LIPID MAPS MASS SPECTROMETRY METHODS CHAPTERS DISCLAIMER: These chapters were written for the sole purpose of guiding qualified, professional scientists in the indicated laboratory procedures. Some of the procedures involve the use of chemicals or equipment that may be dangerous, particularly if improperly performed or if carried out by personnel that are not appropriately trained in laboratory procedures. The authors, editors, institutions, publisher, and associated companies have no responsibility whatsoever for any injuries, harm, damage to property or any monetary losses associated with the use of the procedures described in these chapters. The end user accepts all responsibility for use of the procedures described in these chapters. This work is provided on the LIPID MAPS website with the written permission of the Publisher and the entire volume may be viewed from the website http://www.sciencedirect.com/science/bookseries/00766879. CHAPTER ONE Qualitative Analysis and Quantitative Assessment of Changes in Neutral Glycerol Lipid Molecular Species Within Cells Jessica Krank,* Robert C. Murphy,* Robert M. Barkley,* Eva Duchoslav,† and Andrew McAnoy* Contents 1. Introduction 2 2. Reagents 3 2.1. Cell culture 3 2.2. Standards 3 2.3. Extraction and purification 3 3. Methods 4 3.1. Cell culture 4 4. Results 7 4.1. Qualitative analysis 7 4.2. Quantitative analysis 11 5. Conclusions 19 Acknowledgments 19 References 19 Abstract Triacylglycerols (TAGs) and diacylglycerols (DAGs) are present in cells as a complex mixture of molecular species that differ in the nature of the fatty acyl groups esterified to the glycerol backbone. In some cases, the molecular weights of these species are identical, confounding assignments of identity and quantity by molecular weight. Electrospray ionization results in the forma- þ tion of [MþNH4] ions that can be collisionally activated to yield an abundant product ion corresponding to the loss of ammonia plus one of the fatty acyl groups as a free carboxylic acid. A method was developed using tandem mass * Department of Pharmacology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado { Applied Research Group, MDS Sciex, Concord, Ontario, Canada Methods in Enzymology, Volume 432 # 2007 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)32001-6 All rights reserved. 1 2 Jessica Krank et al. spectrometry (MS) and neutral loss scanning to analyze the complex mixture of TAGs and DAGs present in cells and to quantitatively determine changes in TAGs and DAGs molecular species containing identical fatty acyl groups in an experi- mental series. Eighteen different deuterium-labeled internal standards were synthesized to serve to normalize the ion signal for each neutral loss scan. An example of the application of this method was in the quantitative analysis of TAG and DAG molecular species present in RAW 264.7 cells treated with a Toll-4 receptor ligand, Kdo2-lipid A, in a time course study. 1. Introduction Diacylglycerols and triacylglycerols represent major classes of glyceryl lipids that are present in all mammalian cells. Class analysis of these compounds has largely centered around total DAG or TAG content based upon a spectrophotometric-linked enzymatic assay; however, it is possible to deter- mine individual molecular species of both of these classes of glyceryl lipids by MS. The major challenge in analysis of DAGs and TAGs is the total number of molecular species present within a cell, which results in multiple components appearing at the same molecular weight or truly isobaric compounds. Thus, the measurement of only the molecular ion species by MS, either the ammo- nium adduct ion or the alkali attachment ion (Han and Gross, 2001; McAnoy et al.,2005), would be insufficient to uniquely identifying each component. Qualitative analysis can be carried out even when faced with complex mix- tures of these neutral lipids if one employs tandem MS in three steps (MS3) (McAnoy et al., 2005). However, quantitative analysis is confounded by the isobaric nature of multiple species, and several strategies have been developed through which changes in molecular species can be assessed. The method described here employs a series of experiments using deuterium- labeled internal standards where the deuterium atoms are placed in the glycerol backbone, and the abundance of each glyceryl lipid is compared to the signal for the internal standard. Alternative methods are available to assess TAG and DAG molecular species based upon chromatographic separa- tion of individual molecular species (Phillips et al., 1984) and analysis by gas chromatographs-mass spectrometers (GC-MS) (Myher et al.,1988)or liquid chromatographs-mass