Tandem mass spectrometric analysis of fatty acyl groups of galactolipid molecular species from wheat flour

Ž .Microchemical Journal 68 2001 143�155

Tandem mass spectrometric analysis of fatty acylgroups of galactolipid molecular species from wheat

flour

Young Hwan Kim�, Ji Hye Gil, Jongki Hong, Jong Shin Yoo

Mass Spectrometry Analysis Team, Korea Basic Science Institute, P.O. Box 41, Taejon 305-600, South Korea

Abstract

Our previous work demonstrated that the structures of two and one molecular species of digalactosyl andmonogalactosyl diacylglycerols from wheat flour, respectively, were determined by collision-induced dissociationŽ . Ž� ��. Ž .CID of sodium-adducted molecules M�Na desorbed by fast atom bombardment FAB . However, many morecomponents in their HPLC separations were identified. Then fractionated components were structurally determined

Ž .by CID tandem mass spectrometry MS�MS , including the fatty acid composition and the double-bond positions inthe fatty acyl groups. Furthermore, the relative positions of two fatty acid chains on the glycerol backbone could beassigned by the ratio of the intensity of two specific product ions observed in the CID spectra. The product ionŽ� ��.M�Na-R COOH due to the neutral loss of the fatty acyl group at the sn-2 position via free fatty acid was more2

Ž� ��.abundant than the one M�Na-R COOH due to the loss of the fatty acyl group at the sn-1 position. The1regiospecificity of two fatty acyl linkages was also confirmed by the results which were obtained from the FAB mass

Žspectra of sn-2 acyl lysogalactolipids. These compounds were synthesized by a specific enzyme, Lipase XI from.Rhizopus arrhizus , which cleaved specifically ester bond between glycerol backbone and sn-1 fatty acyl group. � 2001

Elsevier Science B.V. All rights reserved.

Keywords: Diacylglycerols; Wheat flour; Collision-induced dissociation; Tandem mass spectrometry

Abbre�iations: CID, collision-induced dissociation; DGDG, digalactosyl diacylglycerol; FAB, fast atom bombardment; HPLC,high-performance liquid chromatography; MGDG, monogalactosyl diacylglycerol; MGMG, monogalactosyl monoacylglycerol;

� ��3-NBA, 3-nitrobenzyl alcohol; M�Na , sodium-adducted molecule; MS�MS, tandem mass spectrometry; TLC, thin-layerchromatography

� Corresponding author. Tel.: �82-42-865-3433; fax: 82-42-865-3419.Ž .E-mail address: [email protected] Y.H. Kim .

0026-265X�01�$ - see front matter � 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S 0 0 2 6 - 2 6 5 X 0 0 0 0 1 4 1 - 7

( )Y.H. Kim et al. � Microchemical Journal 68 2001 143�155144

1. Introduction

Galactolipids such as monogalactosyl diacyl-Ž .glycerol MGDG and digalactosyl diacylglycerol

Ž .DGDG are distributed extensively in the thyl-akoid membrane of the chloroplasts of higher

� �plants 1 . These compounds play an importantrole in maintaining the structural integrity of

� �chloroplast membrane 2 and the optimal func-� �tioning of the photosynthetic apparatus 3 . In

addition, acyl lipids of thylakoids are recognizedas having a functional role in the activity of

� �several integral membrane protein complexes 4 .These diverse structural and functional roles maybe due to the structural characteristics that has ahydrophobic moiety of two long-chain fatty acidsand a hydrophilic part of a polar head group ofmonogalactose or digalactose as shown in Fig. 1.

However, galactolipids purified from biologicalsources are often mixtures of several molecularspecies which differ in the chain length of twofatty acids and the double-bond position and de-

� �gree of unsaturation in the fatty acyl groups 5 .Hence, it is difficult and time-consuming to de-termine the structure of the fatty acyl groups ofeach molecular species in the mixtures. However,mass spectrometry has been used to solve thisproblem. Until now, there are two mass spec-trometry-based methods that are generally usedto determine the structure of the fatty acyl groupof glycerolipids. In the first method, the fatty acidmethyl ester derivatives prepared by methanolysisof glycerolipids are identified by matching reten-tion times and mass spectra to those of the au-thentic standards using gas chromatography�mass

Ž . � �spectrometry GC�MS 6 . In the second method,Ž .a collision-induced dissociation CID spectralŽ �.pattern of the carboxylate ion RCOO pro-

duced by negative-ion fast atom bombardmentŽ . Ž .FAB mass spectrometry MS is compared with

� �that of the authentic standard 7,8 . However, asmentioned above, the samples from biologicalsources are often mixtures of compounds withdifferent fatty acid groups. Therefore, it is usuallyimpossible to find which of the free carboxylateions or ester derivatives are produced from whichcomponent of the mixtures.

