Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry.

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.

Nanoflow liquid chromatography-tandem mass spectrometry for the characterization of intact phosphatidylcholines from soybean, bovine brain, and liver.

Nanoflow liquid chromatography-electrospray ionization tandem-mass spectrometry (nanoLC-ESI-MS-MS) was applied for the characterization of intact phosphatidylcholine (PC) lipid molecules using a homemade reversed phase capillary column with a pulled tip for direct ESI at positive ion mode. Prior to the analytical column, a short capillary trapping column was utilized for on-line pre-concentration via microcross connection. Separation of intact phosphatidylcholines in the nanoflow LC column was carried out using a binary gradient elution method at 300 nL/min. The structures of the eluted PC components were determined by analysis of the typical fragment ions of PC molecules obtained from collision-induced dissociation (CID) after each precursor scan in mass spectrometry. In the current study, nanoflow LC-ESI-MS-MS analysis of PC molecules demonstrated the ability to obtain clear structural information, such as alkyl chain lengths and the degree of unsaturation with a protonated molecule ([M + H]+) and its characteristic fragment ions ([M + H-RCH2COOH]+, [M + H-RCH=C=O]+, and [M + H-184]+). Results from the nanoflow LC-ESI-MS experiment showed the limit of detection at 3.5 fmol for the 14:0/14:0-PC standard. This technique then was applied to intact PC extracts from soybean, bovine brain, and liver without derivatization and resulted in the identification of 28, 25, and 39 phosphatidylcholines, respectively. The LC-MS-MS method has been shown to be useful for the analysis of low concentration PC molecules in biological samples.

Quantitative lipidomic analysis of plasma and plasma lipoproteins using MALDI-TOF mass spectrometry.

Knowledge of the plasma lipid composition is essential to clarify the specific roles of different lipid species in various pathophysiological processes. In this study, we developed an analytical strategy combining high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and off-line coupling with matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF/MS) to determine the composition of plasma and major lipoproteins at two levels, lipid classes and lipid species. We confirmed the suitability of MALDI-TOF/MS as a quantitative measurement tool studying the linearity and repeatability for triglycerides (TG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, data obtained with this method were correlated with other lipid classes and species measurements using currently available technologies. To establish the potential utility of our approach, human plasma very low density- (VLDL), low density- (LDL) and high density- (HDL) lipoproteins from 10 healthy donors were separated using ultracentrifugation, and compositions of nine lipid classes, cholesteryl esters (CE), TG, free cholesterol (FC), PE, phosphatidylinositol (PI), sulfatides (S), PC, lysophosphatidylcholine (LPC) and sphingomyelin (SM), analyzed. In total, 157 lipid species in plasma, 182 in LDL, 171 in HDL, and 148 in VLDL were quantified. The lipidomic profile was consistent with known differences in lipid classes, but also revealed unexpected differences in lipid species distribution of lipoproteins, particularly for LPC and SM. In summary, the methodology developed in this study constitutes a valid approach to determine the lipidomic composition of plasma and lipoproteins.

Preparation of 13 or 9-hydroperoxy-9Z,11E (9E,11E) or 10E,12Z (10E,12E)-octadecadienoic phosphatidylcholine hydroperoxide.

Increasing evidence of phospholipid peroxidation in the pathophysiology of various diseases demonstrates the need for pure phosphatidylcholine hydroperoxide (PCOOH) reference as a standard for quantification, and for biological studies in this field. Our previous study showed the usability of reaction between hydroperoxide and vinyl ether for preparation of PCOOH. However, the reaction has sometimes provided brown byproducts. Here, we report a method to improve reaction conditions, and demonstrate the production of 30 mg of PCOOH (a pure mixture of 13- and 9-hydroperoxyoctadecadienoyl PCOOH isomers) from 100 mg of phosphatidylcholine without byproduct formation. The resultant PCOOH will be useful as a standard for quantification studies, as well as for the evaluation of PCOOH pathogenicity.

A system establishment compatibility profiles for artificial oxygen carriers and other substances.

Worldwide, great efforts are being made to develop a clinically useful artificial oxygen carrier. Toxicological and immunological compatibility is generally tested using animal experiments but inflammatory parameters in particular show large species-specific differences. Therefore, we developed an in vitro system using human components to establish a compatibility profile of unknown compounds. The test system comprises induction of hemolysis, activation of complement (C3a), induction/suppression of cytokine production, influence on cell proliferation, direct toxicity on peripheral leukocytes, and phagocytosis of the material under test and of microbes. The test system will be described, along with results of various perfluorocarbon emulsions. When testing lecithin-based perfluorodecalin (PFD) emulsions, and comparing them to Pluronic-based PFD emulsions, we could show that Pluronic-based emulsions were virtually untoxic to peripheral human leukocytes. They neither inhibited cell proliferation nor caused any hemolysis, but caused mild to moderate inhibition of endotoxin-induced cytokine production. At the same time, lecithin-based PFD emulsion caused substantial cytotoxicity in phagocytic cells like monocytes (60-100% after 24 h incubation) and granulocytes (10-20% after 24 h incubation). They also suppressed endotoxin-induced cytokine production in monocytes to more than 98% and inhibited cell proliferation of an endothelial (ECV 304) and a monocytic cell line (MonoMac6) to more than 95%.