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 of dissociation: a and J. Mass Spectrom. Scopus Google Scholar) or electron impact excitation of ions from organics (EIEIO) R.B. impact excitation of ions from an to Chem. Scopus Google Scholar) for phosphatidylcholines J.L. T. structural characterization of phosphatidylcholines using electron impact excitation of ions from Chem. PubMed Scopus Google Scholar) and T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). precursor lipid ions by an a ion T. Campbell J.L. dissociation in a ion Chem. PubMed Scopus Google Scholar) with a electron This ions that lipid acyl chain the number and locations of double in the fatty acid and we applied to key structural at the molecular species of identified and developed an in with to TAGs by EIEIO. This in the characterization of the TAG in TAG without the need for authentic TAGs from using a of with or These from while the from TAG standards, including TAG and TAG obtained from of TAG in their carbon-carbon double bond positions, TAG was obtained from TAG standards containing acyl including glycerol or glycerol or and glycerol or from of the TAG standards used without The of these at in the glycerol was by a method by of glycerol and fatty acids from and 70: Scopus Google Scholar). and in from from and molecular from and obtained from and was in from to of in with glycerol of the and the molecular in a with a The was for using a at The from the the liquid was by The was in as from which a was using to a of standards of edible including and from in and from a oil and a in These oils in the to a of total lipid without oil and oil from a and A and a of the from in and to individual of and was well for in a with a was to to and to the liquid and the was for the was by a and This was to The of the oil was from the of the mass of with the with the The was in as These in the with of used a mass system an reaction T. Campbell J.L. dissociation in a ion Chem. PubMed Scopus Google Scholar) for this which was the same as in on analysis T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). The of method on lipid analysis using a ion source was J.L. T. structural characterization of phosphatidylcholines using electron impact excitation of ions from Chem. PubMed Scopus Google Scholar). of in this was and was The of for lipid was T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Duchoslav E. Campbell J.L. Ekroos K. mobility shotgun Chem. 2014; PubMed Scopus Google Scholar). of to of and of for and for The reported T. Campbell J.L. dissociation in a ion Chem. PubMed Scopus Google Scholar) was used in (i.e., electron energy at or electron ion T. Campbell J.L. dissociation in a ion Chem. PubMed Scopus Google Scholar) for reaction was used in this as the T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). used as in the and the The mass in and on a mass on Mass and Google Scholar). was and precursor in this groups of ions by by by the mass the ion source and the Mass of the was For the of the TAG species that we in this we have a modified by Pham H.T. M. K. Mitchell T.W. Blanksby S.J. and ozone-induced dissociation assignment of acyl chain position in Chem. 2016; PubMed Scopus Google Scholar) and based on lipidomics H. M. F. for lipid derived from mass spectrometry.J. Lipid Res. 2013; Full Text Full Text PDF PubMed Scopus Google Scholar). we TAGs with positions of acyl chains using TAG regioisomerism at however, is a as to the and we the TAG to a TAG with a sn-2 but and Pham H.T. M. K. Mitchell T.W. Blanksby S.J. and ozone-induced dissociation assignment of acyl chain position in Chem. 2016; PubMed Scopus Google Scholar) we TAG standards using TAG the composition of such species. In studies J.L. T. structural characterization of phosphatidylcholines using electron impact excitation of ions from Chem. PubMed Scopus Google Scholar, T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar), we have the of for structural information for different of lipids (i.e., However, TAGs are different that these lipids, as are easily For example, in order to TAG ions for MS lipidomics on with such as or Despite this TAG species fragmented easily and TAGs more species of the of chain ions of the species. In the TAGs to primarily at the in chain TAG ions used the of this to EIEIO, TAGs acyl chain and and acyl chain and as ion in the mass in the the intact TAG precursor ion and the acyl chain can a of acyl chain fragments. to studies J.L. T. structural characterization of phosphatidylcholines using electron impact excitation of ions from Chem. PubMed Scopus Google Scholar, T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar), carbon-carbon bond in the acyl chains was by an ion and ion The of these for the and chain and for the acyl chain with the precursor TAG ions the dissociation for TAGs was to and using the and a reaction of T. Campbell J.L. characterization using electron impact excitation of ions from organics (EIEIO) and mass spectrometry.J. Lipid Res. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). The of TAGs various levels of information that can be used to characterize their with the intact of the TAG, we can a of the lipid TAG is the TAG in by of intact Beyond this of we could also on these TAGs to and approximate the acyl chain regioisomerism and such are to other conventional However, more structural information for TAGs these with regioisomeric assignment of acyl The structural characterization of TAGs by by the in the acyl chain it is that the most information is on acyl chain the number of double bonds, and acyl chain the acyl chain and is of the in as we that within the acyl are and and also (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), in in to the acyl chain located at the sn-2 These are by bond as in The other of are the with the of the more the The in the that the ions with the containing a while the an of that as the the acyl chain at the sn-2 position of TAGs, so the the acyl chains at the and For example, a of at which was to a acyl chain at the and other are in the at A was in of at that the