Recently, we reported that intense product ions

observed in the high-mass region of the positive-ion CID spectra of FAB-desorbed sodium-adducted molecules of galactolipids provide infor-mation about the fatty acid composition and the

� �double-bond position in the fatty acyl groups 9 .However, this analysis was limited to one and twocomponents of MGDG and DGDG, respectively,since the abundance of sodium-adductedmolecules of the minor species were insufficientin their FAB mass spectra. Here, as an extensionof the previous study, we will report the structuralanalysis of the fatty acyl groups of individualmolecular species fractionated by HPLC separa-tion of MGDG and DGDG isolated from wheatflour. Additionally, it will be shown that the re-giospecificity of the two acyl linkages on the glyc-erol backbone can be assigned by the ratio of theintensity of two specific product ions observed in

� ��the CID spectra of M�Na ions.

2. Materials and methods

2.1. Materials

MGDG and DGDG from wheat flour and stan-dard fatty acids were obtained from Sigma

Ž .Chemical Co. St. Louis, MO, USA . Informationon the fatty acid composition and the double-bondposition of each molecular species in mixtures ofMGDG and DGDG was not available from thecommercial source. All of the used solvents wereof analytical grade and purchased from BaxterŽ .Muskegon, MI, USA .

2.2. HPLC

Chromatography was carried out on Hewlett-ŽPackard Model HP 1090 series II Palo Alto, CA,

.USA and UV detector was operated at 205 nm.MGDG and DGDG were separated into individ-ual molecular species on a 250 � 4.6 mm

ŽAltex�Beckman Ultrasphere ODS column Beck-.man, Fullerton, CA, USA . Individual species were

eluted isocratically with a mobile phase ofŽ .methanol�water�acetonitrile 90.5:7:2.5, v�v�v

� �at a flow rate of 2.0 ml�min 10 . The fraction ofindividual species collected from the column was

( )Y.H. Kim et al. � Microchemical Journal 68 2001 143�155 145

Fig. 1. The structures of MGDG and DGDG isolated fromwheat flour. The fatty acyl groups designated R and R are1 2attached at the sn-1 and sn-2 positions of the glycerol back-bone, respectively. Also, the sugar moiety corresponding tothe polar head group is ether-linked on the sn-3 position ofthe glycerol.

dried under a stream of N and reconstituted in a2Ž .mixture of chloroform and methanol 1:1, v�v

for FAB-MS and MS�MS analyses.

2.3. Preparation of sn-2 acyl lysogalactolipids

A solution of each galactolipid and Lipase XIŽ .from Rhizopus arrhizus, Sigma: 1800 unit in the

Ž .presence of Triton X-100 2.5 mg in boric acid-Ž .borax buffer 0.63 ml, pH 7.7 was stirred at 37�C

for 1 h for MGDG or 2.5 h for DGDG, respec-� �tively 11 . The reaction solution was adjusted to

pH 4 with acetic acid and then quenched byŽ .adding 2 ml of chloroform�methanol 2:1, v�v .

The extracted organic phase was washed by 1 ml� �of 0.2 M H PO �1 M KCl 12 . The aqueous3 4

phase was re-extracted into chloroform and theresulting organic phases were combined, driedand redissolved in a minimal volume of chloro-

Ž .form�methanol 2:1, v�v . The resulting reaction

products were separated into lysogalactolipids andŽ .fatty acids by thin-layer chromatography TLC

˚Žon a silica gel plate K6 Silica Gel 60 A; What-.man, Hillsboro, OR, USA with chloroform�

Ž .methanol�water 65:25:4, v�v�v . Sn-2 acyl lysocomponents were identified by spraying the TLCplate with 0.01% primuline in a mixture of ace-

Ž .tone and water 4:1, v�v and then by illuminatingwith UV light. They were located on the lowestpart of the TLC plate.