spectrometers (LC-MS) using electrospray ioni- zation (ESI) or atmospheric pressure chemical ionizationet (Ba,ly.rdwell 1996; Hsu and Turk, ).1 99T9he application of the method presented here will be exemplified by glyceryl lipid analysis in the RAW 264.7 cell line. These macrophage-like cells were originally derived from tumors induced in male BALB/c mice by the Abelson murine leukemia virus and respond to the Toll-4 receptor agonist Kdo2-lipid A (Raetz et al.,2006). Analysis of Glyceryl Lipids 3 2. Reagents 2.1. Cell culture RAW 264.7 cells were obtained from American Type Culture Collection (Manassas, VA). Tissue culture reagents included high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Cellgro, Herndon, VA), fetal calf serum (Hyclone, Logan, UT), Dulbecco’s Phosphate-Buffered Saline (D-PBS; Cellgro, Herndon, VA), and penicillin/streptomycin (Cellgro, Herndon, VA). The cells were carried in culture in either 150-cm2 tissue culture flasks with filter caps or 100-mm2 tissue culture dishes (Fisher Scientific, Fair Lawn, NJ). 2.2. Standards All deuterium-labeled glyceryl lipids were [1,1,2,3,3]-d5 in the glycerol backbone. The following lipids were obtained from Avanti Polar Lipids (Alabaster, AL): Kdo2-lipid A; d5-DAG mixture containing d5-14:0/14:0 DAG, d5-15:0/15:0 DAG, d5-16:0/16:0 DAG, d5-17:0/17:0 DAG, d5-19:0/19:0 DAG, d5-20:0/20:0 DAG, d5-20:2/20:2 DAG, d5-20:4/ 20:4 DAG, and d5-20:5/20:5 DAG; d5-TAG mixture containing d5-14:0/16:1/14:0 TAG, d5-15:0/18:1/15:0 TAG, d5-16:0/18:0/16:0 TAG, d5-17:0/17:1/17:0 TAG, d5-19:0/12:0/19:0 TAG, d5-20:0/20:1/ 20:0 TAG, d5-20:2/18:3/20:2 TAG, d5-20:4/18:2/20:4 TAG, and d5-20:5/22:6/20:5 TAG. 2.3. Extraction and purification High performance liquid chromatography (HPLC)–grade ammonium ace- tate (NH4OAc), chloroform (CHCl3), ethyl acetate, hexanes, isooctane, isopropanol, methanol, and methylene chloride (CH2Cl2) were obtained from Fisher Scientific (Fair Lawn, NJ). Optima-grade toluene and water also were obtained from Fisher Scientific (Fair Lawn, NJ). Discovery DSC-Si silica, and Discovery DSC-NH2 amino propyl solid phase extrac- tion cartridges, and the vacuum manifold were obtained from Supelco (Bellefonte, PA). Other equipment included a benchtop vortexer (Vortex Genie 2, VWR Scientific, West Chester, PA), Fluoropore 0.2-mmFG membrane filters (Millipore, Billerica, MA), 96-well polypropylene plates (Eppendorf, Westbury, NY), and Quant-iT DNA analysis kit (Molecular Probes, Eugene, OR). 4 Jessica Krank et al. 3. Methods 3.1. Cell culture RAW 264.7 cells were grown in T-150 flasks containing 30 ml of complete growth medium consisting of 500 ml of high-glucose DMEM supplemen- ted with 10% FBS and 1% penicillin/streptomycin. The cells were main- tained in a humidified incubator at 37 with a 5% CO2 atmosphere. They were split such that 2 Â 106 cells were seeded in a new flask each time the cells reached 80% confluency. Briefly, when the cells reached this con- fluency (approximately every 2 to 3 days), the old growth medium was removed, and 10 ml of fresh medium, which had been previously warmed to 37, was added. The cells were then scraped from the flask using a large disposable cell scraper. A 10-ml aliquot was counted using a hemocytometer. The volume of media determined to contain 2 Â 106 cells was then added to a new T-150 flask, and the volume was adjusted to 30 ml with complete growth medium. The flasks were then placed back into the incubator. For quantitative analysis, three additional T-150 flasks were seeded and allowed to grow until they were approximately 80% confluent. Once conflu- ent, the medium was removed, 10 ml of fresh medium was added to each flask, and the cells were scraped as before. The scraped cells from the three flasks were then combined, and a 10 ml aliquot was removed and counted. Cells (5 Â 106) were then added to each of the 15 100-mm2 tissue culture dishes. The volume of media in the dishes was adjusted to 5 ml with complete growth media. The dishes were then placed in the incubator and allowed to grow for 30 hr. 3.1.1. Kdo2-lipid A preparation A working solution of Kdo2-lipid A, the active component of bacterial endotoxin (Raetz, 1990), was prepared at a concentration of 100 mg/ml in DPBS. This solution was sonicated for 5 min before each experiment to achieve a uniformly opalescent suspension. 3.1.2. Stimulation and harvesting RAW 264.7 cells were treated with Kdo2-lipid A to examine the changes in the neutral glyceryl lipids upon activation of the Toll-4 receptor. Cells were stimulated by the addition of 5 ml of the Kdo2-lipid A working solution for a final concentration of 100 ng/ml. They were then incubated for 0.5, 1, 2, 4, 8, 12, and 24 hr.