Structural identification of triacylglycerol isomers using electron impact excitation of ions from organics (EIEIO)

Electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) was applied to triacylglycerols (TAGs) for in-depth molecular structure analysis using MS. In EIEIO, energetic electrons (∼10 eV) fragmented TAG ions to allow for regioisomeric assignment of identified acyl groups at the sn-2 or sn-1/3 positions of the glycerol backbone. In addition, carbon-carbon double bond locations within the acyl chains could also be assigned by EIEIO. Beyond the analysis of lipid standards, this technique was applied to edible oils and natural lipid extracts to demonstrate the power of this method to provide in-depth structural elucidation of TAG molecular species. Electron-induced dissociation or electron impact excitation of ions from organics (EIEIO) was applied to triacylglycerols (TAGs) for in-depth molecular structure analysis using MS. In EIEIO, energetic electrons (∼10 eV) fragmented TAG ions to allow for regioisomeric assignment of identified acyl groups at the sn-2 or sn-1/3 positions of the glycerol backbone. In addition, carbon-carbon double bond locations within the acyl chains could also be assigned by EIEIO. Beyond the analysis of lipid standards, this technique was applied to edible oils and natural lipid extracts to demonstrate the power of this method to provide in-depth structural elucidation of TAG molecular species. Triacylglycerols (TAGs) are among the most abundant lipids in the human body, located primarily within adipose. TAGs are also found in plants and are concentrated in seeds as a source of energy during embryonic development. It is from these various seeds that a majority of edible oils are derived. The physiological role of TAGs is to serve as a reserve of fatty acids for energy generation via β-oxidation. However, these same stored fatty acids may also serve as precursors, either directly or indirectly, to bioactive oxidized metabolites such as eicosanoids that play a key role in mediating the cell-signaling pathways that control inflammation (1.Bistrian B.R. Novel lipid sources in parenteral and enteral nutrition.Proc. Nutr. Soc. 1997; 56: 471-477Crossref PubMed Google Scholar). High levels of TAG in plasma have been implicated in a host of pathophysiologies, including arteriosclerosis, nonalcoholic fatty liver disease, and metabolic syndrome (2.Ekroos K. Jäni M. Tarasov K. Hurme R. Laaksonen R. Lipidomics: a tool for studies of atherosclerosis.Curr. Atheroscler. Rep. 2010; 12: 273-281Crossref PubMed Scopus (82) Google Scholar). Understanding the exact roles of individual TAG molecules in these diseases is not clear, due mainly to the difficulty in characterizing these TAGs at the molecular level. For example, while current MS technology can account for the total number of carbon atoms in a TAG, as well as the approximate acyl chain composition (3.Ekroos K. Lipidomics: Technologies and Applications. Wiley, New York2012Crossref Scopus (29) Google Scholar), other critical information such as the exact regioisomerism of fatty acids along the glycerol backbone, the position(s) of the acyl chain double bonds, the stereochemistry of the double bonds, and so forth, cannot currently be obtained using conventional bioanalytical techniques. For example, a common method of TAG analysis is the chromatographic detection of FFAs or their derived methyl or ethyl esters, which are obtained by hydrolysis from TAGs (4.Park P.W. Goins R.E. In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods.J. Food Sci. 1994; 59: 1262-1266Crossref Scopus (405) Google Scholar). Although convenient, this method does not allow for consideration of the structural aspects of the intact TAG molecular species (i.e., regioisomeric structural information). This information is critical to understand the dynamic functions of TAGs, their digestion, and their metabolism. A conventional TAG characterization method in MS is based on low-energy (5.McAnoy A.M. Wu C.C. Murphy R.M. Direct qualitative analysis of triacylglycerols by electrospray mass spectrometry using a linear ion trap.J. Am. Soc. Mass Spectrom. 2005; 16: 1498-1509Crossref PubMed Scopus (151) Google Scholar, 6.Renaud J.B. Overton S. Mayer P.M. Energy and entropy at play in competitive dissociations: the case of uneven positional dissociation of ionized triacylglycerides.Int. J. Mass Spectrom. 2013; 352: 77-86Crossref Scopus (16) Google Scholar) or high-energy collision-induced dissociation (CID) (7.Cheng C. Gross M.L. Pittenauer E. Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry.Anal. Chem. 1998; 70: 4417-4426Crossref PubMed Scopus (145) Google Scholar, 8.Kubo A. Satoh T. Itoh Y. Hashimoto M. Tamura J. Cody R.B. Structural analysis of triacylglycerols by using a MALDI-TOF/TOF system with monoisotopic precursor selection.J. Am. Soc. Mass Spectrom. 2013; 24: 684-689Crossref PubMed Scopus (40) Google Scholar). These methods provide TAG structural information, including acyl chain lengths and double bond number; however, the double bond positions and the acyl chain regioisomeric arrangement are not easily defined due to the limited information derived from the resultant fragments. HPLC has also been used to resolve regioisomers of TAGs (9.Leskinen H. Suomela J-P. Pinta J. Kallio H. Regioisomeric structure determination of α- and γ-linolenoyldilinoleoylglycerol in blackcurrant seed oil by silver ion high-performance liquid chromatography and mass spectrometry.Anal. Chem. 2008; 80: 5788-5793Crossref PubMed Scopus (29) Google Scholar), but it requires long analysis times, authentic standards as references, and extensive method development, potentially making this method unattractive to a high-throughput lab. A recent study of TAG regioisomers was reported using differential mobility spectrometry (DMS) without HPLC (10.Maccarone A.T. Duldig J. Mitchell T.W. Blanksby S.J. Duchoslav E. Campbell J.L. Characterization of acyl chain position in unsaturated phosphatidylcholines using differential mobility-mass spectrometry.J. Lipid Res. 2014; 55: 1668-1677Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Despite advances in the goal to characterize lipid molecular species, including using ozone-induced dissociation (OzID) (11.Thomas M.C. Mitchell T.W. Harman D.G. Deeley J.M. Nealon J.R. Blanksby S.J. Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions.Anal. Chem. 2008; 80: 303-311Crossref PubMed Scopus (265) Google Scholar, 12.Poad B.L. Pham H.T. Thomas M.C. Nealon J.R. Campbell J.L. Mitchell T.W. Blanksby S.J. Ozone-induced dissociation on a modified tandem linear ion-trap: observations of different reactivity for isomeric lipids.J. Am. Soc. Mass Spectrom. 2010; 21: 1989-1999Crossref PubMed Scopus (115) Google Scholar) or a Paternò-Büchi reaction (13.Ma X. Chong L. Tian R. Shi R. Hu T.Y. Ouyang Z. Xia Y. Identification and quantitation of lipid C=C location isomers: a shotgun lipidomics approach enabled by photochemical reaction.Proc. Natl. Acad. Sci. USA. 2016; 113: 2573-2578Crossref PubMed Scopus (214) Google Scholar, 14.Stinson C.A. Xia Y. A method of coupling the Paternò-Büchi reaction with direct infusion ESI-MS/MS for locating the C=C bond in glycerophospholipids.Analyst. 2016; 141: 3696-3704Crossref PubMed Google Scholar) to determine double bond positions, an efficient method to characterize lipid structural details has been lacking. Recently, a study using OzID found that TAG molecular species containing C18:1, n-7 versus C18:1, n-9 correlated with clinical variables of dyslipidemia and are proinflammatory (15.Ståhlman M. Pham H.T. Adiels M. Mitchell T.W. Blanksby S.J. Fagerberg B. Ekroos K. Borén J. Clinical dyslipidaemia is associated with changes in the lipid composition and inflammatory properties of apolipoprotein-B-containing lipoproteins from women with type 2 diabetes.Diabetologia. 2012; 55: 1156-1166Crossref PubMed Scopus (75) Google Scholar). These results highlight the need for a method to fully characterize TAG molecular species in order to relate TAG structural details with human disease. In order to acquire more in-depth structural information for lipids, we recently developed a near-complete structural analysis using electron-induced dissociation (16.Zubarev R.A. Haselmann K.F. Budnik B. Kjeldsen F. Jensen F. Towards an understanding of the mechanism of electron-capture dissociation: a historical perspective and modern ideas.Eur. J. Mass Spectrom. (Chichester, Eng.). 