2.4. Mass spectrometry

FAB mass spectra were taken with the firstŽ .MS-1 of the two mass spectrometers of a JMS-HX110A�110A tandem mass spectrometerŽ .JEOL, Tokyo, Japan with mass resolution of

Ž .1000 10% valley using a JMS-DA9000 data sys-� �tem 13 . The ion source was operated with accel-

erating voltage at 10 kV and �10 kV dependingon the positive- and negative-ion modes, respec-tively. Ions were produced by FAB using thecesium ion gun operated at 22 kV. Approximately10 �g of each sample was dissolved in chloro-

Ž .form�methanol 1:1, v�v . Then 1 �l of the solu-tion was mixed with 1 �l of 3-nitrobenzyl alcoholŽ .3-NBA, Sigma saturated with NaI in the posi-tive-ion mode on the FAB probe tip. Tri-

Ž .ethanolamine BDH, Poole, Dorset, UK was usedin the negative-ion mode.

Ž .Tandem mass spectrometry MS�MS was car-ried out using the four-sector instrument with theE B E B configuration. CID of the precursor1 1 2 2

Ž .ions selected by MS-1 E B occurred in the1 1collision cell located between B and E and1 2floated at 3.0 kV and �3.0 kV in the positive-and negative-ion modes, respectively. Both MS-1Ž . Ž .E B and MS-2 E B were operated as dou-1 1 2 2ble-focusing instruments. The collision gas, he-lium, was introduced into the collision chamber ata pressure sufficient to reduce the precursor ionsignal by 70%. Signal averaging with two scanswas carried out. The resolution of MS-1 wasadjusted so that only the 12 C-species of the pre-cursor ions to be analyzed was transmitted. MS-2was operated at a resolution of 1000.


3. Results and discussion

3.1. Monogalactosyl diacylglycerol

The positive-ion FAB mass spectrum of MGDGfrom wheat flour was acquired using 3-NBA satu-rated with NaI as a matrix. The spectrum displays

� ��abundant peaks corresponding to M�Na ionsof the individual molecular species present asshown in Fig. 2a. Two peaks observed at m�z 777and 801 in the positive-ion FAB mass spectrumare interpreted as sodium-adducted molecules oftwo MGDG species containing two acyl groupscorresponding to total fatty acid compositionsŽ .carbon atoms: double bonds of C34:2 and C36:4,respectively. However, the zoomed high-mass re-

� ��gion shown in Fig. 2a revealed M�Na ions ofother minor species with different fatty acyl com-position in addition to major molecular species.Several peaks observed in the HPLC separationof MGDG as shown in Fig. 3a confirm this inter-pretation. The results of the FAB mass analysesof the fractions collected from the column canconclude that the molecular species correspond-ing to the peaks of M1, M2, M3, M4 and M5shown in Fig. 3a have total fatty acid composi-tions of C36:6, C36:5, C36:4, C34:2 and C36:3,respectively.

The positive-ion CID tandem mass spectrum of� �� Ž .M�Na at m�z 777 ion of the molecularspecies labeled as M4 in HPLC separation isshown in Fig. 2b. Even though the CID tandemmass spectrum of sodium-adducted moleculelooks very complicated, it can be interpreted sys-tematically. The nomenclature proposed by

� �Costello and co-worker 14 was adopted withminor changes and addition of the new notationfor the cleavages of the fatty acyl groups as shownin Fig. 2. For the cleavages of the fatty acylchains, the subscript number in the symbol repre-

Ž .sents the relative position sn-1 or sn-2 of thecleavage in the fatty acyl group and the super-script number the cleaved bond position relative

� �to the carbonyl carbon of the fatty acyl group 9 .Based on this analysis, we could divide the productions into two types: one generated by the cleav-

Ž .age of the head group i.e. sugar moiety , and theother by the cleavage of the fatty acyl chains. All

of the product ions observed in the CID spectrum� ��of M�Na ion correspond to sodium attached

ions. In the low-mass region of the positive-ionCID spectrum, the product ions characteristic ofthe head group are observed including those dueto the cleavage of the sugar ring and the glyco-sidic bonds. These product ions were explained in

� �detail previously 9 .In the remaining region of the CID spectrum,

the product ions due to the cleavage of the fattyacid chains are observed. The main fragmentationpathway corresponding to concomitant elimina-tion of two fatty acyl moieties yields a prominent

