Shotgun Analysis of Diacylglycerols Enabled by Thiol–ene Click Chemistry

Diacylglycerols (DAGs) are a subclass of neutral lipids actively involved in cell signaling and metabolism. Alteration in DAG metabolism has been associated with onset and progression of several human-related diseases. The structural diversity of DAGs and their low concentrations in biological samples call for the development of methods that are capable of sensitive identification and quantitation of each DAG species as well as rapid profiling when a biochemical pathway is perturbed. In this work, the thiol–ene click chemistry has been employed to introduce a charge-tag, namely, cysteamine (CA), at a carbon–carbon double bond (CC) of unsaturated DAGs. This one-pot photochemical derivatization is fast (within 1 min), universal (monotagging) for DAGs varying in fatty acyl chain lengths and the number of CCs, and suitable for small sample volume (e.g., 1–50 μL plasma). Because of the presence of the amine group in CA, tagged DAGs showed at least 10 times increase in response to electrospray ionization as compared to conventional ammonium adduct formation. Low-energy collision-induced dissociation of CA tagged DAGs allowed confident assignment of fatty acyl composition. A neutral loss scan based on characteristic 95 Da loss (a combined loss of CA and H₂O) of tagged DAGs has been established as a sensitive means for unsaturated DAG detection (limit of detection = 100 pM) and quantitation from mixtures. The analytical utility of CA tagging was demonstrated by shotgun analysis of unsaturated DAGs in human plasma, including samples from type 2 diabetes mellitus patients

Universal Multilayer Assemblies of Graphene in Chemically Resistant Microtubes for Microextraction

Graphene is a new kind of two-dimensional carbon nanomaterial with excellent properties and is promising for solid-phase microextraction (SPME). Plastic microtubes such as poly­(tetrafluoroethylene) (PTFE) and poly­(ether ether ketone) are ideal substrates for in-tube SPME. However, immobilization of graphene layers onto these materials is still a problem due to their nature of chemical resistance. In order to solve the problem, we proposed a novel method based on universal mussel-inspired polydopamine (PD) and layer-by-layer assembly of graphene in this work. To make a graphene assembly layer inside PTFE, the strategy includes two major steps. First, a PD layer is made on the PTFE surface by noncovalent interaction. Second, multilayer graphene is assembled on the PD layer by covalent interaction. By repeating these two steps, a functional graphene oxide (FGO)-modified PTFE tube with a controllable number of layers can be obtained. Morphology of the multilayer structure of graphene has been confirmed by scanning electronic microscopy. Formation of the covalent layer has also been characterized by Foourier transform infrared and X-ray photoelectron spectroscopy. It is very interesting that (FGO-PD)₃-PTFE shows exceptional efficiency for SPME. Enrichment from 1082- to 2331-fold was achieved for six polyaromatic hydrocarbons (PAHs). An online SPME-HPLC-fluorescent detection method has been developed on the basis of (FGO-PD)₃-PTFE. For qualitative analysis of PAHs, the method has low limits of detection of 0.05–0.1 pg/mL, which is significantly lower (up to 1000 times) than that reported in literature. The method shows wide linear range (0.3–200 pg/mL), good linearity (<i>R</i>² ≥ 0.9968), and good reproducibility (relative standard deviation < 3.4%). The method has been applied to determine PAHs in environmental samples. Good recoveries were obtained, ranging from 85.1% to 96.7%

LipidOA: A Machine-Learning and Prior-Knowledge-Based Tool for Structural Annotation of Glycerophospholipids