2002; 8: 337-349Crossref Scopus (228) Google Scholar) or electron impact excitation of ions from organics (EIEIO) (17.Cody 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) for phosphatidylcholines (PCs) (18.Campbell 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 (94) Google Scholar) and SMs (19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Singly protonated precursor lipid ions generated by an ESI were introduced into a branched ion trap (20.Baba T. Campbell J.L. LeBlanc 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) wherein they were reacted with a 10 eV electron beam. This produced information-rich fragment ions that revealed lipid class, respective acyl chain lengths, the number and locations of double bonds in the fatty acid chains, and regioisomer specificity. Here, we applied EIEIO to reveal key structural details, at the molecular species level of TAGs. We identified and developed an optimized analytical strategy in conjunction with diagnostic rules to analyze TAGs by EIEIO. This resulted in the complete characterization of the TAG lipidome in undefined TAG mixtures without the need for authentic standards. TAGs were analyzed from solutions prepared using a mixture of HPLC-grade dichloromethane (DCM)-methanol (50:50, v/v) with 0.2 mM sodium acetate or 10 mM ammonium acetate. These solvents were purchased from Caledon Laboratory Chemicals (Georgetown, Ontario, Canada), while the salts were purchased from Sigma-Aldrich Canada Co. (Oakville, Ontario, Canada). Synthesized TAG standards, including TAG 16:1/18:1(n-9Z)/16:1 and TAG 16:1/16:1/18:1(n-9Z), were obtained from Larodan Fine Chemicals AB (Malmo, Sweden). Another group of TAG isomers differing only in their carbon-carbon double bond positions, TAG 18:1(n-12Z)/18:1(n-9Z)/18:1(n-12Z), was obtained from Avanti Polar Lipids Inc. (Alabaster, AL). Finally, TAG standards containing polyunsaturated acyl chains, including tridocosahexaenoyl glycerol [TAG(22:6(n-3Z,n-6Z,n-9Z,n-12Z,n-15Z,n-18Z), or DHA-TAG], tri-α-linolenoyl glycerol [TAG(18:3(n-3Z,-n6Z,n-9Z), or ALA-TAG], and tri-γ-linolenoyl glycerol [TAG(18:3(n-6Z,n-9Z,n-12Z), or GLA-TAG] were purchased from Nu-Chek Prep Inc. (Elysian, MN). All of the TAG standards were used without further purification. The working solutions of these standard samples were prepared at 1 µg/ml in the standard working solvent. Tri-linolenoyl glycerol (CLA-TAG) was synthesized in-house followed by a method described by Medina et al. (21.Medina A.R. Cerdán L.E. Giménez A.G. Páez A.C. González M.J.I. Grima E.M. Lipase-catalyzed esterification of glycerol and polyunsaturated fatty acids from fish and microalgae oils.J. Biotechnol. 1999; 70: 379-391Crossref Scopus (88) Google Scholar). Conjugated linolenic acids, CLA(9E,11E), CLA(9Z,11E), and CLA(10E,12Z), suspended in ethanol were purchased from Cayman Chemical (Ann Arbor, MI). Immobilized lipase from Candida antarctica and molecular sieves, 4 Å, were purchased from Sigma Aldrich. Glycerol and HPLC-grade hexane were obtained from Fisher Scientific Co. (Ottawa, Ontario, Canada) and Caledon Laboratory Chemicals, respectively. Ethanol was evaporated in nitrogen gas flow from each CLA to obtain FFA of 40 mg. Each CLA were diluted in hexane (900 µl) with glycerol (4 µl), beads of the lipase (10 mg), and the molecular sieves (100 mg) in a 5 ml glass vial with a Teflon cap. The mixture was incubated for 20 h using a shaking bath (300 rpm) at 50°C. The beads were removed from the solution after centrifugation, then the liquid phase was evaporated by nitrogen gas flow. The residue was suspended in chloroform as stock (nominal concentration ∼10 mg/ml), from which a stock solution was prepared using standard working solvent to a concentration of ∼10 µg/ml. Analytical standards of edible oils, including olive oil, coconut oil, and linseed oil, were purchased from Sigma-Aldrich. Certified ultrapremium extra virgin olive oil, Cobrançosa (Portugal) harvested in 2014, and extra virgin olive oil, Solon (Greece), were purchased from a specialty olive oil store and a grocery store in Toronto, respectively. These oils were diluted in the working solvent to a concentration of ∼100 µg/ml total lipid without purification. Avocado oil and fish oil were extracted in-house from a fresh avocado fruit and wild sock-eye salmon fillet, respectively. A ripe avocado and a filet of the salmon were purchased from grocery stores in Toronto. Deionized water (975 µl), methanol (2,000 µl), and DCM (900 µl) were added to individual samples of avocado and salmon (both ∼0.5 g). Each mixture was shaken well for 1 min in a glass vial with a Teflon cap. Liquid phase was moved to another vial to remove solid. Additional deionized water (1,000 µl) and DCM (900 µl) were added to the liquid and gently mixed. After the mixture was centrifuged for 10 min (1,200 rpm), the DCM phase (bottom layer) was collected by a pipette and evaporated under nitrogen. This dual phase purification process was repeated to each residue. The amount of the extracted oil was determined from the increase