Ž .ion at m�z 243 E . The two product ions result-ing from the concurrent cleavages of the bond �or � to the carbonyl group of one fatty acyl chainand the ester bond of the other fatty acyl chain

Ž2 . Ž3 .are observed at m�z 301 D or 315 D ,1,2 1,2respectively. The formation of five- and six-mem-bered lactone rings seems to be responsible forhigh abundance of these ions. Finally, the consec-utive fragmentation of the remaining fatty acylgroup of the 3I ion generated by the cleavage ofthe bond � to carbonyl group of one fatty acylchain produces the K ion at m�z 373 via McLaf-

� �ferty-type rearrangement 15 .A more important spectral feature is the obser-

vation of homologous series appearing in thehigh-mass region. These are due to the neutrallosses of C H , C H or C H by then 2 n�2 n 2 n n 2 n -2fragmentation occurring along the two fatty acylchains, depending on the presence of the doublebonds. This series begins with the commonproduct ion of m�z 761 due to the loss of CH4from the alkyl termini of the two fatty acyl groupsand ends with each abundant 3I ion for each fattyacyl group, as shown in Fig. 2b. These productions give immediate information about the fattyacid composition and the location of the doublebond on the fatty acyl chain to be explained.

Fig. 4a shows the high-mass region of the posi-tive-ion CID spectrum of the sodium-adductedmolecule corresponding to MGDG species labeledas M1 in HPLC separation. The negative-ion CID

Žspectrum of deprotonated linolenic acid 9,12,15-.octadecatrienoic acid with m�z 277 is also shown

for ease of explanation as shown in Fig. 4b. In the� ��CID spectrum of M�Na ion of m�z 797, two


Ž . Ž .Fig. 2. a Positive-ion mass spectrum obtained by fast atom bombardment of MGDG in 3-NBA matrix saturated with NaI; and b� �� Ž .CID tandem mass spectrum of M�Na ion at m�z 777 of MGDG species corresponding to the peak labeled as M4 in HPLC

� ��separation. The fragmentation pathways observed in the CID of M�Na ion was also shown.

abundant product ions are observed at m�z 519Ž . Ž .G and 535 H . They result from the neutral lossof the fatty acyl group as free fatty acidŽ . Ž .�RCOOH and aldehyde �RCOH , respec-tively. Thus, these ions provide information about

the fatty acid composition. Two product ions atŽ1,5 . Ž0,2 .m�z 663 X and 677 X are generated by

the fragmentation of the sugar moiety, as shownŽin Fig. 2. The concomitant loss of two groups i.e.

.hydroxy and methoxy groups , which are linked to


Ž . Ž .Fig. 3. Separation of the molecular species of a MGDG and b DGDG by HPLC.

the C-4 and C-5 positions of the galactose ring,via 1,2-elimination also yields the product ion atm�z 749. The rest of the peaks are due to aseries of the charge-remote fragmentation via1,4-elimination along fatty acid chains, as is

� �� observed for M�H ion of free fatty acid 16Ž .Fig. 4b . Thus, the positions of double bonds infatty acid chains can be identified by comparingthese product ions with those of the deprotonatedmolecular ion of standard fatty acid. The charge-

remote fragmentation occurring along the alkylchain of a saturated fatty acid results in a seriesof the product ions generated by the loss ofC H with the neighboring peaks in the seriesn 2 n�2separated by 14 u. Presence of a double bond inthe chain reduces the neighboring peak separa-tion to 12 u. The neutral loss pattern in thehomologous ion series appearing at m�z 781,

Ž767, 755 �, where � represents the double-bond. Ž . Ž .position , 741, 727, 715 � , 701, 687, 675 � , 661,


Fig. 4. Comparison of the peak pattern due to the charge-remote cleavages along the hydrocarbon chain of MGDG and standardŽ . Ž . � �� Ž .linolenic acid 9,12,15-octadecatrienoic acid . a High-mass region of the positive-ion CID spectrum of M�Na ion at m�z 797

Ž . Ž . � ��of MGDG minor species C18:3�C18:3 labeled as M1 in HPLC separation and b the negative-ion CID spectrum of M�H ionŽ .at m�z 277 of linolenic acid. An equal sign above a peak indicates the double-bond position.