The Paternò–Büchi (PB) reaction is a carbon–carbon double bond (CC)-specific derivatization reaction that can be used to pinpoint the location(s) of CC(s) in unsaturated lipids and quantitate the location of isomers when coupled with tandem mass spectrometry (MS/MS). As the data of PB-MS/MS are increasingly generated, the establishment of a corresponding data analysis tool is highly needed. Herein, LipidOA, a machine-learning and prior-knowledge-based data analysis tool, is developed to analyze PB-MS/MS data generated by liquid chromatography–mass spectrometry workflows. LipidOA consists of four key functional modules to realize an annotation of glycerophospholipid (GPL) structures at the fatty acyl-specific CC location level. These include (1) data preprocessing, (2) picking CC diagnostic ions, (3) de novo annotation, and (4) result ranking. Importantly, in the result-ranking module, the reliability of structural annotation is sorted via the use of a machine learning classifier and comparison to the total fatty acid database generated from the same sample. LipidOA is trained and validated by four PB-MS/MS data sets acquired using different PB reagents on mass spectrometers of different resolutions and of different biological samples. Overall, LipidOA provides high precision (higher than 0.9) and a wide coverage for structural annotations of GPLs. These results demonstrate that LipidOA can be used as a robust and flexible tool for annotating PB-MS/MS data collected under different experimental conditions using different lipidomic workflows

MS³ Imaging Enables the Simultaneous Analysis of Phospholipid CC and <i>sn</i>-Position Isomers in Tissues

Mass spectrometry (MS) imaging of lipids in tissues with high structure specificity is challenging in the effective fragmentation of position-selective structures and the sensitive detection of multiple lipid isomers. Herein, we develop an MS3 imaging method for the simultaneous analysis of phospholipid CC and sn-position isomers by on-tissue photochemical derivatization, nanospray desorption electrospray ionization (nano-DESI), and a dual-linear ion trap MS system. A novel laser-based sensing probe is developed for the real-time adjustment of the probe-to-surface distance for nano-DESI. This method is validated in mouse brain and kidney sections, showing its capability of sensitive resolving and imaging of the fatty acyl chain composition, the sn-position, and the CC location of phospholipids in an MS3 scan. MS3 imaging of phospholipids has shown the capability of differentiation of cancerous, fibrosis, and adjacent normal regions in liver cancer tissues

Photochemical Tagging for Quantitation of Unsaturated Fatty Acids by Mass Spectrometry

Fatty acid (FA) profiling provides phenotypic information and is increasingly used in a broad range of biological and biomedical studies. Quantitation of unsaturated FAs with confident carbon–carbon double bond (CC) location assignment is both sample and time consuming using traditional gas chromatography mass spectrometry analysis. In this study, we developed a rapid, sensitive, and quantitative method for profiling unsaturated FAs without using chromatographic separations. This method was based on a combination of in-solution photochemical tagging of a CC in FAs and a subsequent gas-phase detagging via tandem (neutral loss scan) mass spectrometry. It enabled quantitation of unsaturated FAs from various biological samples (blood, plasma, and cell lines). More importantly, quantitative information on FA CC location isomers, which was traditionally overlooked, could now be obtained and applied to studying FA changes between normal and cancerous human prostate cells

Zero-Order Release of Gossypol Improves Its Antifertility Effect and Reduces Its Side Effects Simultaneously

Gossypol was considered a promising male contraceptive but finally failed due to two side effects: hypokalemia and the irreversibility of its contraceptive effect. Here we demonstrate that sustained zero-order release could be a solution for these problems. The in vitro release of gossypol from gossypol/PEG layer-by-layer films follows a perfect zero-order kinetics. In vivo tests indicate that the films can maintain the plasma drug concentration constant in male SD rats for ∼20 days for a 30-bilayer film. The plasma drug concentration is 2 orders of magnitude lower than the peak plasma drug concentration when administered orally and the daily dose is >50-fold lower than the commonly used contraceptive oral dose. However, significant antifertility effects were still observed. Furthermore, hypokalemia was not observed, and the antifertility effects can be reversed after a recovery period. The results suggest that zero-order release can significantly improve the desired antifertility effect of gossypol and, meanwhile, significantly reduce its side effects. We envision the drug could be developed to be an effective, safe, and reversible male contraceptive by zero-order release

Development of a Miniature Mass Spectrometry System for Point-of-Care Analysis of Lipid Isomers Based on Ozone-Induced Dissociation