of the mass of each vial with the evaporated residue compared with the empty vials. The washed residue was suspended in chloroform as stock. These stocks were diluted in the standard working solvent with concentration of 100 µg/ml. We used a TOF mass spectrometer system equipped an electron-ion reaction device (ExD cell) (20.Baba T. Campbell J.L. LeBlanc 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) for this work, which was the same instrument as in our previous report on SM analysis (19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The detail of ionization method on lipid analysis using a Turbo VTM ion source (Sciex) was described previously (18.Campbell 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 (94) Google Scholar). Infused flow rate of working solution in this work was 0.3 ml/h, and ESI voltage was +5,500 V. The detail of DMS (SelexION® Technology, Sciex) for lipid application was described before (19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 22.Lintonen T.P.I. Baker P.R.S. 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). Separation voltage (SV) of 3,900 V peak to peak, modifier gas of 2-propanol, and DMS temperature of 200°C were continuously applied. Counter nitrogen gas flow for resolution enhancement (DR) (0–40 psi) and compensation voltage (CoV) were optimized for each measurement. The previously reported ExD device (20.Baba T. Campbell J.L. LeBlanc 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 EIEIO condition (i.e., electron kinetic energy at 10 eV, typically). Quasi-flow-through mode or simultaneous electron ion injection mode (20.Baba T. Campbell J.L. LeBlanc 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) for 150 ms reaction time was used in this work as the optimized product yield condition (19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). We used helium as cooling gas in the ExD and the Q2 collision cell. The TOF mass analyzers were operated in high-sensitivity mode (23.Loboda A.V. Chernushevich I.V. Bloomfield N. Improved resolution and substantially higher sensitivity on a quadrupole-TOF mass spectrometer.57th Conference on Mass Spectrometry and Allied Topics ThPY628. 2009; Google Scholar). Accumulation time was given between 1 and 5 min per single precursor m/z in this work. Roughly separated groups of ions by DMS were further separated by m/z by the Q1 mass filter placed between the ion source and the ExD device. Mass window of the m/z separation was ∼1 m/z unit. For the identification of the TAG species that we analyzed in this work, we have adopted a modified nomenclature first suggested by Marshall et al. (24.Marshall D.L. Pham H.T. Bhujel M. Chin J.S.R. Yew J.Y. Mori K. Mitchell T.W. Blanksby S.J. Sequential collision- and ozone-induced dissociation enables assignment of relative acyl chain position in triacylglycerols.Anal. Chem. 2016; 88: 2685-2692Crossref PubMed Scopus (44) Google Scholar) and based on commonly accepted lipidomics terminology (25.Liebisch G. Vizcaíno J.A. Köfeler H. Trötzmüller M. Griffiths W.J. Schmitz G. Spener F. Wakelam M.J. Shorthand notation for lipid structures derived from mass spectrometry.J. Lipid Res. 2013; 54: 1523-1530Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Briefly, we identify TAGs with known sn positions of given acyl chains using slashes (e.g., TAG 16:0/18:0/18:1 implies known regioisomerism at all sn positions); however, where there is a question as to the sn-1 and sn-3 substitution, we use the underscore terminology (e.g., TAG 18:1_/18:0/_16:0 refers to a TAG with a known sn-2 substitution, but interchangeable sn-1 and sn-3 substitution). As Marshall et al. (24.Marshall D.L. Pham H.T. Bhujel M. Chin J.S.R. Yew J.Y. Mori K. Mitchell T.W. Blanksby S.J. Sequential collision- and ozone-induced dissociation enables assignment of relative acyl chain position in triacylglycerols.Anal. Chem. 2016; 88: 2685-2692Crossref PubMed Scopus (44) Google Scholar) state, we identify synthesized TAG standards using TAG A/B/C nomenclature given the known composition of such species. In previous studies (18.Campbell 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 (94) Google Scholar, 19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), we have demonstrated the utility of EIEIO for providing detailed structural information for different types of polar lipids (i.e., bearing polar head groups). However, TAGs are somewhat different that these lipids, as they are nonpolar, bearing no easily ionizable moiety themselves. For