structural determination of synthetic monoacetyldiglycerides by tandem mass spectrometry of...

Structural Determination of SyntheticMonoacetyldiglycerides by Tandem MassSpectrometry of Sodium-adducted Molecules

Young Hwan Kim1*, So-Yeop Han2*, So-Hye Cho2, Jong Shin Yoo1 and Gil-Ja Jhon2*1Mass Spectrometry Group, Korea Basic Science Institute, PO Box 41, Taejon 305–600, Korea2Department of Chemistry, Ewha Women’s University, Seodaemoon-Gu, Seoul 120–750, Korea

A variety of synthetic monoacetyldiglycerides (MADGs), which show hematopoietic stem cell stimulatingactivity, were investigated using high-energy collision-induced dissociation (CID) tandem mass spectro-metry (MS/MS) coupled with fast-atom bombardment (FAB). CID of sodium-adducted molecules,[M � Na]�, generated product ions highly informative on their structure. In particular, two type of ions(named E2,3and J) and the relative intensity ratio (G2/G1) of two product ions (named G1 and G2) by the lossof free fatty acids (-Rn' COOH) provided definitive information about the position of two long-chain acylgroups on the glycerol backbone. In addition, charge-remote fragmentation (CRF) along the longhydrocarbon chains of two fatty acyl groups determined the position of each double bond. Thus, this directand rapid method can be applied to the structural determination of individual molecular species in amixture of MADGs isolated from biological origins. Copyright # 1999 John Wiley & Sons, Ltd.

Received 4 January 1999; Revised 17 January 1999; Accepted 18 January 1999

Metal ions have recently been employed as useful additivesin mass spectrometry (MS) because of the significantlyimproved structural information by metal ion complexes ofsome biologically important compounds.1,2 As an alter-native to chemical derivatization, the adducts betweenbiomolecules and metal ions are easily produced by fast-atom bombardment (FAB) becausein situ ‘derivatization’occurs when a sample is simply dissolved in a FAB matrixthat is saturated with a metal ion salt. Since these adductions localize a positive charge on the specific site of thecompounds, collision-induced dissociation (CID) of themetal ion complexes also produces ions of structuralimportance by charge-remote fragmentation (CRF). Tan-dem mass spectrometry (MS/MS) of metal-adducted ionshas been exploited for structure elucidation of biomoleculessuch as fatty acids and related compounds,3–5 peptides,6,7

carbohydrates,8–10 antibiotics11 and glycolipids.12,13

Recently, the monoacetyldiglycerides (MADGs), a kindof triacylglycerols (TAGs), have been shown to haveremarkable biological functions such as hematopoietic stemcell stimulating activity† Because TAGs are important inbiology, many analytical methods involving mass spectro-metry have been used to determine their structure.14–26

Among these methods, recent work conducted by Chengetal.23 allows the complete characterization of the structuresof these molecules, using CRF of alkali metal-adductedions. Also, in our previous work, we reported that CID ofFAB-produced sodium-adducted molecules of glycoglycer-olipids and glycerophospholipids provide very usefulinformation on the position of the acyl groups on theglycerol backbone as well as the polar head group, fatty acidcomposition and the double-bond position in the fatty acylchains.27,28 As an extension of our work on the structuraldetermination of glycerolipids, in this paper, we reportstructural elucidation of synthetic MADGs, including theregiospecificity of fatty acyl linkages and the double-bondpositions in the polyunsaturated acid chain, by FAB-CID-MS/MS of sodium-adducted molecules.

EXPERIMENTAL

Materials

All six rac-monoacetyldiglycerides were synthesized fromglycerol and appropriate acids.29 Their structures are shownin Fig. 1. The general synthetic procedure is as follows. Thefatty acid (Sigma, St. Louis, MO, USA) was added toglycerol in acetone at 0°C. After stirring the reactionmixture with dicyclohexylcarbodiimide (DCC; Aldrich,Milwaukee, WI, USA) and dimethylaminopyridine(DMAP; Aldrich) at 0°C for 12h, pure C1-monoacylatedglycerol was obtained by flash-column chromatography.Treatment of monoacylated glycerol with acetic anhydridein the presence of DMAP atÿ78°C for 30 min followed bycolumn purification afforded the C3-acetylated product.The monoacetylmonoglyceride was then acylated with theappropriate fatty acid in the presence of DCC and DMAP indichloromethane at 0°C for 4 h. After column purification,the target 3-acetyl-1,2-diacylglyceride was obtained ingood yield. Thus, the six target monoacetyldiglycerides