Disorder of lipid homeostasis is closely associated with a variety of diseases. Although mass spectrometry (MS) approaches have been well developed for the characterization of lipids, it still lacks an integrated and compact MS system that is capable of rapid and detailed lipid structural characterization and can be conveniently transferred into different laboratories. In this work, we describe a novel miniature MS system with the capability of both ozone-induced dissociation (OzID) and collision-induced dissociation (CID) for the assignment of sites of unsaturation and sn-positions in glycerolipids. A miniature ozone generator was developed, which can be operated at a relatively high pressure. By maintaining high-concentration ozone inside the linear ion trap, OzID efficiency was significantly improved for the identification of CC locations in unsaturated lipids, with reaction times as short as 10 ms. Finally, the miniature OzID MS system was applied to the analysis of CC locations and sn-positions of lipids from biological samples. Direct sampling and fast detection of changes in phospholipid isomers were demonstrated for the rapid discrimination of breast cancer tissue samples, showing the potential of the miniature OzID MS system for point-of-care analysis of lipid isomer biomarkers in complex samples

Efficient Sample Preparation System for Multi-Omics Analysis via Single Cell Mass Spectrometry

Mass spectrometry (MS) has become a powerful tool for metabolome, lipidome, and proteome analyses. The efficient analysis of multi-omics in single cells, however, is still challenging in the manipulation of single cells and lack of in-fly cellular digestion and extraction approaches. Here, we present a streamlined strategy for highly efficient and automatic single-cell multi-omics analysis by MS. We developed a 10-pL-level microwell chip for housing individual single cells, whose proteins were found to be digested in 5 min, which is 144 times shorter than traditional bulk digestion. Besides, an automated picoliter extraction system was developed for sampling of metabolites, phospholipids, and proteins in tandem from the same single cell. Also, 2 min MS2 spectra were obtained from 700 pL solution of a single cell sample. In addition, 1391 proteins, phospholipids, and metabolites were detected from one single cell within 10 min. We further analyzed cells digested from cancer tissue samples, achieving up to 40% increase in cell classification accuracy using multi-omics analysis in comparison with single-omics analysis. This automated single-cell MS strategy is highly efficient in analyzing multi-omics information for investigation of cell heterogeneity and phenotyping for biomedical applications

Metabonomics Indicates Inhibition of Fatty Acid Synthesis, β‑Oxidation, and Tricarboxylic Acid Cycle in Triclocarban-Induced Cardiac Metabolic Alterations in Male Mice

Triclocarban (TCC) has been identified as a new environmental pollutant that is potentially hazardous to human health; however, the effects of short-term TCC exposure on cardiac function are not known. The aim of this study was to use metabonomics and molecular biology techniques to systematically elucidate the molecular mechanisms of TCC-induced effects on cardiac function in mice. Our results show that TCC inhibited the uptake, synthesis, and oxidation of fatty acids, suppressed the tricarboxylic acid (TCA) cycle, and increased aerobic glycolysis levels in heart tissue after short-term TCC exposure. TCC also inhibited the nuclear peroxisome proliferator-activated receptor α (PPARα), confirming its inhibitory effects on fatty acid uptake and oxidation. Histopathology and other analyses further confirm that TCC altered mouse cardiac physiology and pathology, ultimately affecting normal cardiac metabolic function. We elucidate the molecular mechanisms of TCC-induced harmful effects on mouse cardiac metabolism and function from a new perspective, using metabonomics and bioinformatics analysis data

Guaianolide Sesquiterpene Lactones, a Source To Discover Agents That Selectively Inhibit Acute Myelogenous Leukemia Stem and Progenitor Cells

Small molecules that can selectively target cancer stem cells (CSCs) remain rare currently and exhibit no common structural features. Here we report a series of guaianolide sesquiterpene lactones (GSLs) and their derivatives that can selectively eradicate acute myelogenous leukemia (AML) stem or progenitor cells. Natural GSL compounds arglabin, an anticancer clinical drug, and micheliolide (MCL), are able to reduce the proportion of AML stem cells (CD34⁺CD38^–) in primary AML cells. Targeting of AML stem cells is further confirmed by a sharp reduction of colony-forming units of primary AML cells upon MCL treatment. Moreover, DMAMCL, the dimethylamino Michael adduct of MCL, slowly releases MCL in plasma and in vivo and demonstrates remarkable therapeutic efficacy in the nonobese diabetic/severe combined immunodeficiency AML models. These findings indicate that GSL is an ample source for chemical agents against AML stem or progenitor cells and that GSL is potentially highly useful to explore anti-CSC approaches