Structural Identification of Glycerolipid Molecular SpeciesIPT

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Analytical Biochemistry 267, 260–270 (1999)Article ID abio.1998.3041, available online at http://www.idealibrary.com on

solated from Cyanobacterium Synechocystis sp.CC 6803 Using Fast Atom Bombardmentandem Mass Spectrometry

oung Hwan Kim,*,†,1 Jong-Soon Choi,‡ Jong Shin Yoo,* Young-Mok Park,‡ and Myung Soo Kim†Mass Spectrometry and ‡Biomolecule Analysis Groups, Korea Basic Science Institute, Taejon 305-600, Korea; andDepartment of Chemistry, Seoul National University, Seoul 151-742, Korea

eceived May 29, 1998

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Our previous works have demonstrated that fasttom bombardment tandem mass spectrometry can bevaluable tool in determining the complete structure

f glycoglycerolipids and glycerophospholipids. Colli-ion-induced dissociation of sodium-adducted molec-lar ions ([M 1 Na]1 or [M 2 H 1 2Na]1) generatesiverse product ions informative on the double-bondosition in fatty acyl groups as well as the polar headroup and fatty acid composition. Here we report thathis direct and rapid method can be applied to thetructural determination of individual molecular spe-ies of each glycerolipid class purified from the totalipid extract of cyanobacterium Synechocystis sp. PCC803. Especially, based on the preference for the loss ofhe fatty acyl group positioned at the sn-2, it wasroved that all of the molecular species of diacylglyc-rolipids contained a palmitoyl group exclusivelyt the sn-2 position. Additionally, lysoglycerolipids,onoacyl forms of four major membrane diacylglyc-

rolipids, were first isolated together from a fresh ex-ract. Using fast atom bombardment mass spectrome-ry and tandem mass spectrometry, it was found thatach lysoglycerolipid had a molecular species withnly palmitic acid as a fatty acyl group. Thus, theseompounds could be synthesized by specific enzyme-atalyzed hydrolysis of the sn-1 fatty acyl group of theorresponding diacylglycerolipids. © 1999 Academic Press

Cyanobacteria have been used as model organismsor the study of oxygenic photosynthesis because theyre similar to eukaryotic plants in performing photo-

1 To whom correspondence should be addressed at Mass Spectrom-try Group, Korea Basic Science Institute, P.O. Box 41, Taejon 305-00, Korea. Fax: 82-42-865-3419. E-mail: yhkim@comp.kbsi.re.kr.

60

ynthesis with two types of photochemical reactionsnd having chlorophyll a as the major photosyntheticigment (1, 2). Furthermore, the sequence of the entireenome of unicellular cyanobacterium Synechocystisp. PCC 6803 was determined recently (3). This can beelpful in understanding the entire genetic system in-olved in oxygenic photosynthesis. It was also observedhat Synechocystis sp. PCC 6803 had phototactic glid-ng motility, the ability to move toward a light source,n a solid surface of BG11 agar (4–7). Recently, usingomputerized videomicroscope techniques, qualitativend quantitative analyses of the phototactic glidingotility in Synechocystis sp. were studied in detail

J.-S. Choi et al., submitted for publication). Based onhe study of the gliding movement of the Cytophaga-lexibacter group of gliding bacteria, it was found thatnusual sulfonolipids, which are major cell-envelopeomponents, were required for gliding motility (8, 9).hus, we investigated the total lipid extracts from Syn-chocystis sp. PCC 6803 and ATCC 27184, mutant de-cient in gliding, to examine the unusual lipids, usingast atom bombardment (FAB)2 coupled with four-sec-or tandem mass spectrometry (MS/MS). But distinctifferences between the lipid compositions of bothtrains were not found (Y. H. Kim et al., unpublishedork). However, in the course of the analyses of total

ipids, it was proved that the FAB-MS/MS techniqueas very useful to the structural determination of glyc-rolipids. The usefulness of FAB ionization has beenemonstrated for their structural identification (10–

2 Abbreviations used: FAB, fast atom bombardment; MS/MS, tan-em mass spectrometry; SQDG, sulfoquinovosyl diacylglycerol;GDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacyl-

lycerol; PG, phosphatidylglycerol; CID, collision-induced dissocia-ion; SQMG, sulfoquinovosyl monoacylglycerol; MGMG, monogalac-osyl monoacylglycerol; DGMG, digalactosyl monoacylglycerol; LPG,ysophosphatidylglycerol.