*Correspondence to: Y. H. Kim, Mass Spectrometry Group, KoreaBasic Science Institute, PO Box 41, Taejon 305–600, Korea.E-mail: [email protected]. Han and G.-J. Jhon, Dept. of Chemistry, Ewha Women’sUniversity, Seoul 120-750, Korea.E-mail: [email protected] and [email protected]† Pharmaceutical composition containing extracts ofCervus nipponantlers having growth-stimulation activities of hematopoietic cells andmegakaryocyt will be discussed elsewhere.Contract/grant sponsor: Korea Research Foundation; Contract/grantnumber: 1998-015-D00187; 1998-015-D00198.Contract/grant sponsor: Women’s University Research Fund; Con-tract/grant number: KOSEF 961-0302-018-2; MOST.

CCC 0951–4198/99/060481–07 $17.50 Copyright# 1999 John Wiley & Sons, Ltd.

RAPID COMMUNICATIONS IN MASS SPECTROMETRYRapid Commun. Mass Spectrom.13, 481–487 (1999)

could be synthesized in three steps from glycerol in overallyields of 14–53%. Purity of all compounds was verifiedby 1H, 13C, 2D COSY NMR, IR, and high-resolution FAB-MS. The latter results are summarized in Table 1. Allsolvents and reagents were the highest grade commerciallyavailable.

Mass spectrometry

All mass spectrometric analyses were performed using aJMS-HX110/110A tandem mass spectrometer (JEOL,Tokyo, Japan), a four-sector instrument of E1B1E2B2

configuration described previously.11 Briefly, the ionsource was operated at 10 kV accelerating voltage in thepositive-ion mode with a mass resolution of 1000 (10 %valley). Ions were produced by FAB using a cesium ionbeam operated at 2 keV. Approximately 10mg of eachsample were dissolved in chloroform/methanol (1:1, v/v)and mixed with 1ml of 3-nitrobenzyl alcohol (3-NBA;Sigma) with and without Nal on the FAB probe tip. CID ofthe sodium-adducted molecule selected with the first mass

spectrometer (MS-1) occurred in the collision cell locatedbetween B1 and E2 and floated at 3.0 kV. The resultantproduct ions were analyzed by the B/E linked scan methodin the second mass spectrometer (MS-2). The collision gas,helium, was introduced into the collision chamber at apressure sufficient to reduce the precursor ion signal by70%. Signal averaging with two scans was carried out.MS-1 was operated with the resolution adjusted so that onlythe 12C-species of the precursor ions were transmitted.MS-2 was operated at a mass resolution of 1000.

RESULTS AND DISCUSSION

The typical positive-ion FAB mass spectrum of mono-acetyldiglyceride (MADG) shown in Fig. 2(a) displays avery weak peak of the protonated molecule ([M� H]�, m/z637) and abundant fragment ions. The intense peaks atm/z577, 381 and 355 correspond to the diglyceride ions([M � H-Rn'COOH]�) due to loss of acetyl, palmitoyl andoleoyl groups as free fatty acids, respectively. Other ionspecies corresponding to [Rn'CO]� and [Rn'CO� 74]� arealso observed.14 Thus, these fragment ions may be useful indetermining the fatty acid composition. However, MADGsisolated from biological sources are often mixtures of manymolecular species which differ in chain length and theposition and degree of unsaturation of the fatty acyl groups.In this case it is impossible to determine from the fragmentions observed in the FAB mass spectrum, which of thecomponents of the mixture contain which fatty acyl groups.In our previous studies, it was reported that FAB ofglycoglycerolipids and glycerophospholipids in a matrixsaturated with Nal produced prominent sodium-adductedmolecules ([M� Na]� or [M ÿ H� 2Na]�).27,28Similarly,FAB of MADG in a matrix of 3-NBA saturated with Nalproduced an intense peak of the sodium-adducted molecule([M � Na]�, m/z659) as shown in Fig. 2(b).

