In-depth sphingomyelin characterization using electron impact excitation of ions from organics and mass spectrometry

Electron impact excitation of ions from organics (EIEIO), also referred to as electron-induced dissociation, was applied to singly charged SM molecular species in the gas phase. Using ESI and a quadrupole TOF mass spectrometer equipped with an electron-ion reaction device, we found that SMs fragmented sufficiently to identify their lipid class, acyl group structure, and the location of double bond(s). Using this technique, nearly 200 SM molecular species were found in four natural lipid extracts: bovine milk, porcine brain, chicken egg yolk, and bovine heart. In addition to the most common backbone, d18:1, sphingosines with a range of carbon chain lengths, sphingadienes, and some sphinganine backbones were also detected. Modifications in natural SMs were also identified, including addition of iodine/methanol across a carbon-carbon double bond. This unparalleled new approach to SM analysis using EIEIO-MS shows promise as a unique and powerful tool for structural characterization. Electron impact excitation of ions from organics (EIEIO), also referred to as electron-induced dissociation, was applied to singly charged SM molecular species in the gas phase. Using ESI and a quadrupole TOF mass spectrometer equipped with an electron-ion reaction device, we found that SMs fragmented sufficiently to identify their lipid class, acyl group structure, and the location of double bond(s). Using this technique, nearly 200 SM molecular species were found in four natural lipid extracts: bovine milk, porcine brain, chicken egg yolk, and bovine heart. In addition to the most common backbone, d18:1, sphingosines with a range of carbon chain lengths, sphingadienes, and some sphinganine backbones were also detected. Modifications in natural SMs were also identified, including addition of iodine/methanol across a carbon-carbon double bond. This unparalleled new approach to SM analysis using EIEIO-MS shows promise as a unique and powerful tool for structural characterization. SMs comprise a class of lipids within the sphingolipid category that are found ubiquitously in the human body, but are enriched in brain lipids where they play a crucial role in insulating nerve cell axons. This class of lipids is also found in relatively high concentrations in cell membrane microdomains, termed lipid rafts, which are believed to be involved in diverse cell functions, such as cell trafficking, cell signaling, lipid-protein interactions, and apoptosis [for a general review of SM, see (1Slotte J.P. Biological functions of sphingomyelins.Prog. Lipid Res. 2013; 52 ([Erratum. 2013. Prog. Lipid Res.52: 681.].): 424-437Crossref PubMed Scopus (192) Google Scholar, 2Chakraborty M. Jiang X.C. Sphingomyelin and its role in cellular signaling.Adv. Exp. Med. Biol. 2013; 991: 1-14Crossref PubMed Scopus (46) Google Scholar)]. The use of ESI MS to fully characterize the diverse SM molecular species has proven to be challenging (3Shaner R.L. Allegood J.C. Park H. Wang E. Kelly S. Haynes C.A. Sullards M.C. Merrill Jr, A.H. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.J. Lipid Res. 2009; 50: 1692-1707Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 4Deimler R.E. Sander M. Jackson G.P. Radical-induced fragmentation of phospholipid cations using metastable atom-activated dissociation mass spectrometry (MAD-MS).Int. J. Mass Spectrom. 2015; 390: 178-186Crossref PubMed Scopus (33) Google Scholar). Structural elucidation of SM using collision-induced dissociation (CID), a commonly used fragmentation method for lipid analysis, is limited due the low-energy collisions (<100 eV) and generally only provides information regarding lipid class, carbon chain length, and the total number of double bonds that comprise the lipid backbone and the acyl group (3Shaner R.L. Allegood J.C. Park H. Wang E. Kelly S. Haynes C.A. Sullards M.C. Merrill Jr, A.H. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.J. Lipid Res. 2009; 50: 1692-1707Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Molecular species-specific information such as backbone type (sphinganine, sphingosine, and sphingadiene with different carbon chain lengths) and the structure of the amide-linked acyl chains (i.e., carbon chain length and the number and stereo-configuration of double bonds) are not provided in a typical CID MS/MS spectrum. The analysis of glycerophospholipids by CID also provides limited structural information and requires polarity switching to identify both the phospholipid class and the fatty acid composition (5Stutzman J.R. Blanksby S.J. McLuckey S.A. Gas-phase transformation of phosphatidylcholine cations to structurally informative anions via ion/ion chemistry.Anal. Chem. 2013; 85: 3752-3757Crossref PubMed Scopus (24) Google Scholar, 6Ekroos K. Ejsing C.S. Bahr T. Karas M. Simons K. Shevchenko A. Charting molecular composition of phosphatidylcholines by fatty acid scanning and ion trap MS3 fragmentation.J. Lipid Res. 2003; 44: 2181-2192Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 7Pham H.T. Maccarone A.T. Thomas M.C. Campbell J.L. Mitchell T.W. Blanksby S.J. Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: the next step towards “top-down” lipidomics.Analyst. 2014; 139: 204-214Crossref PubMed Google Scholar). In order to fully characterize the structure of lipids, a different fragmentation technique is needed. Recently, Campbell and Baba (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google Scholar) reported a “nearly” complete identification method of phosphatidylcholines (PCs) using electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) (9Cody R.B. Freiser B.S. Electron impact excitation of ions from organics: an alternative to collision induced dissociation.Anal. Chem. 1979; 51: 547-551Crossref Scopus (121) Google Scholar) in a branched radio-frequency electron-ion reaction device (10Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar) (referred to as ExD cell in this work). Using EIEIO to fragment glycerophospholipids, information regarding lipid class (or head group), acyl chain length, the number and location of double bonds, and the regioisomeric structure were obtained in a single experiment in the positive ion mode. Using an alternative fragmentation method, Deimler, Sander, and Jackson (4Deimler R.E. Sander M. Jackson G.P. Radical-induced fragmentation of phospholipid cations using metastable atom-activated dissociation mass spectrometry (MAD-MS).Int. J. Mass Spectrom. 2015; 390: 178-186Crossref PubMed Scopus (33) Google Scholar) recently reported a radical induced dissociation of PCs using bombardment of metastable helium atoms (MAD-MS). These reports on novel approaches to structural elucidation of glycerophospholipids suggest that characterization of other lipid categories, such as SM, may be improved by alternative fragmentation strategies to CID. In this study, we applied EIEIO to SM structural characterization using a branched ion trap (10Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar) installed in a quadrupole TOF mass spectrometer with a differential mobility spectrometry (DMS) cell (SelexIONTM Technology) installed between the ion source and the mass spectrometer (11Schneider B.B. Covey T.R. Coy S.L. Krylov E.V. Nazarov E.G. Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.Int. J. Mass Spectrom. 2010; 298: 45-54Crossref PubMed Scopus (130) Google Scholar, 12Baker P.R.S. Armando A.M. Campbell J.L. Quehenberger O. Dennis E.A. Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.J. Lipid Res. 2014; 55: 2432-2442Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Lintonen T.P. Baker P.R. Suoniemi M. Ubhi B.K. Koistinen K.M. Duchoslav E. Campbell J.L. Ekroos K. Differential mobility spectrometry-driven shotgun lipidomics.Anal. Chem. 2014; 86: 9662-9669Crossref PubMed Scopus (116) Google Scholar). The DMS was used to isolate SM from other interfering lipid classes, namely PCs and triacylglycerols (TAGs), prior to analysis. This is particularly important because SMs and PCs share the same head group (i.e., phosphocholine) and are roughly in the same mass range. Using DMS to isolate the SM, we obtained nearly 200 EIEIO-based MS/MS spectra on individual SM molecular species in various biological lipid extracts without isobaric interference from other lipid classes or categories, which generated in-depth structural details, including the sphingolipid backbone length and number of double bonds, acyl chain structure, including carbon chain length, and the number and locations of double bond(s). Additionally, the fragments appeared in the product ion spectrum that enabled differentiation between sphinganines (SMs that have no carbon-carbon double bond in the sphingoid backbone), sphingosines (SMs that have one carbon-carbon double bond in the sphingoid backbone), and sphingadienes (SMs containing two carbon-carbon double bonds in the sphingoid backbone). Consequently, EIEIO yields “near-complete” characterization of SM molecular species. All lipid standards and lipid mixtures were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and are identified here using the guidelines of Liebisch et al. (14Liebisch G. Vizcaíno J.A. Köfeler H. Trötzmüller M. Griffiths W.J. Schmitz