0003-2697/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

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261IDENTIFICATION OF GLYCEROLIPIDS IN Synechocystis PCC 6803

4). Actually, glycerolipids purified from biologicalources are often mixtures of many molecular species,hich differ in chain length of fatty acyl groups, and

he double-bond position and degree of unsaturation inhe fatty acyl groups. Hence, it is difficult and time-onsuming to separate a naturally occurring mixture oflycerolipids into each molecular species. Recently, weeported that tandem mass spectrometry of sodium-dducted molecular ions ([M 1 Na]1 or [M 2 H 1Na]1) which were produced by FAB of glycoglycero-ipids (15) and glycerophospholipids (16) provided use-ul information on the double-bond position in the fattycyl chains as well as the polar head group and fattycid composition. Thus, this method can be widely ap-lied to the structural analysis of molecular species ofarious glycerolipids isolated from the mixture of bio-ogical origin. In this paper, we describe direct andapid structural identification of the molecular speciesf glycerolipids purified from the total lipid extract ofyanobacterium Synechocystis sp. PCC 6803, including

IG. 1. The structures of the glycerolipids isolated from cyanobac-erium Synechocystis sp. PCC 6803.

ulfoquinovosyl diacylglycerol (SQDG), monogalactosyliacylglycerol (MGDG), digalactosyl diacylglycerolDGDG), and phosphatidylglycerol (PG), known as four

ajor membrane lipids in cyanobacteria. In addition,our minor lysoglycerolipids corresponding to theironoacylglycerol types were first isolated together

rom a fresh extract and their structural analyses werelso performed.

ATERIALS AND METHODS

hemicals and Materials

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol,-palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol,nd g-linolenic acid were obtained from Sigma (St.ouis, MO). All solvents used were of analytical gradend purchased from Baxter (Muskegon, MI).

edia and Growth Conditions

The cyanobacterium Synechocystis sp. PCC 6803 wasrown in BG11 medium described by Kratz and Myers17). The medium was supplemented with 10 mM oflucose. The cells were cultivated at 28°C for 3 to 4ays and illuminated continuously at an intensity of000 lux with a fluorescent lamp. The culture wasarvested in the mid-log phase of growth and by cen-rifugation at 12,000gmax and 4°C for 15 min.

solation of Glycerolipids

The harvested cells were homogenized in 300 ml ofhloroform:methanol:water (1:1:1, v/v/v). The total lip-ds were extracted by the method of Bligh and Dyer18). The final extract in chloroform phase was fullyvaporated and the residue was reconstituted in 2 ml ofhloroform:methanol (1:1, v/v). According to theethod of Douce et al. (19), the extract was purified by

wo-dimensional thin-layer chromatography (2D-TLC)n a silica plate (Whatman, Hillsboro, OR). The sol-ent systems used were chloroform:methanol:water65:25:4, v/v/v) as a solvent for dimension I and chlo-oform:acetone:methanol:acetic acid:water (10:4:2:2:1,/v/v/v/v) for dimension II. Glycerolipids were visual-zed by spraying the 2D-TLC plate with 0.01% primu-in in a mixture of acetone and water (4:1, v/v) and thenlluminating with ultraviolet light. All of the eightpots were identified and scraped from the plate. Thenach spot was eluted with chloroform:methanol (1:1,/v) for FAB-MS and MS/MS analyses.

ass Spectrometry

All mass spectrometric analyses were performed us-ng a JMS-HX110/110A tandem mass spectrometerJEOL, Tokyo, Japan) of a four-sector instrument withn E1B1E2B2 configuration as previously described in

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etail (20). Briefly, the ion source was operated at 10V accelerating voltage in the positive-ion mode withhe mass resolution of 1000 (10% valley). Ions were

TAB

Structural Identification of Glycerolipid Mo

Lipid class MW[M 1 Na]1 or

[M 2 H 1 2Na]1

MGDG 752.6 775.5754.6 777.5756.6 779.5

DGDG 914.6 937.6916.6 939.6918.6 941.7

SQDG 792.5 837.5794.5 839.5818.5 863.5820.5 865.5

PG 744.5 789.5746.5 791.5748.5 793.5

MGMG 492.3 515.4DGMG 654.3 677.2SQMG 556.2 601.0LPG 484.2 529.1

a Individual molecular species of diacylglycerolipids are designaespectively (i.e., C18:1/C16:0 5 sn-1/sn-2). The fatty acyl groups are16:0 (palmitoyl), C16:1 (palmitoleoyl), C18:1 (oleoyl), C18:2 (linole

elative quantity of each molecular species to the most abundant specb The intensity ratio of G1 and G2 ions observed in the CID spectra

QDG and PG.c Not measured due to overlap.