The attachment of a sodium ion to the molecule localizesthe positive charge more strongely than that of a proton andthus its CID induces systematic fragmentation via charge-remote cleavage. Thus, structural information about theprecursor ions can easily be obtained from interpretation oftheir CID spectra The CID spectrum of [M� Na]� (m/z659) of 1-oleoyl-2-palmitoyl-3-acetyl-rac-glycerol (C18:1/C16:0-MADG) is shown in Fig. 3(a). The ‘number ofcarbon atoms’: ‘number of double bonds’ denotes the

Table 1. Accurate mass of each precursor ion and G2/G1 ratio obtained from high-resolution FAB mass measurement and CID of the[M � Na]� precursor of the synthetic monoacetyldiglyceride (MADG) molecular species, respectively

Fatty acid[M�Na]�, m/z

G2/G1MADG compositiona Calculated Observedb Ratioc

1 C18:1/C16:0 659.5227 659.5228 1.96� 0.102 C16:0/C18:1 659.5227 659.5223 2.60� 0.083 C18:2/C16:0 657.5070 657.5079 2.00� 0.094 C16:0/C18:2 657.5070 657.5079 2.07� 0.035 C16:0/C18:3 655.4914 655.4908 2.42� 0.046 C16:0/C20:4 681.5070 681.5078 2.57� 0.14

a Individual molecular species of MADG are designated with the acyl groups atsn-1 andsn-2 positions listed in order, respectively (i.e., C16:0/C18:1 =sn-1/sn-2). Thefatty acyl groups are symbolized by the convention, carbon number:double-bond number (i.e., C16:0 (palmitoyl), C18:1 (oleoyl), C18:2 (linoleoyl), C18:3 (linolenoyl),C20:4 (arachidonoyl)).b For FAB exact mass measurements, the high voltage scanning method at resolution 10000 (10% valley) was employed.c The mean value and standard deviation of the G2/G1 ratio were measured from three replicate experiments which were performed under the same conditions.

Figure 1. Structures of synthetic monoacetyldiglycerides investigated.

Rapid Commun. Mass Spectrom.13, 481–487 (1999) Copyright# 1999 John Wiley & Sons, Ltd.

482 DETERMINATION OF MONOACETYLDIGLYCERIDES BY FAB-CID-MS/MS

composition of each fatty acyl group on the glycerolbackbone. For example, C18:1/C16:0 means that the fattyacyl groups at thesn-1 andsn-2 positions are oleoyl andpalmitoyl groups, respectively. The high-energy CIDspectrum of [M� Na]� of MADG shows spectral patternsvery similar to those observed in the CID spectra of sodium-adducted molecules of glycoglycerolipids27 and glycero-phospholipids28 except for the product ions generated by thefragmentation of their polar head groups. The fragmentationpathways are illustrated in Fig. 4 and the nomenclature usedis taken from Ref. 27. In Fig. 3(a), the abundant3I1 and3I2

ions atm/z449 and 475 correspond to thea,b-unsaturatedcarbonyls generated by the cleavage of bonds betweenb andg carbons of oleoly and palmitoyl groups, respectively, via1,4-elimination of CRF.30 Therefore, these ions provide

information about the compositions of fatty acyl groups atthe sn-1 andsn-2 positions.

Three product ions, which are observed atm/z377, 403and 599 and denoted as G1, G2 and G3, respectively,correspond to the sodiated diglyceride ions ([M� Na-Rn'COOH]�). Thus, these ions also confirm the composi-tions of two long-chain acyl groups as well as one acetylgroup. In addition, the intensity ratio of two G1 and G2 ionscan be used to determine the relative positions of the twofatty acyl groups atsn-1 andsn-2. In Fig. 3(a), the G2 ion atm/z 403, due to loss of the palmitoyl group at thesn-2position, is about 2.1 times more abundant than the G1 ion atm/z 377, due to the loss of the oleoyl group at thesn-1position. On the contrary, in the CID spectrum of[M � Na]� of 1-palmitoyl-2-oleoyl-3-acetyl-rac-glycerol

Figure 2. Positive-ion FAB mass spectra of 1-oleoyl-2-palmitoyl-3-acetyl-rac-glycerol (C18:1/C16:0-MADG) in amatrix of 3-nitrobenzyl alcohol (a) without Nal and (b) with Nal.

