Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers
2008; Elsevier BV; Volume: 50; Issue: 8 Linguagem: Inglês
10.1194/jlr.d800051-jlr200
ISSN1539-7262
AutoresRebecca L. Shaner, Jeremy C. Allegood, Hye Jung Park, Elaine Wang, Samuel Kelly, Christopher A. Haynes, M. Cameron Sullards, Alfred H. Merrill,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoSphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. JLR LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations. Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. JLR LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations. Sphingolipids are an amazingly complex family of compounds found in eukaryotes as well as some prokaryotes and viruses. They are involved in many aspects of cell structure, metabolism, and regulation (1Lahiri S. Futerman A.H. The metabolism and function of sphingolipids and glycosphingolipids.Cell. Mol. Life Sci. 2007; 64: 2270-2284Crossref PubMed Scopus (258) Google Scholar, 2Merrill Jr., A.H. Wang M.D. Park M. Sullards M.C. 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Structure-specific, quantitative methods for analysis of sphingolipids by liquid chromatography-tandem mass spectrometry: "inside-out" sphingolipidomics.Methods Enzymol. 2007; 432: 83-115Crossref PubMed Scopus (97) Google Scholar), especially when tandem mass spectrometry (MS/MS) with electrospray ionization (ESI) is combined with liquid chromatography (LC) to minimize overlap of isomers such as glucosylceramide (GlcCer) and galactosylceramide (GalCer) (15Merrill Jr., A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.Methods. 2005; 36: 207-224Crossref PubMed Scopus (456) Google Scholar, 34Sullards M.C. Allegood J.C. Kelly S. Wang E. Haynes C.A. Park H. Chen Y. Merrill Jr., A.H. Structure-specific, quantitative methods for analysis of sphingolipids by liquid chromatography-tandem mass spectrometry: "inside-out" sphingolipidomics.Methods Enzymol. 2007; 432: 83-115Crossref PubMed Scopus (97) Google Scholar). Quantitative analysis of lipids by mass spectrometry requires internal standards to control for variability in recovery from the biological material and factors that can affect ion yield. The ideal internal standard is a stable isotope-labeled version of each analyte of interest, but it is impractical for these to be used for the large numbers of compounds examined in a lipidomic study. Therefore, an alternative is to identify internal standards that are similar in structure and ionization and fragmentation characteristics for the categories of compounds under investigation. This study evaluates an internal standard cocktail (now commercially available) developed for the LIPID MAPS Consortium (34Sullards M.C. Allegood J.C. Kelly S. Wang E. Haynes C.A. Park H. Chen Y. Merrill Jr., A.H. Structure-specific, quantitative methods for analysis of sphingolipids by liquid chromatography-tandem mass spectrometry: "inside-out" sphingolipidomics.Methods Enzymol. 2007; 432: 83-115Crossref PubMed Scopus (97) Google Scholar) that contains uncommon chain-length sphingoid bases (C17) for sphingosine (So), sphinganine (Sa) and the 1-phosphates (S1P and Sa1P) and C12:0 fatty acid analogs of Cer (and, as discussed in the text, the cocktail initially also had C25:0-Cer), ceramide 1-phosphate (Cer1P), SM and mono- and dihexosylCer (HexCer and diHexCer). Examples are also given for how the types of analytes can be expanded by supplementation with additional internal standards. Because it is common in mass spectrometry for laboratories to have different categories of instruments available, this article describes the optimization and validation of the sphingolipid internal standard cocktail by electrospray tandem mass spectrometry on two types of instruments, a triple quadrupole mass spectrometer and a quadrupole linear-ion trap mass spectrometer, both operating in the triple quadrupole modes. The analytes of interest were identified and quantified by LC ESI-MS/MS using multiple reaction monitoring (MRM), a technique where the eluate is repetitively scanned for selected precursor-product ion pairs to enhance the sensitivity and specificity of the analysis. The method is applicable to relatively small samples (e.g., a Petri dish of cells), as exemplified by analysis of RAW264.7 cells, a mouse macrophage cell line. The LIPID MAPS™ internal standard cocktail (initially Sphingolipid Mix I, catalog number LM-6002; later replaced by Sphingolipid Mix II, catalog number LM-6005 as explained under "Results") was provided by Avanti Polar Lipids (Alabaster, AL) in sealed ampules and certified (35Moore J.D. Caufield W.V. Shaw W.A. Quantitation and standardization of lipid internal standards for mass spectroscopy.Methods Enzymol. 