IG. 2. Positive-ion FAB mass spectrum of MGDG derived from Syevealing [M 1 Na]1 and [M 2 H 1 2Na]1 ions of several molecular

roduced by fast atom bombardment using the cesiumon gun operated at 22 kV. Approximately 10 mg of eachample dissolved in chloroform:methanol (1:1, v/v) was

1

lar Species in Synechocystis sp. PCC 6803

[M 2 H]2 Structure of fatty acyl groupsa G2/G1b

C18:3g/C16:0 (100) 1.43C18:2/C16:0 (47) 1.59C18:1/C16:0 (41) 1.52

C18:3g/C16:0 (100) 1.25C18:2/C16:0 (45) 1.30C18:1/C16:0 (25) 1.32

791.5 C16:1/C16:0 (16) 1.85793.5 C16:0/C16:0 (19) N.M.c

817.5 C18:2/C16:0 (64) 1.42819.6 C18:1/C16:0 (100) 1.07743.2 C18:3g/C16:0 (47) 1.14745.3 C18:2/C16:0 (100) 1.23747.4 C18:1/C16:0 (53) 1.38

C16:0C16:0

555.2 C16:0483.2 C16:0

with the acyl groups at sn-1 and sn-2 positions listed in order,bolized by the convention, carbon number:double bond number (i.e.,), C18:3g (g-linolenoyl)). The number in parentheses indicates thein each glycerolipid class, regarding the abundance of isotopic peaks.[M 1 Na]1 ions for MGDG and DGDG and [M 2 H 1 2Na]1 ions for

hocystis sp. PCC 6803. (Inset) The zoomed in 770,m/z,805 region,ecies.

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263IDENTIFICATION OF GLYCEROLIPIDS IN Synechocystis PCC 6803

ixed with 1 ml of 3-nitrobenzyl alcohol (Sigma) on theAB probe tip. Collision-induced dissociation (CID) ofhe sodium-adducted molecular ions selected with therst mass spectrometer (MS-1) occurred in the collisionell located between B1 and E2 and floated at 3.0 kV.he resultant product ions were analyzed by the B/Ecan method of the second mass spectrometer (MS-2).he collision gas, helium, was introduced into the col-

ision chamber at a pressure sufficient to reduce therecursor ion signal by 70%. Signal averaging with twocans was carried out. MS-1 was operated at the reso-ution adjusted so that only the 12C-species of the pre-ursor ions was transmitted. MS-2 was operated at aesolution of 1000.

ESULTS AND DISCUSSION

ajor Membrane Diacylglycerolipids: MGDG, SQDG,DGDG, and PG

Cyanobacteria have a glycerolipid composition veryimilar to that of the chloroplasts of leaves, the major

IG. 3. FAB tandem mass spectrum of [M 1 Na]1 ion (m/z 775) corroposed pathways are shown above the spectrum. The nomenclaturhanges. All fragments contain sodium, i.e., the ion labeled Bn correspbserved here for the Cn ion indicates that it has the lactone form thaefinition of the alcohol type ion, Cn

1 5 [Cn 1 2H]1. For the cleavageshe relative position (sn-1 or sn-2) of the cleavage in the fatty acyl ghe carbonyl carbon of the fatty acyl group.

lycerolipids being MGDG, SQDG, DGDG, and PG (21,2). The molecular structures of these lipids are shownn Fig. 1. Generally, they are extracted as complex

ixtures of many molecular species, as can be seenrom the FAB mass spectrum of MGDG shown in Fig., when isolated from biological samples. These speciessually differ in the chain lengths of their fatty acylroups and/or the number of double bonds in two fattycyl groups. The characteristics of their positive-ionAB mass spectra are the absence of the protonatedolecular ion ([M 1 H]1), but the presence of the

odium-adducted molecular ion ([M 1 Na]1 or [M 21 2Na]1). Although the source of sodium ion is not

