Lipid Annotation by Combination of UHPLC-HRMS (MS), Molecular Networking, and Retention Time Prediction: Application to a Lipidomic Study of In Vitro Models of Dry Eye Disease

Abstract

Annotation of lipids in untargeted lipidomic analysis remains challenging and a systematic approach needs to be developed to organize important datasets with the help of bioinformatic tools. For this purpose, we combined tandem mass spectrometry-based molecular networking with retention time (tR) prediction to annotate phospholipid and sphingolipid species. Sixty-five standard compounds were used to establish the fragmentation rules of each lipid class studied and to define the parameters governing their chromatographic behavior. Molecular networks (MNs) were generated through the GNPS platform using a lipid standards mixture and applied to lipidomic study of an in vitro model of dry eye disease, i.e., human corneal epithelial (HCE) cells exposed to hyperosmolarity (HO). These MNs led to the annotation of more than 150 unique phospholipid and sphingolipid species in the HCE cells. This annotation was reinforced by comparing theoretical to experimental tR values. This lipidomic study highlighted changes in 54 lipids following HO exposure of corneal cells, some of them being involved in inflammatory responses. The MN approach coupled to tR prediction thus appears as a suitable and robust tool for the discovery of lipids involved in relevant biological processes.

Nicotinamide Mononucleotide Alleviates Hyperosmolarity-Induced IL-17a Secretion and Macrophage Activation in Corneal Epithelial Cells/Macrophage Co-Culture System

“…Through analyzing human corneal epithelial cells (CEC) exposed to hyperosmosis stress (HS) and control status, more than 150 unique phospholipids and sphingolipids were annotated and this lipid histochemical study highlighted the relationship between lipid changes in corneal cells exposed to HS and inflammatory responses. 4 Besides, in a previous study, it was found that HS would lead to an excessive reactive oxygen species release and lead to decreased autophagy and decreased cell activity in DED. 5 Protection of CEC in the pathological status would help in the management of DED and this research approach provided us various potential therapies for DED.…”

Proteomic Analysis of Tears and Conjunctival Cells Collected with Schirmer Strips Using timsTOF Pro: Preanalytical Considerations

“…The members of this molecular family are key mediators in intercellular and intracellular processes in both inflammatory processes and cell death phenomena [86,87]. Interestingly, these bioactive lipids and their modulation were also described in studies of an in vitro DED model [88,89]. This metabolic involvement is supported by the identification of several enzymes.…”

Bisphenol A Impairs Lipid Remodeling Accompanying Cell Differentiation in the Oligodendroglial Cell Line Oli-Neu

“…Chromatographic separation was achieved on an Acquity ® CSH C18 column (100 mm × 2.1 mm, 1.7 μm). UHPLC-HRMS data acquisition and analysis were adapted from a previously described protocol [ 17 ]. Data dependent acquisition was performed to provide tandem mass spectra of the five most intense ions detected on a first mass spectrum.…”