2007; 432: 351-367Crossref PubMed Scopus (24) Google Scholar) to be over 95% pure and within 10% of the specified amount (25 µM). It was composed of the 17-carbon chain length sphingoid base analogs C17-sphingosine, (2S,3R,4E)-2-aminoheptadec-4-ene-1,3-diol (d17:1-So) 3This nomenclature designates the backbone sphingoid base by number of hydroxyls (e.g., d = di-) and carbon atoms:double bonds; if there is an amide-linked fatty acid, it is designated by "C" followed by the number of carbon atoms:double bonds.; C17-sphinganine, (2S,3R)-2-aminoheptadecane-1,3-diol (d17:0-Sa); C17-sphingosine 1-phosphate, heptadecasphing-4-enine-1-phosphate (d17:1-So1P); and C17-sphinganine 1-phosphate, heptadecasphinganine-1-phosphate (d17:0-Sa1P); and the C12-fatty acid analogs of the more complex sphingolipids C12-Cer, N-(dodecanoyl)-sphing-4-enine (d18:1/C12:0); C12-Cer 1-phosphate, N-(dodecanoyl)-sphing-4-enine-1-phosphate (d18:1/C12:0-Cer1P); C12-sphingomyelin, N-(dodecanoyl)-sphing-4-enine-1-phosphocholine (d18:1/C12:0-SM); C12-glucosylceramide, N-(dodecanoyl)-1- β -glucosyl-sphing-4-eine (d18:1/C12:0-GlcCer); and C12-lactosylceramide, N-(dodecanoyl)1- β -lactosyl-sphing-4-eine (d18:1/C12:0-LacCer); as well as one very-long-chain Ceranalog, C25-Cer, N-(pentacosanoyl)-sphing-4-enine (d18:1/C25:0), which was added initially but later removed for reasons described under "Results." The other chain-length subspecies of these sphingolipids, which were compared with the internal standards, as well as internal standards for sulfatides (d18:1/C12:0-sulfatide, ST) and GalCer (d18:1/C12:0-GalCer), were obtained from Avanti and Matreya (Pleasant Gap, PA). When the dihydro- (i.e., sphinganine backbone) versions of the standards were not commercially available, they were synthesized by reduction of the backbone double bond using hydrogen gas and 10% Pd on charcoal (Aldrich-Sigma, St. Louis, MO) (36Schwarzmann G. A simple and novel method for tritium labeling of gangliosides and other sphingolipids.Biochim. Biophys. Acta. 1978; 529: 106-114Crossref PubMed Scopus (215) Google Scholar) and verification that the conversion was complete by LC ESI-MS/MS. The HPLC grade solvents (acetonitrile, # EM-AX0145; chloroform, # EM-CX1050; hexane, # JT9304-33; and methanol, # EM-MX0475, as well as formic acid (ACS grade, # EM-FX0440-7), were obtained from VWR (West Chester, PA), and acetic acid (ACS grade, # A38C-212) was obtained from Fischer (Pittsburg, PA). RAW264.7 cells, a macrophage-like cell line derived from tumors induced in male BALB/c mice by the Abelson murine leukemia virus (37Raschke W.C. Baird S. Ralph P. Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus.Cell. 1978; 15: 261-267Abstract Full Text PDF PubMed Scopus (629) Google Scholar), were obtained from the American Type Culture Collection (Manassas, VA) (cat# TIB-71; lot# 3002360), stored as frozen stocks to ensure the cells were never passaged more than 20 times, and cultured according to LIPID MAPS protocols (www.lipidmaps.org), as briefly summarized here. The cells were grown in 60 mm plastic culture dishes in DMEM supplemented with 10% FBS, 4 mM L-glutamine, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. RAW264.7 cells were cultured at 37°C, 95% relative humidity, and 5% CO2 in a ThermoForma Steri-cult CO2 incubator. Cells that were at 80% confluence were rinsed with phosphate buffered saline (PBS), scraped from the dish, and seeded at 2.5 × 106 cells in 5 ml of media in 60 mm dishes, and analyzed after 24 h. The cells were quantified by DNA assay using the Quant-iT DNA Assay Kit, Broad Range (Molecular Probes, cat #Q-33130). The cells were washed twice with PBS, with the dishes tilted to aid in the removal of as much liquid as possible. Then the cells were scraped from the dish in the residual PBS (typically < 0.2 ml) 4If the cells are recovered in a larger volume, the volumes of the other steps must be increased proportionately; or in some cases, the cells can be