ade clear, an intense sodium-adducted molecular ionresent in the FAB mass spectra has an advantagever the protonated molecular ion as the precursor ionn the MS/MS analysis. Above all, the attachment of aodium ion (or ions) to the polar head group in [M 1a]1 (or [M 2 H 1 2Na]1) localizes a positive chargeore strongly than does that of H1 in [M 1 H]1. Thus,

onding to the most abundant species (C18:3g/C16:0) of MGDG. Theroposed by Costello and co-workers (23, 24) was adopted with minors to [Bn 1 Na 2 H]1, which can be expressed as [Bn(Na)]1. The mass

rresponds to Cn 1 Na 2 3H or better Cn(Na)-2H with respect to theirthe fatty acyl chains, the subscript number in the symbol representsp and the superscript number the cleaved bond position relative to

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he CID spectrum of a sodium-adducted molecular ionndicates a more systematic pattern than that of arotonated molecular ion, due to the charge-remoteragmentation, and then can be interpreted more eas-ly to analyze the structure of the lipid molecular spe-ies. As can be seen from the inset of Fig. 2, the relativeuantities of molecular species present in an extractedlycerolipid class can be approximately evaluated fromhe relative abundances of sodium-adducted molecularons in the FAB mass spectrum as summarized inable 1. As a result, it was observed that the change ofrowth conditions such as growth medium, tempera-ure, and intensity of light affected the relative abun-ance of each species to some extent. The CID-MS/MSpectrum of a sodium-adducted molecular ion is char-cterized by diverse product ions, as can be seen fromhe MS/MS spectrum of MGDG shown in Fig. 3. Theragmentation pathways are also illustrated in theame figure. Among these ions, the A, B9, and X ionsenerated by the cleavages through the sugar residuere very informative on the structure of the head groupf glycoglycerolipids. Also, the B, C-2H, Y-2H, and Yons rise from cleavages of the C-O-C bonds betweenhe sugar and glycerol units of glycoglycerolipids or

TAB

Fragmentation Assignment of Product Ions ObMolecular Ions of the Most Abundant Specie

AssignmentbMGDG

[M 1 Na]1 5 775

0,4A 83 (6.5)3,5A 97 (8.3)1,5A 157 (7.9)B9 169 (13.4)B 185 (44.0)C-2H 201 (9.4)E 243 (85.2)2D1,2 301 (56.3)3D1,2 315 (63.9)G1 497 (34.7)H1 513 (7.9)G2 519 (49.5)H2 535 (14.1)3I1 569 (33.6)3I2 591 (100)Y-2H —Y —1,5X 641 (18.8)0,2X 655 (7.2)

a The number in parentheses represents abundance relative to thb The mass observed here for the Cn-ion indicates that it has the

espect to the definition of the alcohol type ion, Cn1 5 [Cn 1 2H]1

imilarly, Yn ions correspond to Yn(Na) with respect to the alcohol-typndicates the keto form, sodiated as Yn(Na)-2H.

c DGDG also generates product ions corresponding to the cleavaget m/z 245(6.2), 259(14.9), 319(10.1), 331(14.3), 347(66.6), and 363(2

d SQDG also generates an SO3Na21 ion at m/z 126(87.8).

nterglycosidic bonds of sugar moiety of DGDG. Thetructural distinctions between the head groups ofhree glycoglycerolipids yield only a minor spectralifference in the low-mass region of their CID spectra,ut the remaining parts are very similar to each other,s summarized for each major molecular species inable 2. The intense E ion and two abundant D ionsontain intact sugar and give information about theolecular weight of the head group. In addition, the

ompositions of two fatty acyl groups are determinedmmediately from a pair of the prominent G ions cor-esponding to the loss of each fatty acyl group as a freeatty acid. In the case of the major species (m/z 775) of

GDG shown in Fig. 3, these ions are observed at m/z97 and 519. Thus, the molecular species contains C18:3nd C16:0 acids as fatty acyl groups. This result is alsoonfirmed by other pairs of H and I ions. Especially, thentensity ratio of two G1 and G2 ions can be used toetermine the relative position of the two fatty acylroups. This result will be discussed later in detail.More importantly, there is an abundant series of

igh-mass product ions formed by charge-remote cleav-ges of fatty acyl chains. These are generated by theoss of CnH2n12 via 1,4-elimination as observed for [M 2

