Ion mobility-mass spectrometry (IM-MS) has proven to be a highly informative technique for the characterization of lipids from cells and tissues. We report the combination of hydrophilic-interaction ...liquid chromatography (HILIC) with traveling-wave IM-MS (TWIM-MS) for comprehensive lipidomics analysis. Main lipid categories such as glycerolipids, sphingolipids, and glycerophospholipids are separated on the basis of their lipid backbones in the IM dimension, whereas subclasses of each category are mostly separated on the basis of their headgroups in the HILIC dimension, demonstrating the orthogonality of HILIC and IM separations. Using our previously established lipid calibrants for collision cross-section (CCS) measurements in TWIM, we measured over 250 CCS values covering 12 lipid classes in positive and negative modes. The coverage of the HILIC-IM-MS method is demonstrated in the analysis of Neuro2a neuroblastoma cells exposed to benzalkonium chlorides (BACs) with C10 or C16 alkyl chains, which we have previously shown to affect gene expression related to cholesterol and lipid homeostasis. We found that BAC exposure resulted in significant changes to several lipid classes, including glycerides, sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines. Our results indicate that BAC exposure modifies lipid homeostasis in a manner that is dependent upon the length of the BAC alkyl chain.
Collision cross section (CCS) measurement of lipids using traveling wave ion mobility-mass spectrometry (TWIM-MS) is of high interest to the lipidomics field. However, currently available calibrants ...for CCS measurement using TWIM are predominantly peptides that display quite different physical properties and gas-phase conformations from lipids, which could lead to large CCS calibration errors for lipids. Here we report the direct CCS measurement of a series of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in nitrogen using a drift tube ion mobility (DTIM) instrument and an evaluation of the accuracy and reproducibility of PCs and PEs as CCS calibrants for phospholipids against different classes of calibrants, including polyalanine (PolyAla), tetraalkylammonium salts (TAA), and hexakis(fluoroalkoxy)phosphazines (HFAP), in both positive and negative modes in TWIM-MS analysis. We demonstrate that structurally mismatched calibrants lead to larger errors in calibrated CCS values while the structurally matched calibrants, PCs and PEs, gave highly accurate and reproducible CCS values at different traveling wave parameters. Using the lipid calibrants, the majority of the CCS values of several classes of phospholipids measured by TWIM are within 2% error of the CCS values measured by DTIM. The development of phospholipid CCS calibrants will enable high-accuracy structural studies of lipids and add an additional level of validation in the assignment of identifications in untargeted lipidomics experiments.
Ion mobility-mass spectrometry (IM-MS) can provide orthogonal information, i.e., m/z and collision cross section (CCS), for the identification of drugs and drug metabolites. However, only a small ...number of CCS values are available for drugs, which limits the use of CCS as an identification parameter and the assessment of structure–function relationships of drugs using IM-MS. Here, we report the development of a rapid workflow for the measurement of CCS values of a large number of drug or drug-like molecules in nitrogen on the widely available traveling wave IM-MS (TWIM-MS) platform. Using a combination of small molecule and polypeptide CCS calibrants, we successfully determined the nitrogen CCS values of 1425 drug or drug-like molecules in the MicroSource Discovery Systems’ Spectrum Collection using flow injection analysis of 384-well plates. Software was developed to streamline data extraction, processing, and calibration. We found that the overall drug collection covers a wide CCS range for the same mass, suggesting a large structural diversity of these drugs. However, individual drug classes appear to occupy a narrow and unique space in the CCS–mass 2D spectrum, suggesting a tight structure–function relationship for each class of drugs with a specific target. We observed bimodal distributions for several antibiotic species due to multiple protomers, including the known fluoroquinolone protomers and the new finding of cephalosporin protomers. Lastly, we demonstrated the utility of the high-throughput method and drug CCS database by quickly and confidently confirming the active component in a pharmaceutical product.
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•Lipidomic alterations in E. coli with defected PG and CL synthesis are characterized by HILIC-IM-MS.•In the absence of phosphatidylglycerol synthase, PE-derived lipids with ...cyclopropane fatty acids are decreased.•Knock-out of cardiolipin synthases A, B, and C results in an accumulation of PG-derived lipids, including acylPGs.•HILIC-IM-MS can resolve 1- and 2-lysoPEs in both the chromatographic and IM dimensions.•The calibrated CCS values of cardiolipins, acylPGs and N-acylPEs are reported for the first time.