Ž3 .647, 633, 619, 605 and 591 I in Fig. 4a isidentical with that appearing at m�z 261, 247,

Ž . Ž . Ž .235 � , 221, 207, 195 � , 181, 167, 155 � , 141,127, 113, 99, 85 and 71 in Fig. 4b. This suggeststhat the positions of three double bonds in thefatty acyl groups of the MGDG species locate atthe 9th, 12th and 15th carbon away from thecarbonyl carbon of fatty acyl chain. These posi-tions are the same as those of the linolenic acid.The intensities of the corresponding peaks in theseries in Fig. 4a,b are also comparable except for

Ž3 .the m�z 591 ion I generated by the neutralloss of C H . Extra stabilization of the product15 26

ion with � ,�-unsaturated carbonyl structure bythe charge-remote cleavage of the bond at the�-position to the carbonyl group is responsible forhigh abundance of this ion. Thus, in addition tothe mass difference between neighboring ions,the relative peak intensities are also helpful inthe determination of the double-bond positions inthe fatty acid chain.

However, the species, which has two fatty acylgroups with different composition, indicates muchmore complex pattern than that of above spectrabecause of the charge-remote cleavages of twodifferent fatty acyl chains. As shown in Fig. 5a, in


the collision spectrum of the species with C34:2composition, the two peaks at m�z 497 and 521corresponding to the G and G ions provide2 1information about the individual fatty acid com-position of C18:2 and C16:0, respectively. How-

Ž .ever, it is impossible at the moment vide infra toŽdetermine which of the two fatty acids is R Fig.1

.1 . For the moment, we will invoke the generalrule that for ‘eukaryotic’ lipid, the sn-2 position isoccupied primarily by 18 carbon fatty acids, whilethe sn-1 position may contain either 18 or 16

� �carbon fatty acids 17,18 . Mass spectral evidencefor the relative positions of two fatty acyl groupswill be discussed shortly. Homologous ions gener-

ated by the charge-remote fragmentation of fattyacid chains in this case can be divided into twogroups; m�z 691, 677, 663, 649, 635, 621, 607 and

Ž3 . �593 I due to a saturated fatty acyl group i.e.1Ž .� Ž .palmitoyl group C16:0 , and m�z 693 � , 679,

Ž . Ž3 .665, 653 � , 639, 625, 611, 597, 583 and 569 I2due to a doubly unsaturated fatty acyl groupŽ .C18:2 . The ions at m�z 761, 747, 733, 719 and705 are common to cleavage of both fatty acylgroups. Thus, the C18:2 fatty acyl group attachedat the sn-2 position is 9,12-octadecadienoyl groupŽ .i.e. linoleoyl group . Similarly, the other speciesof m�z 799 which is identified with two unsatu-

Ž .rated fatty acyl groups C18:2 and C18:3 from

Fig. 5. Comparison of the spectral pattern due to the charge-remote cleavages along the hydrocarbon chain of MGDG speciesŽ . Ž . Ž . Ž .labeled as: a M4 C16:0�C18:2 and b M2 C18:2�C18:3 in HPLC separation.


Table 1Structural identification of the fatty acyl groups of MGDG and DGDG molecular species purified by HPLC from wheat flour

� � b� � � �Lipid class HPLC M�Na M�H Structure of fatty acyl G �G2 1apeak groups

MGDG M1 797.3 773.4 C18:3�C18:3 �

M2 799.3 775.4 C18:2�C18:3 1.51M3 801.4 777.5 C18:2�C18:2 �

M4 777.4 753.4 C16:0�C18:2 1.85M5 803.4 779.4 C18:2�C18:1

DGDG D1 961.5 937.5 C18:2�C18:3 1.43D2 963.4 939.5 C18:2�C18:2 �

D3 939.6 915.5 C16:0�C18:2 1.75D4 965.5 941.5 C18:2�C18:1

a Individual molecular species of galactolipids are designated with the acyl groups at sn-1 and sn-2 positions listed in order,Ž .respectively i.e. C18:1�C16:0�sn-1�sn-2 . The fatty acyl groups are symbolized by the convention, carbon number:double bond

� Ž . Ž . Ž . Ž .�number i.e. C16:0 palmitoyl , C18:1 oleoyl , C18:2 linoleoyl , C18:3 linolenoyl .b � ��The intensity ratio of G and G ions observed in the CID spectra of M�Na ions. Because the species corresponding to1 2

