This study aimed to investigate the human proteome profile of samples collected from whole (W) Schirmer strips (ScS) and their two parts—the bulb (B) and the rest of the strip (R)—with a comprehensive proteomic approach using a trapped ion mobility mass spectrometer, the timsTOF Pro. Eight ScS were collected from two healthy subjects at four different visits to be separated into three batches, i.e., 4W, 4B, and 4R. In total, 1582 proteins were identified in the W, B, and R batches. Among all identified proteins, binding proteins (43.4%) and those with catalytic activity (42.2%) constituted more than 80% of the molecular functions. The most represented biological processes were cellular processes (31.2%), metabolic processes (20.8%), and biological regulation (13.1%). Enzymes were the most represented protein class (41%), consisting mainly of hydrolases (47.5%), oxidoreductases (22.1%), and transferases (16.7%). The bulb (B), which is in contact with the conjunctiva, might collect both tear and cell proteins and therefore promote the identification of more proteins. Processing B and R separately before mass spectrometry (MS) analysis, combined with the high data acquisition speed and the addition of ion-mobility-based separation in the timsTOF Pro, can bring a new dimension to biomarker investigations of a limited sample such as tear fluid.

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 (t_R) 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 t_R 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 t_R prediction thus appears as a suitable and robust tool for the discovery of lipids involved in relevant biological processes.

Metabolomics in clinical toxicology aim at reliably identifying and semi-quantifying a broad array of endogenous and exogenous metabolites using dedicated analytical methods. Here, we developed a three-step-based workflow to investigate the metabolic impact of the antidepressant drug venlafaxine in a poisoned patient who developed life-threatening cardiac failure managed with extracorporeal membrane oxygenation. Both targeted quantitative and untargeted semi-quantitative metabolomic analyses using liquid chromatography hyphenated to high-resolution tandem mass spectrometry were performed to determine the plasma kinetics of venlafaxine, O-desmethyl-venlafaxine, and N-desmethyl-venlafaxine and to identify sixteen different venlafaxine-derived metabolites including one unknown (i.e., venlafaxine conjugated to a hexosyl-radical), respectively. Correlations between the quantitative metabolomic data and annotated endogenous metabolites suggested impaired amino acid and lipid metabolism, Krebs cycle, and kynurenine pathway. This preliminary study represents a first step towards a more extensive application of toxicometabolomics in clinical toxicology and a useful workflow to identify the biomarkers of toxicity.

Identification of xenobiotics and their phase I/II metabolites in poisoned patients remains challenging. Systematic approaches using bioinformatic tools are needed to detect all compounds as exhaustively as possible. Here, we aimed to assess an analytical workflow using liquid chromatography coupled to high-resolution mass spectrometry with data processing based on a molecular network to identify tramadol metabolites in urine and plasma in poisoned patients. The generated molecular network from liquid chromatography coupled to high-resolution tandem mass spectrometry data acquired in both positive and negative ion modes allowed for the identification of 25 tramadol metabolites in urine and plasma, including four methylated metabolites that have not been previously reported in humans or in vitro models. While positive ion mode is reliable for generating a network of tramadol metabolites displaying a dimethylamino radical in their structure, negative ion mode was useful to cluster phase II metabolites. In conclusion, the combined use of molecular networks in positive and negative ion modes is a suitable and robust tool to identify a broad range of metabolites in poisoned patients, as shown in a fatal tramadol-poisoned patient.

Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.