2

ved in FAB-CID-MS/MS of Sodium-Adductedn MGDG, DGDG, and SQDG, Respectively

Product ions,a m/z

DGDGc

[M 1 Na]1 5 937SQDGd

[M 2 H 1 2Na]1 5 865

83 (6.2) 169 (18.4)97 (8.1) 183 (16.6)

157 (5.2) 243 (19.1)169 (11.0) 255 (7.8)185 (22.1) 271 (61.2)201 (18.2) 287 (31.4)405 (100) 329 (100)463 (48.7) 387 (38.4)477 (51.6) 401 (54.7)659 (36.7) 583 (42.8)675 (8.8) 599 (5.6)681 (45.8) 609 (45.9)697 (20.5) 625 (14.2)731 (30.2) 655 (46.7)753 (84.4) 681 (54.5)773 (10.7) —775 (12.7) —803 (38.6) —817 (12.3) —

ost intense product ion. All product ions contain sodium.one form and corresponds to Cn1Na23H or better Cn(Na)-2H withthe CID of [M 1 H]1 of glycoconjugates by Costello et al. (23, 24).ns designated as Yn

1 5 [Yn 1 2H]1 (23, 24). The Yn 2 2H designation

he second sugar moiety. They are 0,4A2, 3,5A2, 1,5A2, B92, B2, and C2-2H), respectively.

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265IDENTIFICATION OF GLYCEROLIPIDS IN Synechocystis PCC 6803

]2 of a free fatty acid (25). The spectral pattern ofhese product ions gives immediate information abouthe presence and location of the double bond in fattycyl chains. Figure 4a shows the expansion of the com-lex CID spectrum of a major [M 1 Na]1 ion (m/z 937)f DGDG. This molecular species contains C18:3 and16:0 acids because the G1 and G2 ions are observed at/z 659 and 681, respectively. The positive-ion CID

pectrum of a sodium-adducted molecular ion, [M 21 2Na]1, of g-linolenic acid (6,9,12-octadecatrienoic

cid) is also shown in Fig. 4b for a comparative expla-ation. The homologous product ions in Fig. 4a can beivided into two groups: m/z 851, 837, 823, 809, 795,81, 767, and 753 (3I2) due to the palmitoyl chainC16:0) and 853, 839, 825, 813, 799, 785, 773, 759, 745,

IG. 4. Comparison of the peak pattern due to the charge-remote ccid (6,9,12-octadecatrienoic acid). (a) High-mass region of the CID sppecies (C18:3g/C16:0) of DGDG. (b) CID spectrum of [M 2 H 1 2Na]he double-bond position. All product ions contain sodium.

nd 731 (3I1) due to the unsaturated acyl chain (C18:3).he ions at m/z 921, 907, 893, 879, and 865 are common

o the cleavage of both fatty acyl groups. The spectralattern except for the unusual intensity of the ion at/z 773 is very similar to that of corresponding product

ons observed in the CID spectrum of [M 2 H 1 2Na]1

on (m/z 323) of free g-linolenic acid. The high intensityf the peak at m/z 773 in comparison with that of theorresponding ion at m/z 159 is due to the overlap with1-2H ion which is always observed in the CID spec-

rum of DGDG. Thus, the major molecular species ofGDG contains g-linolenic and palmitic acids as two

atty acyl groups. This structural analysis is also ap-licable to other molecular species of DGDG, MGDG,nd SQDG. The results are summarized in Table 1.

age along the hydrocarbon chain of DGDG and standard g-linolenicrum of [M 1 Na]1 ion (m/z 937) corresponding to the most abundantn (m/z 323) of g-linolenic acid. An equal sign above a peak indicates

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The sole glycerophospholipid found in cyanobacteria,G, has a phosphate group ether-linked to glycerol ashe head group. In the sodium-adducted molecular ion,M 2 H 1 2Na]1, a positive charge is localized at thehosphate group due to the attachment of two sodiumons. This is confirmed by the presence of the productons formed by the fragmentation of the phosphoglyc-rol head group, such as PO3Na2