Copyright# 1999 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom.13, 481–487 (1999)

DETERMINATION OF MONOACETYLDIGLYCERIDES BY FAB-CID-MS/MS 483

(C16:0/C18:1-MADG), shown in Fig. 3(b), the G2 ion atm/z377 is more abundant than the G1 ion atm/z403. This trendof the product ion due to the loss of thesn-2 fatty acyl groupalways being more abundant than the one corresponding tothe loss of thesn-1 fatty acyl group is identified from theCID spectra of other synthetic MADGs. These results aresummarized in Table 1. The larger abundance of the G2 ionis likely due to the fact that a proton from either thesn-1 orthe sn-3 moiety can be removed in the fragmentationprocess, as shown in Fig. 4. However, the loss of R1'COOHcan only involve the secondary proton found atsn-2. In thecase of MADGs isolated from biological origins, amolecular species is often a mixture of two regioisomerswhich exchange each other’s fatty acyl group at thesn-1 andsn-2 positions. Thus, the quantity of a regiospecific isomerin the mixture may be calculated from the G2/G1 ratios oftwo regiospecifically pure standard MADGs and themolecular species. However, to obtain reliable G2/G1 ratiosthey should be measured under the same conditions, i.e.source tuning, precursor ion attenuation, MS/MS interfacetuning, etc. On the other hand, two types of ions (named E2,3

and J) corresponding to sodiated propenyl ester with ansn-1acyl group and vinyl ester with ansn-2 acyl group,respectively, also contain information on the regiospecifi-city of the acyl linkages.23 They are observed atm/z345 and305 (see Fig. 3(a)) while in Fig. 3(b) they are observed atm/z319 and 331, respectively. The common ion (named asK) at m/z253 observed in the CID spectra of all MADGsinvestigated is due to the consecutive fragmentation of otherfatty acyl groups by a McLafferty rearrangement, throughthe 3I ions. The CID spectra of all the synthetic MADGsinvestigated are tabulated in Table 2.

More importantly, there is an abundant series of high-mass product ions formed by CRF of fatty acyl chains.These are generated by the loss of CnH2n�2 via 1,4-elimination, with the neighboring peaks in the seriesseparated by 14 u. The spectral pattern of these productions immediately gives information about the presence andlocation of the double bonds in the fatty acyl chains. Thepresence of a double bond in the chain reduces theneighboring peak separation to 12 u. The homologousproduct ions before them/z 545 ion, in the high-mass

Figure 3. FAB tandem mass spectra of [M� Na]� (m/z 659) of (a) 1-oleoyl-2-palmitoyl-3-acetyl-rac-glycerol(C18:1/C16:0-MADG) and (b) 1-palmitoyl-2-oleoyl-3-acetyl-rac-glycerol (C16:0/C18:1-MADG). The subscript inthe symbol represents the relative position (sn-1, sn-2 or sn-3) of the cleavage in the fatty acyl chain and thesuperscript the cleaved bond position relative to the carbonyl carbon of the fatty acyl group. An equal sign above apeak indicates the double-bond position. The fragmentation pathways for the product ions are illustrated in Fig. 4.



region of Fig. 3(a), can be divided into two groups;m/z533,519, 505, 491, 477, 463 and 449 (3I1) due to the oleoyl chainat thesn-1 position, andm/z531, 517, 503, 489 and 475 (3I2)due to the palmitoyl chain at thesn-2 position. The ions atm/z643, 629, 615, 601, 587, 573, 559 and 545 are commonto the cleavage of both fatty acyl groups. Each group beginswith the product ion atm/z643 corresponding to the loss ofCH4 from the alkyl-terminus side and ends with an abundant3I ion. The only even-mass product ion atm/z560, whichmust be produced by homolytic cleavage of an allylic C–Cbond, is abundant owing to the extra stability of the resultingallyl radical ion.