removed from dishes and centrifuged to reduce the volume, if this does not disrupt membrane integrity, as assessed by a method such as (trypan blue) dye exclusion. using Nalgene cell scrapers (Rochester, NY) and transferred into 13 × 100 mm borosilicate tubes with a Teflon-lined cap (catalog #60827-453, VWR, West Chester, PA). After adding 0.5 ml of CH3OH and 0.25 ml of CHCl3, the internal standard cocktail (500 pmol of each species dissolved in a final total volume of 10 μl of ethanol) were added, and the contents were dispersed using a Branson 1510 ultra sonicator (Sigma) at room temperature for 30 s. This single phase mixture was incubated at 48°C overnight in a heating block, which affords optimal extraction of sphingolipids due to their high phase transition temperatures (3van Echten-Deckert G. Sphingolipid extraction and analysis by thin-layer chromatography.Methods Enzymol. 2000; 312: 64-79Crossref PubMed Google Scholar). After cooling, 75 µl of 1 M KOH in CH3OH was added and, after brief sonication, incubated in a shaking water bath for 2 h at 37°C to cleave potentially interfering glycerolipids. After cooling to room temperature, approximately 3 to 6 µl of glacial acetic acid was added to bring the extract to neutral pH (checked with pH paper to ensure that the extract has been neutralized), 5This differs from previous reports from our laboratory where neutralization of the single phase extract was not necessary, however, we are now fi nding some degradation unless this is done. Whether this is due to contaminants in the test tubes, a shift in the temperature of the Speed Vac, etc., has not been ascertained. and a 0.4-ml aliquot was transferred to a new test tube to serve as the "single-phase extract" (which was centrifuged to remove the insoluble residue, the supernatant collected, the residue reextracted with 1 ml of methanol:CHCl3, 1:2, v:v, centrifuged, and the supernatants combined). To the remainder of the original extract was added 1 ml of CHCl3 and 2 ml of H2O followed by gentle mixing then centrifugation using a table-top centrifuge, and the lower layer (the "organic-phase extract") was transferred to a new tube. The upper phase was extracted with an additional 1 ml of CHCl3, which was also added to the organic-phase extract. The solvents were removed from the single-phase extract and the organic-phase extract using a Savant AES2000 Automatic Environmental Speed Vac. The dried residue was reconstituted in 0.3 ml of the appropriate mobile phase solvent for LC-MS/MS analysis (described herein and the summary in Fig. 1), sonicated for approximately 15 s, then transferred to a microcentrifuge tube and centrifuged at 14,000 to 16,000 g for several min (as needed) before transfer of the clear supernatant to the autoinjector vial for analysis. Summarized in Fig. 1 are the types of LC columns and extraction conditions that were found to be optimal for analysis of different subcategories of sphingolipids that are often encountered in mammalian cells, such as the RAW264.7 cell line. This scheme illustrates, nonetheless, that the investigator has multiple options depending on the nature of the biological sample; for example, if a particular biological sample contains only GlcCer (as is the case for RAW264.7 cells under standard culture conditions), then a shorter amino-column can be used, but if GlcCer and GalCer are both present, an additional run with a longer silica column will also be necessary to distinguish these isomers. Specific LC conditions and instrument parameters for specific analytes are described herein. For all methods, 0.03–0.05 ml were injected onto the column. These compounds were analyzed using the single-phase extract because their recovery into the organic phase of a traditional lipid extraction can be variable. They were separated by reverse phase LC using a Supelco 2.1 (i.d.) × 50 mm Discovery C18 column (Sigma, St. Louis, MO) and a binary solvent system at a flow rate of 1.0 ml/min. If this flow rate does not afford complete desolvation (typically seen as a jagged elution profile), the flow rate can be reduced and/or the ion source gas flow rate can be increased. Prior to injection of the sample, the column was equilibrated for 0.4 min with a solvent mixture of 60% Mobile phase A1 (CH3OH/H2O/HCOOH, 58/41/1, v/v/v, with 5 mM ammonium formate) and 40% Mobile