G. Spener F. Wakelam M.J.O. Shorthand notation for lipid structures derived from mass spectrometry.J. Lipid Res. 2013; 54: 1523-1530Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). The specific lipid molecular species, SM(d18:1,12:0) and PC[16:0,18:1(9Z)] were used for method validation. Commercial extracts of SM derived from porcine brain (brain SM), bovine milk (milk SM), and chicken egg (egg SM) were used to characterize SM from biological sources, and bovine heart extract (BHE) was used as a complex biological extract. The solvent for all working solutions was HPLC-grade dichloromethane:methanol (50/50, v/v) with 10 mM ammonium acetate. These solvents and ammonium acetate were purchased from Caledon Laboratory Chemicals (Georgetown, ON, Canada) and Sigma-Aldrich Canada Co. (Oakville, ON, Canada), respectively. Concentrations of working solution were 250 μg/ml for BHE; 100 μg/ml for brain SM, milk SM and egg SM; and 1 μg/ml for the standard SM and PC samples. All experiments were conducted using a modified TripleTOF® 5600 system that was equipped with both a DMS device and a branched radio-frequency ExD cell (10Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar). The of DMS and its to lipid analysis are (11Schneider B.B. Covey T.R. Coy S.L. Krylov E.V. Nazarov E.G. Planar differential mobility spectrometer as a pre-filter for atmospheric pressure ionization mass spectrometry.Int. J. Mass Spectrom. 2010; 298: 45-54Crossref PubMed Scopus (130) Google Scholar, 12Baker P.R.S. Armando A.M. Campbell J.L. Quehenberger O. Dennis E.A. Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.J. Lipid Res. 2014; 55: 2432-2442Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 13Lintonen T.P. Baker P.R. Suoniemi M. Ubhi B.K. Koistinen K.M. Duchoslav E. Campbell J.L. Ekroos K. Differential mobility spectrometry-driven shotgun lipidomics.Anal. Chem. 2014; 86: 9662-9669Crossref PubMed Scopus (116) Google Scholar). the DMS cell is in the atmospheric pressure between the ESI source and the The DMS were to isolate SM ions from isobaric from PCs and These a DMS cell of as the gas gas for the DMS and addition of to the gas as a the DMS the separation was an the was from to in of MS or EIEIO-MS spectra were The of the ExD cell was reported (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google Scholar, T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar). the ions in the device, helium gas was with a pressure in the ExD cell of This is the same pressure in due to the in the that gas The electron used in the EIEIO experiments was generated by a The was the of the electron in the ExD cell by a in mode. The of the electron was by the to the branched ion trap (10Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar) was used in this to the and of EIEIO fragment In ions were by the electron as ions were the ExD cell (i.e., electron was applied the was and the was All of the ions in the ExD cell the EIEIO reaction were and (i.e., the TOF for mass analysis by the for 1 In is a in which the electron the some SM species, of their EIEIO mass spectra was to of species in the of we an EIEIO mass spectrum without SM ions to the ExD as a only in the ExD cell to from were this of the SM ions were to the ExD cell and EIEIO was in a of fragment ions derived from both the SM and the the EIEIO from the mass improved the analysis of the SM species This characterization of the in the ExD cell was obtained EIEIO In order to on the two of the ExD cell and were to ions to be the collision the electron was and gas was the ExD as a collision In order to to the ion the was to the ExD The ExD cell was by using In EIEIO 10 eV) was applied to a SM(d18:1,12:0) and the spectrum was in fragment ions to complete structural characterization for this SM molecular species. In the the specific of this EIEIO spectrum information on SM structural SM head group fragments and chain SM backbone with of the acyl group and the head group and fragments from both carbon chains with a fragment for the carbon-carbon bond In CID spectra on the same fragment ions and structural which only identified the head group and the chain length of the backbone (3Shaner R.L. Allegood J.C. Park H. Wang E. Kelly S. Haynes C.A. Sullards M.C. Merrill Jr, A.H. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.J. Lipid Res. 2009; 50: 1692-1707Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). CID not fragments containing information on the backbone or the acyl The of the of was for SM analysis using as we on PCs (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google Scholar). EIEIO fragmentation we to to the chain fragment with their radical and the of we from a brain extract as a to to the fragmentation the backbone the head group and the acyl EIEIO spectra were the fragment the of fragments a common of one was the acyl group and backbone between and which a of and information for the acyl was a to between and information was EIEIO spectra were for SMs the acyl chain fragmentation structural PC (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google and electron ionization ionization of was In some was in EIEIO but we a for such we 10 for the SM with to for EIEIO spectra for from a to SM shows the fragmentation reaction of 10 electron was its (i.e., reaction was not improved by the applied on the electron was used in this of the in order to reaction between and ions were the ExD cell for 100 without electron ion the was the electron was The electron or reaction was the of this The ions were from the ExD cell the TOF mass by electron was ion electron ion electron and the head group fragments are in of SMs was was reported for PC (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google in the same The that the reaction between SM and PC is not but the between the ions and be SM analysis be using as has for PCs (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google Scholar) as as for electron capture dissociation of and (10Baba T. Campbell J.L. Le Blanc J.C.Y. Hager J.W. Thomson B.A. Electron capture dissociation in a branched radio-frequency ion trap.Anal. Chem. 2015; 87: 785-792Crossref PubMed Scopus (30) Google Scholar). shows ion and product in which was the method used in this was a product was The EIEIO-based fragmentation the analysis of SM(d18:1,12:0) provides a for with the of the in the EIEIO spectra in The the of a head group in the which is used to identify PCs or SMs by CID In SMs are only from PCs by the that SM ions are of and for PCs due to the this may not be the an SM in a modified an SM be in within the of an interfering PC the of a PC be as an a of The also be by the use of DMS EIEIO provides SM fragment ions and not by which fragments and This in product ion spectra that be to SM, and enabled to specific fragments to SM molecular species in standards as as complex biological lipid extracts without the for separation P.R.S. Armando A.M. Campbell J.L. Quehenberger O. Dennis E.A. Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.J. Lipid Res. 2014; 55: 2432-2442Abstract Full Text Full Text PDF PubMed Scopus (80) Google in EIEIO MS EIEIO of SMs also fragment ions from bond the on the SM backbone These fragment ions in are unique to SM molecular species and are in where common SM are such as sphingosines with of sphingadienes, and The acyl chain are to in EIEIO as they are the most in an EIEIO spectrum. the fragment ion of in is an of such an acyl chain acyl chain in was used for to the acyl we identified four to identify the two chains in this SM molecular species, and backbone and acyl chain fragments are with a SM and its This of was for analysis using SMs were found in a single EIEIO spectrum and as they share share backbone and acyl the of the other EIEIO fragment ions the of in analysis also identified double bond locations from the of the chain using the same that was reported (8Campbell J.L. Baba T. Near-complete structural characterization of phosphatidylcholines using electron impact excitation of ions from organics.Anal. Chem. 2015; 87: 5837-5845Crossref PubMed Scopus (92) Google Scholar) (i.e., mass the double bond location and with in the EIEIO identification of double bond using was relatively for this was not the for where the was not Consequently, an was that an acyl chain with double bonds has one of (or or with two carbon-carbon single bond in of acyl In of sphingadiene the double bond was identified as the common location in and biological PubMed Google Scholar). EIEIO to characterize SMs in complex lipid the of isobaric interference be by using an of In this study, we DMS to the of EIEIO this lipidomics DMS SM from other interfering lipid classes, namely PCs and prior to EIEIO analysis. This is particularly the total lipid of using The mass spectrum lipid classes and categories, including and with SM to the total lipid Using DMS for of SM the SM of the was identified The of the DMS was using the standard as as SMs from brain, egg yolk, and milk In addition to we also SMs the ions The for the DMS is the was to where separation of lipid classes P.R.S. Armando A.M. Campbell J.L. Quehenberger O. Dennis E.A. Three-dimensional enhanced lipidomics analysis combining UPLC, differential ion mobility spectrometry, and mass spectrometric separation strategies.J. Lipid Res. 2014; 