example, in order to form TAG ions for MS analysis, all lipidomics work flows must rely on adduct formation with some added salt, such as sodium or ammonium cations. Despite this difference, TAG species fragmented easily under EIEIO conditions (Fig. 1A–C), just like PCs and SMs measured previously. Sodiated TAGs were more promising EIEIO targets than ammoniated species because of the simplicity of dual chain loss ions of the sodiated species. In contrast, the ammoniated TAGs tended to fragment primarily at the NH4+ group, resulting in uninformative chain loss (supplemental Fig. S1). Hence, sodiated TAG ions were used throughout the remainder of this EIEIO study. When subjected to EIEIO, sodiated TAGs undergo dual acyl chain loss (fragments appear between m/z 300 and 350) and single acyl chain loss (fragments appear between m/z 500 and 650) as major product ion channels. Also appearing in the EIEIO mass spectrum, in the m/z region between the intact TAG precursor ion and the single acyl chain loss product ion, one can find a complete series of acyl chain fragments. Similar to our previous studies (18.Campbell 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 (94) Google Scholar, 19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), each carbon-carbon single bond cleavage in the acyl chains was represented by two ions: an even-electron (nonradical) product ion and its matched radical ion pair (bearing one fewer hydrogen atom than its partner). The relative intensities of these diagnostic peaks were typically ∼1% for the dual and single chain loss fragments and ∼0.1% for the acyl chain fragments compared with the residual precursor TAG ions (base peak, 100%; Fig. 1A–C). Overall, the EIEIO dissociation efficiency for TAGs was comparable to our previous PC and SM experiments when using the simultaneous ion-electron injection mode and a reaction time of 150 ms (19.Baba T. Campbell J.L. Le Blanc J.C.Y. Baker P.R.S. In-depth sphingomyelin characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; 57: 858-867Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The EIEIO spectra of TAGs contain various levels of information that can be used to characterize their structures. Beginning with the intact m/z of the sodiated TAG, we can obtain a brutto level assessment of the lipid [e.g., TAG (50:1) is the TAG investigated in Fig. 1A by virtue of its observed intact m/z]. Beyond this base level of characterization, we could also perform CID on these TAGs to reveal rudimentary and approximate knowledge about the acyl chain regioisomerism and unsaturation; such assessments are available to many other conventional MS-based analyses. However, EIEIO provides much more structural information for TAGs than do these methods, beginning with regioisomeric assignment of acyl chains. The structural characterization of TAGs by EIEIO begins by surveying the spectrum in the dual acyl chain loss region (m/z 250–400); it is here that the most information is contained on acyl group chain length, the number of double bonds, and acyl chain regioisomerism. Conversely, the single acyl chain loss region contains fewer characteristic peaks and is generally of lesser utility. Using the spectra in Fig. 1C as examples, we observe that within the dual acyl group loss region there are two distinct peak groups: singlets (e.g., m/z 331.25) and paired doublets (e.g., m/z 345.26 and 347.24). High-energy CID experiments also yield similar fragments (7.Cheng C. Gross M.L. Pittenauer E. Complete structural elucidation of triacylglycerols by tandem sector mass spectrometry.Anal. Chem. 1998; 70: 4417-4426Crossref PubMed Scopus (145) Google Scholar, 8.Kubo A. Satoh T. Itoh Y. Hashimoto M. Tamura J. Cody R.B. Structural analysis of triacylglycerols by using a MALDI-TOF/TOF system with monoisotopic precursor selection.J. Am. Soc. Mass Spectrom. 