phase B1 (CH3OH/HCOOH, 99/1, v/v, with 5 mM ammonium formate), and after sample injection (typically 50 μl), the A1/B1 ratio was maintained at 60/40 for 0.5 min, followed by a linear gradient to 100% B1 over 1.8 min, which was held at 100% B1 for 5.3 min, followed by a 0.5 min wash of the column with 60:40 A1/B1 before the next run. The elution times for these analytes (Fig. 2) are discussed under "Results." In cases where there is significant carryover of Cer1P on this LC column (i.e., over 1%, which occurs with reverse phase columns obtained from some suppliers as well as with some lots of the columns described in this article), Cer1P can be analyzed instead using a Supleco 2.1 (i.d.) × 50 mm Discovery C8 column (Sigma, St. Louis, MO), with the column heated to 60°C and a binary solvent system [based on reference (38Boath A. Graf C. Lidome E. Ullrich T. Nussbaumer P. Bornancin F. Regulation and traffic of ceramide 1-phosphate produced by ceramide kinase: comparative analysis to glucosylceramide and sphingomyelin.J. Biol. Chem. 2008; 283: 8517-8526Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar)] at a flow rate of 0.6 ml/min. Prior to the injection, the column is equilibrated for 2 min with a solvent mixture of 70% Mobile phase altA1 (CH3OH/H2O/THF/HCOOH, 68.5/28.5/2/1, v/v/v, with 5 mM ammonium formate) and 30% Mobile phase altB1 (CH3OH/THF/HCOOH, 97/2/1, v/v/v, with 5 mM ammonium formate), and after sample injection (30 µL), the altA1/altB1 ratio is maintained at 70/30 for 0.4 min, followed by a linear gradient to 100% altB1 over 1.9 min, which is held at 100% altB1 for 5.3 min, followed by a 0.5 min wash of the column with 70:30 altA1/altB1 before the next run. These compounds were analyzed using the organic-phase extract and normal-phase LC using a Supelco 2.1 (i.d.) × 50 mm LC-NH2 column at a flow rate of 1.0 ml/min and a binary solvent system as shown in Fig. 2C. Prior to injection, the column was equilibrated for 1.0 min with 100% Mobile phase A2 (CH3CN/CH3OH/HCOOH, 97/2/1, v/v/v, with 5 mM ammonium formate), and after sample injection, Mobile phase A2 was continued for 3 min, followed by a 1.0-min linear gradient to 100% Mobile phase B2 (CH3OH/H2O/HCOOH, 89/6/5, v/v/v, with 50 mM triethylammonium acetate), which was held for 3.0 min, then restored to 100% A2 by a 1.0-min linear gradient, and maintained at 100% A2 for 1 min to reequilibrate the column. In addition to these analytes, Cer1P and sulfatides (ST) can also be analyzed, however, their recoveries are higher in the single-phase extract, which can be reconstituted in the solvent for normal phase chromatography (Mobile phase A2). The elution times for these analytes are discussed under "Results." If the biological sample contains both GlcCer and GalCer (the latter is less widely distributed, and only found in barely detectable amounts in RAW264.7 cells until stimulation with Kdo2-Lipid A, as described under "Results"), these can be resolved using a different normal phase column (Supelco 2.1 (i.d.) × 250 mm LC-Si) and an isocratic elution with Mobile phase A3 (CH3CN/CH3OH/H3CCOOH, 97/2/1, v/v/v, with 5 mM ammonium acetate) at 1.5 ml per min (this can be reduced to 0.75 ml/min if necessary for complete desolvation). After the column is preequilibrated for 1.0 min, the sample (dissolved in Mobile phase A3) is injected, and the column is isocratically eluted for 8 min. In most cases, the GlcCer and GalCer are separated by 0.5–1 min (as shown in Fig. 2D), which should be confirmed during the analysis by interspersing vials with these internal standards throughout the runs. Sulfatides were analyzed using the same normal phase chromatography as described for SM, GlcCer, etc. (Fig. 2C); however, prior to extraction, the samples were spiked with 500 pmol of C12-sulfatide (d18:1/C12-GalSulfate) (from Avanti Polar Lipids). The organic-phase extract can be used for a qualitative screen of whether or not sulfatides are present, but for quantitation, a separate run should be made using the single-phase extract because it has the higher recovery (over 50%). Two systems were used for these analyses: a Perkin Elmer Series 200 MicroPump system coupled to a PE Sciex API 3000 triple quadrupole (QQQ) mass spectrometer (Applied Biosystems, Foster City, CA), and a Shimadzu LC-10 AD
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