Our understanding of phospholipid biosynthesis in Gram-positive and Gram-negative bacteria is derived from the prototypical Gram-negative organism Escherichia coli. The inner and outer membranes of E. coli are largely composed of phosphatidylethanolamine (PE), minor amounts of phosphatidylglycerol (PG) and cardiolipin (CL). We report here the utility of hydrophilic interaction liquid chromatography (HILIC) paired with ion mobility-mass spectrometry (IM-MS) for the comprehensive analysis of the E. coli lipidome. Using strains with chromosomal deletions in the PG and CL synthesis genes pgsA and clsABC, respectively, we show that defective phospholipid biosynthesis in E. coli results in fatty-acid specific changes in select lipid classes and the presence of the minor triacylated phospholipids, acylphosphatidyl glycerol (acylPG) and N-acylphosphatidylethanolamine (N-acylPE). Notably, acylPGs were accumulated in the clsABC-KO strain, but were absent in other mutant strains. The separation of 1-lyso and 2-lyso-phosphatidylethanolamines (lysoPEs) is demonstrated in both the HILIC and IM dimensions. Using our previously validated calibration method, collision cross section values of nearly 200 phospholipids found in E. coli were determined on a traveling wave IM-MS platform, including newly reported values for cardiolipins, positional isomers of lysoPEs, acylPGs and N-acylPEs.
Lipidomics is a rapidly growing field that enables the characterization of the entire lipidome in cells, tissues, or an organism. Changes in lipid metabolism and homeostasis caused by different ...disease states or drug treatments can be probed by lipidomics experiments, which can aid our understanding of normal physiology and disease pathology at the molecular level. While current technologies using liquid chromatography coupled with high-resolution mass spectrometry have greatly increased coverage of the lipidome, there are still limitations in resolving the large number of lipid species with similar masses in a narrow mass window. We recently reported that two orthogonal separation techniques, hydrophilic interaction liquid chromatography (HILIC) and ion mobility (IM), enhance the resolution of lipid species based on headgroup polarity and gas-phase size and shape, respectively, of various classes of glycerolipids, glycolipids, phospholipids, and sphingolipids. Here we describe the application of our HILIC-IM-MS lipidomics protocol to the analysis of lipid extracts derived from either tissues or cells, to identify significant changes in the lipidome in response to an internal or external stimulus, such as exposure to environmental chemicals.
Rate constants for autoxidation propagation of several unsaturated lipids in benzene solution at 37 °C and in phosphatidylcholine liposomes were determined by a linoleate radical clock. This radical ...clock is based on competition between hydrogen atom abstraction by an intermediate peroxyl radical derived from linoleic acid that leads to a trans,cis-conjugated hydroxyoctadecadienoic product and β-fragmentation of the same peroxyl that gives the trans,trans-product hydroxyoctadecadienoic acid. Rate constants determined by this approach in solution relative to linoleic acid (k p = 62 M−1 s−1) were: arachidonic acid (k p = 197 ± 13 M−1 s−1), eicosapentaenoic acid (k p = 249 ± 16 M−1 s−1), docosahexaenoic acid (k p = 334 ± 37 M−1 s−1), cholesterol (k p= 11 ± 2 M−1 s−1), and 7-dehydrocholesterol (k p= 2260 ± 40 M−1 s−1). Free radical oxidations of multilamellar and unilamellar liposomes of various mixtures of glycerophosphatidylcholine molecular species were also carried out. In some experiments, cholesterol or 7-dehydrocholesterol was incorporated into the lipid mixture undergoing oxidation. A phosphatidylcholine bearing a linoleate ester at sn-2 was a component of each liposome peroxidation reaction and the ratio of trans,cis/trans,trans (t,c/t,t)-conjugated diene oxidation products formed from this phospholipid was determined for each oxidation reaction. This t,c/t,t-product ratio from linoleate was used to “clock” liposome constituents as hydrogen atom donors in the lipid bilayer. Application of this lipid bilayer radical clock gives relative autoxidation propagation rate constants of arachidonate (20:4), eicosapentaenoate (20:5), docosahexaenoate (22:6), and 7-dehydrocholesterol to be 115 ± 7, 145 ± 8, 172 ± 13, and 832 ± 86, respectively, a reactivity trend that parallels the one in solution. We also conclude from the liposome oxidations that linoleate peroxyl radicals at different positions on the eighteen-carbon chain (at C-9 and C-13) have different kinetic properties. This is in contrast to the results of solution oxidations of linoleate in which the C-9 and C-13 peroxyl radicals have similar reactivities. We suggest that peroxyl radical β-scission depends on solvent polarity and the polarity of the local environment of peroxyl radicals in liposomal oxidations depends on the position of the peroxyl radical on the 18-carbon chain.