Even for MADG with a polyunsaturated fatty acyl group,the product ions due to CRF along the highly unsaturatedacid chain are observed. The high-mass region of the CIDspectrum of the [M� Na]� precursor ion (m/z681) of 1-palmitoyl-2-arachidonoyl-3-acetyl-rac-glycerol (C16:0/C20:4-MADG) is shown in Fig. 5(a). To compare thisspectrum with that of arachidonic acid, the CID spectrum of[M ÿ H� 2Na]� (m/z349) of arachidonic acid is shown inFig. 5(b). The fragmentation of the [MÿH�2Na]� ion ofarachidonic acid is distinctly indicative of double-bondpositions. In Fig. 5(a), a series of homologous ions due tocharge-remote cleavages along the arachidonoyl chain at the

Figure 4. The fragmentation pathways of the product ions observed in the CID spectrum of [M� Na]� (m/z659) of 1-oleoyl-2-palmitoyl-3-acetyl-rac-glycerol (C18:1/C16:0-MADG).

Table 2. The CID spectral difference of [M� Na]� of synthetic MADGs investigated

Product ions, m/za

Label 1 2 3 4 5 6

E1,2 123(37.5) 123(41.5) 123(24.7) 123(22.9) 123(22.2) 123(30.6)2D1,2 181(39.7) 181(38.0) 181(25.9) 181(24.0) 181(20.3) 181(30.6)3D1,2 195(47.0) 195(47.2) 195(35.8) 195(31.7) 195(31.5) 195(46.2)K 253(25.4) 253(25.1) 253(18.2) 253(15.7) 253(15.9) 253(14.9)J 305(28.0) 331(46.1) 305(26.3) 329(41.6) 327(51.8) 353(62.0)E2,3 345(20.0) 319(21.1) 343(25.6) 319(12.9) 319(18.1) 319(18.7)G1 377(25.7) 403(22.4) 377(25.6) 401(18.7) 399(20.8) 425(16.3)G2 403(53.6) 377(58.7) 401(53.6) 377(39.4) 377(50.4) 377(43.0)G3 599(14.0) 599(10.6) 597(13.9) 597(12.7) 595(12.0) 621(10.8)3I1 449(61.9) 475(100) 449(40.9) 473(100) 471(100) 497(100)3I2 475(100) 449(70.6) 473(100) 449(48.2) 449(43.3) 449(35.9)

a The number in parentheses represents the abundance relative to the most intense product ion. All product ions contain sodium.



sn-2 position in the high-mass region appear atm/z449, 463,477 (not found), 489, 503, 517, 529, 543, 557, 569, 583,597, 609, 623, 637, 651 and 665. Their spectral feature isvery similar to that of the product ions atm/z117, 131, 145(not found), 157, 171, 185, 197, 211, 225, 237, 251, 265,277, 291, 305, 319 and 333 observed in the CID spectrum ofthe [Mÿ H� 2Na]� ion of free arachidonic acid. Hence,these results show that the CID spectral analysis of sodium-adducted molecules of MADG provides information on thepositions of double bonds even when four double bonds arepresent on the fatty acyl group.

CONCLUSIONS

The structural determination of monoacetyldiglycerides,which have an important biological function, was comple-tely performed by FAB-CID-MS/MS. The high-energy CIDspectra of sodium-adducted molecules contain informationabout the regiospecificity of two long-chain fatty acylgroups as well as the fatty acid composition and the double-bond positions in the fatty acyl chain. Thus, this direct and

rapid method has great potential for determining thecomplete structure of individual components present in amixture of MADGs with different fatty acyl groups insamples of biological origin.

Acknowledgements

The financial support for this work from the Korea ResearchFoundation (1998-015-D00187, 1998-015-D00198), KOSEF 961-0302-018-2, and MOST through the Women’s University ResearchFund (1998) is gratefully acknowledged.

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structural determination of synthetic monoacetyldiglycerides by tandem mass spectrometry of...

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