1 (m/z 125),2PO4Na2

1 (m/z 143), [HOP(O)(ONa2)OCH3]1 (m/z

57), [CH3OP(O)(ONa2)OCH 5 CH 2 CH2OH]1 (m/z13), and [HOP(O)(ONa2)OCH2CH(OH)CH2OH]1 (m/z17) as shown in Fig. 5. Also, the intense peak observedt m/z 199 corresponds to disodiated five- or six-embered hydroxycyclophosphane. The fragmentation

athways are illustrated in Fig. 5 also. The remainder

IG. 5. FAB tandem mass spectrum of [M 2 H 1 2Na]1 ion (mynechocystis sp. PCC 6803. (b) 1-Palmitoyl-2-oleoyl-sn-glycero-3-pho

ons contain sodium.

f the peaks in the CID spectrum displays the spectralattern very similar to those of the CID spectra ofodium-adducted molecular ions for glycoglycerolipids.hus, from the analysis of the homologous product ions

n a high-mass region, the species of m/z 793 containsleic and palmitic acids. The results obtained for otherpecies are also summarized in Table 1.In the structural analysis of diacylglycerolipids, it is

lso important to determine the relative position of thewo different fatty acyl groups. Positional distributionf fatty acids in diacylglycerolipids is usually deter-ined by enzymatic hydrolysis of the sn-1 fatty acid

ster with a lipase from Rhizopus arrhizus delemar26) and GC analysis of the released fatty acid andhose of the lyso compound formed. But further sepa-

93) of PG. (a) The molecular species (C18:1/C16:0) isolated fromoglycerol. The fragmentation pathways are also shown. All product

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267IDENTIFICATION OF GLYCEROLIPIDS IN Synechocystis PCC 6803

ation from a mixture of species with more samples iseeded to determine the relative position of the fattycyl groups of each species. To date, several investiga-ions for the positional determination of fatty acylroups in glycerophospholipids have been reported, us-ng the FAB-CID-MS/MS technique (10, 11, 27). Re-ently, in our previous work (16), it was found that theegiospecificity of the acyl chain linkages in glycero-hospholipids could be determined from the ratio ofntensities of two unsaturated lysophosphatidic acidons observed in CID-MS/MS of [M 1 Na]1 ions. Theseroduct ions are generated by the loss of each free fattycid at the sn-1 and sn-2 positions together with aerivative ether-linked to a phosphate group, respec-ively. In addition, it was proved that another pair ofhe product ions (G1 and G2) corresponding to an un-aturated lysophospholipid in the same spectra alsoisplayed a similar trend and could be used as theecondary guideline in the determination of the regio-pecificity. According to these results, the product ionue to the loss of the sn-2 fatty acyl group is alwaysore abundant than one corresponding to the loss of

he sn-1 fatty acyl group. Based on these empiricalules, the regiospecificity of molecular species in PG

IG. 6. FAB tandem mass spectrum of [M 2 H 1 2Na]1 ion (m/z 6ith the fragmentation pathways. All product ions contain sodium.

solated from Synechocystis sp. PCC 6803 was deter-ined in our previous study (16). Additionally, the

ntensity ratio of G1 and G2 ions, which were observedn the CID spectra of [M 2 H 1 2Na]1 precursors (Fig.a), provided the same result for the relative position ofatty acyl groups (see Table 1). A commercially avail-ble PG compound, 1-palmitoyl-2-oleoyl-sn-glycero-3-hosphoglycerol, was examined to confirm this empir-cal rule further. In this case, a G2 ion resulting fromhe loss of oleic acid at the sn-2 position is about 1.5imes more abundant than the G1 ion due to the loss ofalmitic acid at the sn-1 position in the CID spectrumf [M 2 H 1 2Na]1 ion (m/z 793) as can be seen fromig. 5b. According to the results summarized in Table, all species of PG contain palmitic acid (C16:0) at then-2 position. In the case of DGDG, our previous result15) showed that the G2 ion of m/z 659 was about 1.5imes more abundant than G1 ion of m/z 683 in the CIDpectrum of [M 1 Na]1 ion (m/z 939) from wheat flour.his is in good agreement with the result that in wheatlycerolipids, C16 fatty acids are essentially confined tohe sn-1 position (28). However, in the CID spectrum ofhe same precursor ion from cyanobacteria, the producton of m/z 683 is more abundant than that of m/z 659.