M5 and D4 are mixtures of regioisomers, the difference between the abundance of the G and G ions is very small.1 2

the G and G ions at m�z 519 and 521, respec-1 2Ž .tively, contains linoleoyl C18:2 and linolenoyl

Ž . Ž .C18:3 groups see Fig. 5b .�More importantly, the CID spectra of M�

��Na ions also provide information about therelative positions of two different fatty acyl groupon the glycerol backbone. Recently, Kim et al.� �19 investigated the CID fragmentation ofsodium-adducted molecules of syntheticmonoacetyldiglycerides with diverse fatty acidcomposition. It was found that the relative abun-dance of the G and G ions due to the neutral1 2loss of the fatty acyl groups at sn-1 and sn-2positions, respectively, provided information aboutthe regiospecificity of the two acyl linkages. Thereis a trend that the formation of the product ionŽ� ��.M�Na�R COOH due to the loss of the2sn-2 fatty acyl group is always more favorable

Ž� ��.than the one M�Na�R COOH correspond-1ing to the loss of the sn-1 fatty acyl group. Thesame trend was observed in the CID spectra ofMGDG molecular species. The observed resultswere summarized in Table 1. The larger abun-dance of the G ion is likely due to the fact that a2proton from either the sn-1 or sn-3 moiety can be

� �removed in the fragmentation process 19 . How-ever, the loss of R COOH can only involve the1secondary proton found at sn-2. However, in the

Ž .case of the species with oleoyl C18:1 andŽ .linoleoyl C18:2 groups, the preference for the

loss of any fatty acyl group than any other one isnot clear, because of the small difference of theabundance of the two G ions. Therefore, thisspecies is probably a mixture of equal amounts oftwo regioisomers exchanging each other’s fattyacyl group at the sn-1 and sn-2 positions.

To further investigate the regiospecificity of thefatty acyl linkages of MGDG species, sn-2 acyl

Žlysogalactolipids i.e. monogalactosyl monoacyl-.glycerol; MGMG were synthesized by specificŽ .enzyme, Lipase XI from Rhizopus arrhizus which

cleaves ester bond between glycerol backboneand sn-1 fatty acyl group specifically. Fig. 6ashows the FAB mass spectrum of MGMG synthe-sized and the inset in the same figure indicates adistribution of the molecular species present. Thesodium-adducted molecule observed at m�z 539corresponds to the major species with a linoleoylgroup at the sn-2 position. This species is mainly

Ž .derived from MGDG major species M3 with twoŽ .linoleoyl groups C18:2 . The minor species with

linolenoyl and oleoyl groups also appear at m�z537 and 541, respectively. The absence of MGMG

Ž .species at m�z 515 with a palmitoyl group provesthat the corresponding MGDG species observedat m�z 777 has palmitoyl and linoleoyl groups at


Ž . Ž .Fig. 6. a FAB mass spectrum of sn-2 acyl lyso MGDG i.e. MGMG synthesized by a specific enzymatic hydroylsis of the fatty acylŽ . � �� Ž .group at the sn-1 position; and b the CID spectrum of M�Na ion observed at m�z 539 in a .

sn-1 and sn-2 positions, respectively. These re-sults are in good agreement with those obtainedfrom the ratio of the relative abundance of thetwo G and G ions. Fig. 6b displays the CID1 2

� ��spectrum of M�Na ion of the MGMG majorspecies. The spectral pattern is very similar tothat of MGDG except for less abundant E, G,and D ions and more intense H ion.

3.2. Digalactosyl diacylglycerol

The above spectral interpretation could be ex-tended to DGDG molecular species from wheatflour, which carries a disaccharide moiety. Themass spectrum obtained by FAB of DGDG in a

matrix with NaI is shown in Fig. 7a. The predomi-nant peaks at m�z 939 and 963 correspond to thespecies with C34:2 and C36:4 composition, re-spectively. However, the HPLC separation repre-

Ž .sents peaks D1 and D4 corresponding to otherminor components with C36:5 and C36:3 compo-

Ž .sition as well as major components D3 and D2 ,as shown in Fig. 3b. The positive-ion CID tandem