The maximum power point tracking (MPPT) is a strategy that allows imposing the PV array operation point on the maximum power point (MPP) or close to it under any environmental condition. The ...conventional incremental conductance (INC) algorithm is the most popular algorithm. But due to the fixed step size, its response speed is low under the rapid change of the solar irradiation level or load resistance. In this paper, a new MPPT technique is proposed to enhance the response speed. It consists of two stages: (1) the computing stage and (2) the regulating stage. The computing stage includes the coarse positioning operation and fine positioning operation. And an initial value of the duty cycle is generated in the computing stage, according to the characteristics of the DC-DC converter and the characteristics of the I‐V curve. The regulating stage regulates the duty cycle of the DC-DC converter with a small step size, which can improve the tracking efficiency. And the computing stage can enhance the response speed. A simulation comparison of the proposed MPPT technique with other techniques is carried out in MATLAB/Simulink under different scenarios. The simulation results reveal that the response of the proposed algorithm is 4.6 times faster than that of the INC under these scenarios, and the proposed algorithm has higher efficiency.
Free radical chain oxidation of highly oxidizable 7-dehydrocholesterol (7-DHC), initiated by 2,2′-azobis(4-methoxy-2,4-dimethylvaleronitrile), was carried out at 37 °C in benzene for 24 h. Fifteen ...oxysterols derived from 7-DHC were isolated and characterized with 1D and 2D NMR spectroscopy and mass spectrometry. A mechanism that involves abstraction of hydrogen atoms at C-9 and/or C-14 is proposed to account for the formation of all of the oxysterols and the reaction progress profile. In either the H-9 or H-14 mechanism, a pentadienyl radical intermediate is formed after abstraction of H-9 or H-14 by a peroxyl radical. This step is followed by the well-precedented transformations observed in peroxidation reactions of polyunsaturated fatty acids such as oxygen addition, peroxyl radical 5-exo cyclization, and SHi carbon radical attack on the peroxide bond. The mechanism for peroxidation of 7-DHC also accounts for the formation of numerous oxysterol natural products isolated from fungal species, marine sponges, and cactaceous species. In a cell viability test, the oxysterol mixture from 7-DHC peroxidation was found to be cytotoxic to Neuro2a neuroblastoma cells in the micromolar concentration range. We propose that the high reactivity of 7-DHC and the oxysterols generated from its peroxidation may play important roles in the pathogenesis of Smith−Lemli−Opitz syndrome, X-linked dominant chondrodysplasia punctata, and cerebrotendinous xanthomatosis, all of these being metabolic disorders characterized by an elevated level of 7-DHC.
Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive human disease caused by mutations in the gene encoding 7-dehydrocholesterol (7DHC) reductase (DHCR7), resulting in abnormal accumulation of ...7DHC and reduced levels of cholesterol in bodily tissues and fluids. A rat model of the disease has been created by treating normal rats with the DHCR7 inhibitor, AY9944, which causes progressive, irreversible retinal degeneration. Herein, we review the features of this disease model and the evidence linking 7DHC-derived oxysterols to the pathobiology of the disease, with particular emphasis on the associated retinal degeneration. A recent study has shown that treating the rat model with cholesterol plus suitable antioxidants completely prevents the retinal degeneration. These findings are discussed with regard to their translational implications for developing an improved therapeutic intervention for SLOS over the current standard of care.
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