and positions are with acyl chains for this TAG However, a more the other as it of at acyl and at to an acyl the and positions in TAGs are not in using we the such as TAG these and are for acyl chain lengths and are of the TAGs from which This to the for acyl and for as ions for TAG regioisomerism of is in in a Complete of is in the the and the also the number of acyl groups in a TAG For example, for the and and acyl chain at with at the sn-2 position and different acyl chains at and and For the same acyl groups are at and such that the of the that of the These are used in the of information on the number of carbon-carbon double in TAG acyl chains is using a TAG ion is fragmented using conventional acyl chain can be and the of total within acyl chains can be 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 of the locations of these carbon-carbon double is more the of (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, 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). EIEIO, we are to this in TAG molecules the same as we reported for J.L. T. structural characterization of phosphatidylcholines using electron impact excitation of ions from Chem. PubMed Scopus Google Scholar) and and The of is that carbon-carbon are but carbon-carbon double are in a in the mass an and an For acyl chains as a of ions by (i.e., a or For acyl chains containing double bonds, the of the be by for double bond in the fragmented of the acyl double are not in EIEIO, the of the at the double bond a in the of acyl chain by on acyl TAGs, and is in C. in of acyl chain by on acyl TAGs and is in by the double bond the carbon-carbon double bond positions we the of the by the This was using the the of as from the methyl carbon of the acyl chain and the molecular mass of the The of the are in which was from of and For example, in double from and are with and In double from is with these TAGs of acyl their and their acyl chain (i.e., acyl chain of the acyl chains in species the same and from the of are the for the at the also this during the analysis of oil that TAGs such was not the acyl groups not to this is to a with and double (i.e., from a precursor that a at the we the to at the position to from this For of TAGs containing a of different acyl the and analysis more This is acyl chains of a TAG, while their within the same of the precursor For example, the of acyl chain with the of the bond and double bond for a location along the identified acyl chain locations In such the the of a double does not to due to the from acyl bond However, a at the double bond making double bond For example, in the analysis of TAG a in the to at the n-9 In to at the n-9 and positions, with the at the position at n-9 the This is with TAG which an acyl chain with a double bond at n-9 and acyl chains with a double bond at For TAGs of acyl chains double the the and we acyl chain positions as for and and also for the The for the is with the that the electrons of double and or double the analysis of TAG was we that more (i.e., extracts from natural a For we that of be to the of TAGs from species, other molecules the same molecular mass in but different In addition, other TAG species could be as based on their For example, a TAG of and could from the of a TAG that by double bond of TAG is with a 2 at this is of the monoisotopic of the TAG, it is in a with the TAG, of TAG we used M. Duchoslav E. Campbell J.L. Ekroos K. mobility shotgun Chem. 2014; PubMed Scopus Google Scholar, differential mobility as a for mass J. Mass Spectrom. 2010; PubMed Scopus Google Scholar, A.M. Campbell J.L. lipidomics analysis differential ion mobility and mass Lipid Res. 2014; 55: Full Text Full Text PDF PubMed Scopus Google Scholar) to TAGs from species such as oxidized TAGs, and other of the and the of the for from species with molecular a the for analysis of natural lipid extracts using and EIEIO. the is the a of ionized TAGs and other lipid species. of these ions the while the is from to in at are precursor or at this to TAG to for and from of this in a these are a of TAG and for using at to provide and precursor ion the for the TAG precursors, of the are found for TAG precursor to the most precursor ions with the levels of from species such as and oxidized species. 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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) in the with on other extracts in the and oils we oxidized as in the the oil was oxidized The of these oil identified TAG species, as by the TAGs identified in the oil the identified TAG we could the composition which that the FFAs in the and oils as well as the by the of the we also to the TAG in and oils and the of the different species in and the regioisomerism of the oil and oil in a with the acyl chain in the and positions on the and the acyl chain in the sn-2 position on the using the 2 and In is a for an acyl chain to be in the sn-2 while the and positions the and acyl The and oils not in these sn-2 was at and of and In this and (i.e., TAG was abundant in but was abundant in and of the sources of the In the same species, these or In to the conventional this acyl in TAGs more information to the of In the information, results of analysis of other oil oil is an of TAGs with and acyl at sn-2 and of and at and abundant at and at oil is an of long chain with acids C. L. C. of using electrospray with mass spectrometry for the characterization of Mass Spectrom. PubMed Scopus Google Scholar). This was also oxidized acyl was but was of was at The of a natural in is the In MS TAG TAGs identified double bond In the TAGs, species of and double bond number are but double bond in the In the and at the sn-2 In this we have the of and to provide in-depth characterization of 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 also identified 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. This study that is a tool for lipidomics structure analysis of TAGs, as well for and other lipid for this including of the at the position (i.e., as well as double bond

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