55: 2432-2442Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). as was from to TOF MS were for step in the from to a SM species be as a of where DMS of SM In this a to provided separation of SM from other lipid classes and categories, as by the MS spectra only These provided MS/MS that were the of the mass DMS we ion of 1 for a single TOF spectrum and for individual MS/MS DMS to ions the of SM for a of biological lipid extracts was shows an MS spectrum obtained the analysis of brain SM, with the identified SMs from the EIEIO experiments in The reported in identified SM to the by that The identified SM the of the acyl in the same as an of of other SM are in the information SMs in porcine brain SM in with with with with the of in the total lipids, which other identified SMs the of acyl in the isobaric in may not be because of species. double bond locations in and were not in double bond in a new the of in the total lipids, which other identified SMs the of acyl in the isobaric in may not be because of species. double bond locations in and were not in double bond typical spectrum of sphingadiene SM is in which is found in bovine brain The backbone was identified which the backbone is The double bond location was identified the and are the of SMs that one double bond and two double bonds in the acyl respectively. sphingosines and some sphingadienes were in all the but the sphinganine backbone was only in the bovine brain and milk SM The molecular species were d18:1, which is of backbones were in brain, and but only were and were in from to including number of were also in brain, milk, and a in brain, and but was in milk fatty in are generally not are and have in their that fatty and be the chains in SM are in The identified of acyl chains using this EIEIO method to the fatty acid by the Polar Lipids, Inc. in Scholar). The brain SM a of SM molecular species derived from the combinations of different sphingoid backbones and acyl but this also of SM molecular species containing unique The most SM found in ion of and of with the of SM molecular species, is their that are standard SM molecular species. These are SM, because all lipid class for SM are but the acyl group and backbone are with the in The that the have an in their In their EIEIO and CID an of was This mass is the same as that of an The EIEIO spectra from the to of and O. analysis of the chain fragments shows that an and a group are to double bonds in SM The was found the and the group was the head group in all four the double bond in the sphingadiene backbone was modified in the of The source of SM is is that the and group are is used to double bonds in lipid analysis and specific method for and other Lipid Res. Full Text PDF Google Scholar). the structural of SM that with SM extracts were to and from this we not of SM molecular species containing or group the the SM was with SM molecular species with acyl chains containing carbon-carbon double of a by an was as the reaction a double which is used for of double bond number as was also but was the by an we that the SM found in bovine brain extracts is not via a typical addition across a double bond. In the of a biological reaction is that this type of was only in brain, which is to have relatively of in other the of the F. The role of in brain PubMed Scopus Google Scholar). that are the of this are to identify the of the be that this new has the to such In this study, EIEIO spectra were for a to high spectra to EIEIO on SMs with no ion and high analysis, such as be identify the the be the by was using the EIEIO spectra in be as the of all the which is a triple of the standard of the the same of the of SMs in was not because SMs were in the total lipids, the for a typical was to identify SM molecular species. be for species, but also be this because DMS SMs from other lipid classes without the for The of DMS and EIEIO MS is a tool for SM analysis. Lipid class, backbone structure, and acyl chain are identified using fragmentation information in a single EIEIO spectrum. This method also the to identify in SM molecular species. ionization have promise in SMs G. Molecular species of by spectrometry with atmospheric pressure Mass Spectrom. PubMed Scopus (80) Google Scholar, with mass spectrometry and for analysis of and milk A. PubMed Scopus Google their have limited to species and composition from limited information derived from MS/MS be as to no structural information for only of the head group In separation by for to fragmentation from analysis and In the for SMs requires no separation and provides in-depth structural of lipid

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