of SQMG with the palmitoyl group as the fatty acyl group together

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herefore, this precursor ion contains linoleic andalmitic acids at the sn-1 and sn-2 positions, respec-ively. On the other hand, the CID spectrum of [M 1a]1 ion (m/z 777) of MGDG from cyanobacteria

howed that the G2 ion of m/z 521 was more abundanthan the G1 ion of m/z 497, in contrast to that of theame precursor ion from wheat flour. Therefore, thealmitoyl group of this precursor ion is at the sn-2osition. Further investigation for each other molecu-ar species of MGDG and DGDG also proved that aalmitic acid was positioned exclusively at the sn-2.age and co-workers (14) proposed that the relative

ntensity of G1 and G2 ions, which were observed in theID spectrum of [M 2 H]2 ion of SQDG, providedvidence for the relative position of the two fatty acylroups. Based on this rule, the regiospecificity of theatty acyl linkages of each SQDG species was deter-ined from negative-ion CID spectrum reported previ-

usly (15). These results were confirmed further by thentensity ratio of the same type ions, which were alsobserved in the CID spectrum of [M 1 Na]1 ion, asummarized in Table 1. The most characteristic resultor all species investigated is the exclusive localizationf palmitic acid at the sn-2 position. In general, it isell-known that in cyanobacteria, the C16 fatty acids ofiacylglycerolipids are localized mostly at the sn-2 po-

TABLE 3

Fragmentation Assignment of Product Ions Observed inAB-CID-MS/MS of Sodium-Adducted Molecular Ions ofQMG, MGMG, and DGMG, Respectively

Assignment

Product ions,a m/z

SQMGb

[M 2 H 1 2Na]1

MGMG[M 1 Na]1

DGMGc

[M 1 Na]1

0,4A 169 (15.2) 83 (7.9) 83 (6.4)3,5A 183 (8.6) 97 (6.8) 97 (3.9)1,5A 243 (8.8) 157 (6.9) 157 (4.8)B9 255 (5.4) 169 (16.6) 169 (16.0)B 271 (26.1) 185 (37.1) 185 (20.0)C-2H 287 (17.4) 201 (22.3) 201 (12.8)E 329 (15.6) 243 (13.3) 405 (10.9)G 345 (8.4) 259 (13.6) 421 (12.0)H 361 (29.9) 275 (44.6) 437 (44.4)2D1,2 387 (12.0) 301 (10.8) 463 (8.9)3D1,2 401 (5.1) 315 (7.3) 477 (6.4)3I 417 (68.6) 331 (100) 493 (100)Y-2H — — 513 (11.0)Y — — 515 (10.1)1,5X — 381 (16.3) 543 (41.1)0,2X — 395 (4.9) 557 (10.3)

a The number in parentheses represents abundance relative to theost intense product ion. All product ions contain sodium.b SQMG also generates an SO3Na2

1 ion at m/z 126(100).c DGMG also generates product ions corresponding to the cleavage

f the second sugar moiety. They are 0,4A2, 3,5A2, 1,5A2, B92, B2, C2-2H,,5X0, and 0,2X0 at m/z 245(10.8), 259(19.9), 319(8.3), 331(18.1),47(48.5), 363(58.7), 381(10.4), and 395(4.0), respectively.

ition and the C18 fatty acids at the sn-1 position (29–1). Hence, this supports the applicability of the em-irical rules mentioned above to the positionaletermination of the fatty acyl groups of each molecu-ar species in diacylglycerolipids, even though a limitedumber of compounds were investigated.From the structural analysis of the fatty acyl groups

f each molecular species in diacylglycerolipids, Syn-chocystis sp. PCC 6803 is characterized by a highontent of polyunsaturated fatty acids, represented byinoleic (C18:2) and g-linolenic (C18:3g) acids. Accord-ng to the classification of blue–green algae (or cya-obacteria) by Kenyon (32), this strain 6803 belongs tohe third group due to the presence of significant quan-ities of g-linolenic acid.

inor Membrane Monoacylglycerolipids: SQMG,MGMG, DGMG, and LPG

SQMG, MGMG, DGMG, and LPG corresponding tohe monoacyl forms of diacylglycerolipids were firstsolated simultaneously from a fresh extract of Syn-chocystis PCC 6803, where diminutive amounts ofaterials were available. Several years ago, Mu-

akami and co-workers (33) found MGMG and DGMGn the cyanobacterium, Phormidium tenue. However,hey were not observed in a fresh algal extract but onlysolated from extract of the alga stored at 220°C for