� �� Ž .mass spectrum of M�Na ion at m�z 939 ofDGDG component with a total fatty compositionof C34:2 is shown in Fig. 7b. Its fragmentationpattern is similar to the CID spectra of MGDGspecies with the same composition. In the CID of� ��M�Na ion of DGDG, the main differencesare that the product ions generated by the cleav-


Ž . Ž .Fig. 7. a Positive-ion mass spectrum obtained by FAB of DGDG in 3-NBA matrix saturated with NaI; and b CID tandem mass� �� Ž .spectrum of M�Na ion at m�z 939 of DGDG species corresponding to the peak labeled as D3 in HPLC separation. The

� ��fragmentation pathways observed in the CID of M�Na ion are also shown.

ages of second sugar moiety of DGDG appear inthis spectrum and all of the product ions gener-ated by the fragmentation of fatty acyl groups are

Žshifted to higher mass by 162 u corresponding to

.the mass of a monosaccharide residue . Espe-cially, the 0,4A and 3,5A ions observed at m�z2 2245 and 259 provide the information about theŽ .1�6 sugar linkage between the two sugar rings.


Fig. 8. Comparison of the spectral pattern due to the charge-remote cleavages along the hydrocarbon chain of DGDG speciesŽ . Ž . Ž . Ž .labeled as: a D2 C18:2�C18:2 ; and b D4 C18:2�C18:1 in HPLC separation.

As observed in Fig. 8a, in the high-mass region of� �� Ž .the CID spectrum of M�Na ion at m�z 963

of DGDG major species with two C18:2 acylgroups, a series of the product ions indicate thedouble-bond positions. These homologous ionsare observed at m�z 947, 933, 919, 905, 891,

Ž . Ž .879 � , 865, 851, 839 � , 825, 811, 797, 783, 769Ž3 .and 755 I . Thus, these species contain two

linoleoyl groups. In addition, two product ions atŽ . Ž .m�z 799 Y -2H and 801 Y generated by the1 1

cleavage of the interglycosidic bond between thetwo sugar rings are also observed in this region,as shown in Fig. 7. They correspond to the keto-and alcohol-type ions, respectively. In the case ofthe species with different fatty acyl groups, ho-

mologous ions appearing in the CID spectrum ofthe m�z 965 ion shown in Fig. 8b can be dividedinto two groups. The first group corresponds to a

Ž .series of the ions at m�z 879, 865, 851, 839 � ,825, 811, 797, 783, 769 and 755 ions due to a

Ž .singly unsaturated fatty acyl chain C18:1 . Thesecond group corresponds to a series of the ions

Ž . Ž .at m�z 881 � , 867, 853, 841 � , 827, 813, 799,785, 771 and 757 ions due to a doubly unsaturated

Ž .fatty acyl chain C18:2 . The ions at m�z 949,935, 921, 907 and 893 are common to the cleavageof both fatty acyl groups. The neutral losses in thefirst homologous series are identical with those ofoleic acid, while the pattern in the second seriesis identical with that of linoleic acid. Thus, it can


be concluded that this species contains oleic andlinoleic acids. For the components with differentfatty acyl groups, the ratio of the intensity of G1and G ions provides information about the re-2giospecificity of the acyl linkages. The resultsobtained are summarized in Table 1. However,the results obtained by the structural determina-tion of sn-2 acyl lyso components synthesized byspecific enzymatic hydrolysis of sn-1 fatty acylgroup were in accord with those obtained fromthe MGMG. Also, the results for the regiospeci-ficity of the acyl linkages in galactolipids isolatedfrom wheat flour were in good agreement with

� �those reported previously 20 .As demonstrated by these examples, CID-

MS�MS of the sodium-adducted molecules ofMGDG and DGDG offered complete structuralanalysis of the fatty acyl residues and regiospeci-ficity of two fatty acyl linkages as well as the headgroup. The majority of product ions observed in

� ��the CID spectra of M�Na ions of MGDGand DGDG are due to the fragmentation remote

Žfrom the charge site i.e. the binding site of.sodium ion . Moreover, the positive charge is

probably localized on the sugar moiety, since themajority of oxygen atoms capable of forming acomplex with a sodium ion are present on thesugar ring. This result provides explanation onthe variety of the product ions generated by thecharge-remote fragmentation giving structural in-formation of the fatty acyl chains.

References

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Documents

Tandem mass spectrometric analysis of fatty acyl groups of galactolipid molecular species from wheat flour