studies on the structural characterization of NO2-PLs allowed to suggest that both the information can vary depending on the type of mass spectrometer and the dissociation technique used to induce the fragmentation. This is a key issue since it can hinder the identification of these nitrated and nitroxidized derivatives in biological samples. Thus, an understanding of what analytical parameters are best to obtain the most useful information is a key issue for the identification of these modified PLs species. In this way, the aims of this work are (a) the identification of typical fragmentation patterns using different MS instrumental platforms to improve the accurate detection of the nitrated and nitroxidized derivatives of complex lipids obtained from in vitro biomimetic models systems, and (b) the development of lipidomic strategies to improve their identification in complex biological samples using the optimized high-throughput MS-based methodologies. To accomplish our goal, we first evaluate the influence of the normalized collision energy (NCE), under higher-energy collisional dissociation (HCD)- MS/MS conditions in Q-Exactive Orbitrap, in the fragmentation pattern to identify the most suitable acquisition conditions and reporter ions to detect nitrated PLs. This will contribute to identify and quantify these species in biological samples. Nitrated and nitroxidized PLs species were synthesized through an in vitro mimetic system of nitration with nitronium tetrafluoroborate (NO2BF4), which was used to nitrate phosphatidylcholine (PC) and phosphatidylethanolamine (PE) standards. The results showed that the intensity of the typical NL of nitrous acid (HNO2, 47 Da) diminishes with increasing NCE, becoming non-detectable for a higher NCE. However, fragment ions corresponding to the carboxylate anions of the modified fatty acyl chain were identified as potential reporter ions to detect nitrated PLs when using the HCD-MS/MS in lipidomics analysis. This methodology was applied in the analysis of cell lipid extracts treated with nitrated PC, which allowed the detection of NO2-PC and the validation of the developed methodology. The results obtained in this chapter revealed that the relative abundance of reporter ions of nitrated and nitroxidized PLs is significantly affected by the NCE applied. which in fact determines what fragment ions are observed, being a key factor for the successful of the detection of these nitrated PLs in biological samples. As the fragmentation fingerprinting of nitrated PLs obtained under HCDMS/MS conditions revealed to be dissimilar to the one previously reported under collision-induced dissociation (CID)-MS/MS, we evaluated the potential differences on the relative abundance of the typical reporter ions and fragmentation pattern of nitroso and nitro derivatives of different classes of PLs. These species were generated in vitro and their characterization was performed using MS/MS approaches based on two different ion activation methods: CID in a LXQ-Linear Trap (LIT) and HCD in a Q-Exactive Orbitrap. The results presented in this chapter revealed that the major differences between MS/MS spectra from CID-LXQ-LIT and HCD-Orbitrap are related with different relative abundance, and consequently distinct intensity, of specific reporter ions of nitrated PLs. Nitrated and nitroxidized triacylglycerides generated in vitro were also characterized by HCD-MS/MS in a Q-Exactive Orbitrap. Its fragmentation pattern included the typical NL of HNO2 with a high relative abundance, revealing to be more similar with the fragmentation pattern of nitrated PLs obtained in CID-LXQ-LIT. Overall, the results gathered in this thesis provide new insights into the nitrated and nitroxidized derivatives of esterified NO2-FA in PLs and TAG, and the identification of their fragmentation fingerprinting based on advanced HCDMS and MS/MS approaches. This information can be successfully used for targeted analysis of nitrated and nitroxidized lipids in biological samples, in different circumstances, namely in health and/or in disease conditions contributing to determine the biological properties and in vivo signaling actions of these nitrated molecules.