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.

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.

Long-term expression of foreign genes in normal human epidermal keratinocytes after transfection with lipid/DNA complexes.

Normal human epidermal keratinocytes were isolated and cultivated in serum-free medium. The expression of the integrin subunits alpha6 and beta1 indicated that a high number of keratinocytes from the stem cell system was present. These cells were transfected with complexes made of different cationic lipids and marker genes. Effectene showed a 20-fold higher transfection efficiency, compared to Lipofectin and Lipofectamine, and a similar low toxicity. The transfection protocol was optimised. A DNA/lipid ratio of 0.133 showed the highest transfection efficiency. Keratinocytes expressed the marker gene luciferase for 20 days. The maximum expression occurred after 3-4 days, where individual patches of fluorescent keratinocytes were detected. Transfected keratinocytes, cultivated at the air-liquid interface, expressed the marker gene beta-galactosidase for at least 7 weeks.

Canine osteosarcoma: amputation and chemoimmunotherapy.

Canine osteosarcoma is a highly metastatic cancer commonly seen in large breed dogs. At the time of diagnosis, approximately 90% to 95% of the dogs have established micrometastases. Dogs undergoing amputation alone have a median survival time of 3 to 4 months. Amputation followed by cisplatin chemotherapy increases median survival times to 9 to 11 months. When dogs are treated with amputation and cisplatin, followed by immunotherapy (with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine), median survival times increase to 14.4 months, the longest reported median survival time for dogs with osteosarcoma treated by amputation and any form of adjuvant therapy.

Direct chemiluminescence immunoassay (CLIA) for muramyl tripeptide phosphatidyl-ethanolamine in plasma.

A competitive chemiluminescent immunoassay for quantitation of muramyl tripeptide phosphatidyl-ethanolamine (MTP-PE) in plasma has been developed. The assay is based on the use of an acridinium ester-labelled analogue of muramyl tripeptide and a rabbit antiserum. It includes an overnight incubation and a separation with a second antibody covalently coupled to paramagnetic particles. The sensitivity of detection is 0.012 nmol/l, the assay working range is 0.1-5 nmol/l, and the inter-assay CVs are less than or equal to 10%. Using up to 6000-fold sample dilutions, a wide working range (0.1-30,000 nmol/l) is obtained. Rat plasma samples were collected during and one day after intravenous infusion of MTP-PE. Following infusion, the concentrations in plasma declined multiphasically. Half-life time was 0.37 h +/- 0.03 (mean +/- SD, alpha phase) and 1.76 h +/- 0.08 (mean +/- SD, beta phase), clearance and volume of distribution were 0.09 +/- 0.02 l/h x kg (mean +/- SD) and 0.06 +/- 0.01 l/kg (mean +/- SD) respectively. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the problems associated with reagents of limited shelf-life.