2013; 24: 684-689Crossref PubMed Scopus (40) Google Scholar), confirming our assignments here. Each singlet peak (e.g., m/z 305.24 in Fig. 1A; m/z 331.26 in Fig. 1B, C) maps to the acyl chain located at the TAG's sn-2 site. These singlet peaks are formed by two possible bond cleavage patterns as indicated in Fig. 1E. The other set of diagnostic fragments are the doublet peaks, with the lower m/z member of the pair being more intense than the higher m/z peak. The difference in m/z between the doublet peaks (1.98 m/z units) reveals that the two fragment ions differ only slightly, with the lighter fragment containing a methylene (-CH2-) group, while the heavier fragment contains an oxygen atom instead of that -CH2- group (Fig. 1D, F). Just as the singlet peak identifies the acyl chain at the sn-2 position of TAGs, so the doublet peaks identify the acyl chains at the sn-1 and sn-3 positions. For example, Fig. 1B displays a set of doublet peaks at m/z 319.26/321.24, which was related to a 16:0 acyl chain at the sn-1 and sn-3 sites; no other doublets are visible in the EIEIO spectrum, revealing identical substitution at both sn positions. A similar outcome was observed in Fig. 1C, where one set of doublet peaks at m/z 345.26/347.24 show that the sn-1 and sn-3 positions are both substituted with 18:1 acyl chains for this TAG molecule. However, Fig. 1A presents a more complex scenario than the other two EIEIO spectra, as it contains two sets of doublet peaks. One set appears at m/z 319.26/321.24 (a 16:0 acyl chain) and at m/z 345.26/347.24 (related to an 18:1 acyl chain). Because the sn-1 and sn-3 positions in TAGs are not distinguished in chirality using our present EIEIO method, we use the interchangeable “_” notation, such as TAG 16:0_/18:1/_18:1. More importantly, these singlet and doublet peaks are consistent for given acyl chain lengths and are independent of the TAGs from which they originate. This allows us to predict the m/z values for any acyl chain's singlet and doublet peaks for use as diagnostic fragment ions (Table 1; supplemental Table S1).TABLE 1Diagnostic peak m/z for TAG regioisomerism identificationDouble Bond No.Length0123456sn-1, sn-3 Diagnostic peak m/z (sodiated)16319.261317.246315.230313.214321.240319.225317.209315.19418347.293345.277343.261341.246339.230349.272347.256345.240343.225341.20920375.324373.308371.293369.277367.261365.246377.303375.287373.272371.256369.240367.22522403.355401.339399.324397.308395.293393.277391.261405.334403.319401.303399.287397.272395.256393.240sn-2 Diagnostic peak m/z (sodiated)16305.246303.23301.214299.19918333.277331.261329.246327.23325.21420361.308359.293357.277355.261353.246351.2322389.339387.324385.308383.293381.277379.261377.246Complete list of diagnostic peaks is shown in supplemental Table S1. Open table in a new tab Complete list of diagnostic peaks is shown in supplemental Table S1. Interestingly, the peak intensity ratio between the singlet peak and the doublet peaks also represents the number of specific acyl groups in a TAG molecule. For example, Fig. 1A displays equivalent intensities for the singlet peak (m/z 305.24) and two doublet peaks (m/z 319.26/321.24 and m/z 345.27/347.25), indicating unique acyl chain substituents at each sn position, with 16:0 at the sn-2 position (the singlet peak) and different acyl chains at sn-1 and sn-3(16:0 and 18:1). For Fig. 1B, C, the same acyl groups are bonded at sn-1 and sn-3 such that the intensities of the doublet peaks were twice that of the singlet peak. These intensity ratios are ultimately used in the deconvolution of regioisomer constituent calculations (vide infra). As stated previously, obtaining information on the number of carbon-carbon double bonds present in constituent TAG acyl chains is somewhat facile using MS-based methods. Generally, after a TAG ion is fragmented using conventional CID, acyl chain fragment losses can be detected, and the degree of total unsaturation within those acyl chains can be estimated (6.Renaud J.B. Overton S. Mayer P.M. Energy and entropy at play in competitive dissociations: the case of uneven positional dissociation of ionized triacylglycerides.Int. J. Mass Spectrom. 2013; 352: 77-86Crossref Scopus (16) Google Scholar). However, the identification of the specific locations of these carbon-carbon double bonds is much more difficult, requiring the use of ion/molecule reactions (11.Thomas M.C. Mitchell T.W. Harman D.G. Deeley J.M. Nealon J.R. Blanksby S.J. Ozone-induced dissociation: elucidation of double bond position within mass-selected lipid ions.Anal. Chem. 2008; 80: 303-311Crossref PubMed Scopus (265) Google Scholar, 12.Poad B.L. Pham H.T. Thomas M.C. Nealon J.R. Campbell J.L. Mitchell T.W. Blanksby S.J. Ozone-induced dissociation on a modified tandem linear ion-trap: observations of different reactivity for isomeric lipids.J. Am. Soc. Mass Spectrom. 2010; 21: 1989-1999Crossref PubMed Scopus (115) Google Scholar, 13.Ma X. Chong L. Tian R. Shi R. Hu T.Y. Ouyang Z. Xia Y. Identification and quantitation of lipid C=C location isomers: a shotgun lipidomics approach enabled by photochemical reaction.Proc. Natl. Acad. Sci. USA. 2016; 113: 2573-2578