ore than 1 month. Also, SQMG and LPG were notound. The remarkable feature of the FAB mass spec-ra of monoacylglycerolipids is the almost exclusiveresence of the molecular species with a palmitic acidC16:0) as a fatty acyl group. The CID spectrum ofQMG species (i.e., [M 2 H 1 2Na]1 5 601) shown inig. 6 displays a spectral pattern very similar to that ofQDG except for less abundant E, G, and D ions and aore intense H ion. The proposed fragmentation path-ays are shown in the same figure. On the other hand,

he charge-remote cleavages along the hydrocarbonhain of the palmitoyl group also give rise to a series ofroduct ions separated by 14 Da. The CID spectra ofGMG and DGMG indicate almost the same spectral

eature as summarized in Table 3. The CID spectrumf the molecular species ([M 2 H 1 2Na]1 5 m/z 529)f LPG and fragmentation pathways together arehown in Fig. 7a. The intense product ion, which wasbsent in the CID spectrum of PG, is observed at m/z37. This ion provides information about the regio-pecificity of the acyl chain in lysophospholipids. Aetailed discussion will be represented shortly.From the previous result of exclusive localization of a

almitoyl group at the sn-2 position in diacylglycero-ipids, the palmitoyl groups in monoacylglycerolipidshould be positioned at the sn-2, since they would beynthesized by specific enzyme-catalyzed hydrolysis ofhe sn-1 fatty acyl group of the corresponding diacyl-lycerolipids. Furthermore, because of not finding the

sscawatptOasatmf

HslolaAwlvfcdc1s

F6

269IDENTIFICATION OF GLYCEROLIPIDS IN Synechocystis PCC 6803

pecies with a C18 fatty acyl group in their FAB masspectra, nonspecific hydrolysis, which may occur in theourse of isolation and purification under a weaklycidic condition, can be excluded. Murakami and co-orkers (33) found that the compositions of the fattycid residues in MGMG and DGMG were very similaro those of MGDG and DGDG, respectively, at the sn-2osition. However, through the NMR study of them,he fatty acyl group was attached at the sn-1 position.n the basis of this finding, they proposed that mono-cylgalactolipids were presumably formed by regio-elective deacylation at the sn-1 position followed bycyl group migration. However, this acyl group migra-ion may occur during long-time storage, since such aigration in lysophospholipids is generally rapid and

avors predominantly the sn-1 position (34). Recently,

IG. 7. FAB tandem mass spectrum of [M 2 H 1 2Na]1 ion (m/z 529803. (b) 1-Palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol. The fra

an and Gross (35) reported that positive-ion electro-pray ionization tandem mass spectrometry of sodiatedysophospholipid regioisomers results in the presencef multiple diagnostic pairs of product ions, which al-ows rapid and direct discrimination between sn-1-cyl- and sn-2-acyllysophospholipid regioisomers.mong these pairs, the neutral loss of a derivative,hich is ether-linked to a phosphate group in each

ysophospholipid class, results in the kinetically fa-ored formation of a five-membered phosphodiesterrom a sodiated 1-acyl regioisomer compared to theorresponding six-membered phosphodiester from so-iated 2-acyl regioisomer. In the CID spectrum of theommercially available sn-1-LPG regioisomer (i.e.,-palmitoyl-2-hydroxy-sn-glycero-3-phosphoglycerol)hown in Fig. 7b, the disodiated five-membered phos-

f LPG. (a) The molecular species isolated from Synechocystis sp. PCCentation pathways are also shown. All product ions contain sodium.

) ogm

pwpdftLcstl

sretl

R

1

1

1

1

1

1

1

11

1

2

22

22

2

2

2

2

2

3

3

33

3

3

270 KIM ET AL.

hodiester (m/z 437) due to the neutral loss of glycerolas more abundant than disodiated sn-1-acylphos-hatic acid (m/z 455) resulting from the loss of vinyl-iol. But in the CID spectrum of the LPG extractedrom Synechocystis PCC 6803, the intensity ratio of thewo product ions was reversed (see Fig. 7a). Hence, thePG species isolated from a fresh extract of Synecho-ystis PCC 6803 should contain palmitic acid at then-2 position. It can be deduced from this result thathe palmitoyl groups of other lysoglycerolipids are alsoocalized at the sn-2 position.

In conclusion, the usefulness of the technique pre-ented here should for the first time allow direct andapid structural identification of diverse glycerolipidsxtracted from cyanobacteria. It should also be possibleo perform total lipid analysis at the molecular speciesevel.

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