Ami. Chem. 1882, 64, 2965-2971
2865
Fast Atom Bombardment Tandem Mass Spectrometric Identification of Diacyl, AIkylacyl, and Alk- 1-enylacyl Molecular Species of Glycerophosphoethanolamine in Human Polymorphonuclear Leukocytest Kathleen A. Kayganich and Robert C. Murphy' National Jewish Center for Immunology and Respiratory Medicine, 1400 Jackson Street, Denver, Colorado 80206
Fast atom bombardment lonlzation wlth tandem mass spectrometryof both porltlveand negatlveIonsk a usefultednlque for the ldentifkatkn of Intact glycerophosphoethandamlne (OPE), -p provkllng lnformatlon as to polar head group and fatty acyl substituents. I n the ldentlflcatlonof GPE molecular specks, pwttlve k n neutral k#, scannlng for 141 units was attempted to d r m the presence d the phoc phoethanolamlnepolar head group. Thk scan was found to dlscrhrlnate against the abundant subclass of phorphollplds h a m an l-aaIk-l'-enyl Ilnkage, termed plasmabgen8, as well as l-@alkyl ether specles. The neutral loss process k sugge&ed to Involve attack of a carbonyl oxygen from elther sn-1 or sn-2 on the sub3 methylenecarbon wlth lo+r of neutral phorphoethandamlne. Udng FAB/MS/MS alone, It Is nol pogdbleto dlfferentbte between plasmalogen8and other 1 4 alkyi ether molecular specles havlng the same molecular welght. The comblnatlon of mlld add hydrolyrk, whkh selecllvely hydrolyzes the laMk l-@alk-l'-enyl bond, wlth suboequentFAB/MS/MSdhSingukhedrpedecloftWdbtM subclasses. Udng these technlqws and precursor ion scans for the arachkknoyl carboxylate ankn, m/r 303, the arachldonlc add contalnlngglyceropM@oethanolamine molecular specks were Mentifled and the relathe abundance of arachldonoyl plasmalogen, alkylacyl, and 1,2dlacyl OPE molecular specb In the hman polymorphonuclearleukocyte (neutrophl) was detennkred to be 75.4 % ,12.1% ,and 12.5 %, respectively. These values were not rlgnHlcantty dlfferent from that reported In the literature urlng conventlonal methodology.
INTRODUCTION Glycerophospholipids isolated from a biological sample occur as a complex mixture of species, differing not only in the polar head group and fatty acid composition but also in the type of linkage found at the sn-1 position of the glycerol backbone (Chart I). Isolation of singlemolecular speciesfrom biological sources is quite difficult due to the complexity of these mixtures as well as to the similarities in chemical structures. Mixtures of molecular species persist even after extraction and sequential normal-phase and reversed-phase + Abbreviations: FAB,fast atom bombardment;MS/MS, tandemmaw Spectrometry;GPE, glycerophosphoethanolamine;CID, collision-induced dissociation; en-1 and sn-2, stereospecificallynumbered poeitiona 1 and 2 on the sn-GPEbackbone. (Also,the identity of the radyl groups in the en-1 and en-2 poeitions are indicated by the numerical order in which they are written, for example, 16Oe/204 GPE, where 160 represents a 16-carbon radyl group with no double bonds and 204 represents a 20carbonfatty acid with four double bonds. The lower case letter following the sn-1 radyl group indicatee the type of linkage at sn-1, either a, e, or p representing acyl, ether, or vinyl ether, which is commonly referred to 88 plasmalogen). 0003-2700/92/0364-2965$03.00/0
HPLC. Tandem mass spectrometry is particularly useful for complex mixture analysis because it adds a mass spectrometric separation step prior to fiial analysis. Several authors have demonstrated the usefulness of tandem mass spectrometry and fast atom bombardment ionization for the analysis of molecular species of phospholipide.1-3 Recently, we used negative ionization FABIMSIMS to identify the composition of arachidonate-containingmolecular species of glycerophosphocholine in human polymorphonuclear leukocytes (also referred to as the human neutrophil) in order to investigatethe source of arachidonicacid used for production of leukotrienes upon cell activation.* Neutrophil glycerophosphoethanolamine (GPE) also contains a significant reservoir of arachidonic acid, and we have found FAB/MS/ MS to be useful for the analysis of arachidonate-containii GPE molecular species. Despite the specificity of tandem mass spectrometry for identification of molecular species, a difficulty remains in differentiation of isobaric uneaturated 1 - 0 - ~ 1 - 2 - 0 - a c y l ( l - O - ~ l aand ~ l ) l-O-alk-l'-enyl-2-0acyl (plasmalogens or alk-1-enylacyl GPE) species because low-energy collision-induceddissociationof these ether-linked species does not yield product ions characteristic of the substituent at sn-1. These molecular speciesdiffer by location of a single double bond in the sn-1 radyl substituent. Other methods for identifying plasmalogens involve degradation followed by derivatization and separation of the resulting produ~ta.~ The facile acid-catalyzed hydrolysia of the sn-1 position has been the basis for the quantitative determination of plasmalogem through analysis of resultant long-chain fatty aldehydes! We report here the use of tandem mass spectrometry with mild acid hydrolysis, to differentiate between three glycerophospholipid subclasses and to identify the arachidonate-containingmolecularspecies of GPE in human neutrophils. This method of analysis of glycerophosphoethanolamine is rapid and circumvents the need for derivatization and additional chromatography.
METHOD AND EXPERIMENTS Materials. Diethanolamine (J. T. Baker) was used as the FAB liquid matrix. HPLC grade solvents (Fisher)were used for extraction, HPLC, and hydrolysis reactions. Methanolic HCl(2 N)was produced by dissolving a calculatedamount of redistilled acetyl chloride (Aldrich) in methanol. Reversed-phase HPLCpurified 16Op/204GPE and l80a/204GPE isolated from HL60 cells were a kind gift from Dr. C. Leslie (National Jewish Center for Immunologyand RespiratoryMedicine, Denver, CO). (1) Jensen, N. J.; Tomer, K. B.; Grose',M. L. Lipide 1986,21,58CH88. (2) Jensen, N. J.; Tomer, K. B.;Gross, M. L. Lipide1987,22,4@0-489. (3) M k t e r , H.; Budzikiewiu,H. M. Biol. Chem. Hoppe-Seyler 1988, 369,303-308. (4) Kayganich, K.; Murphy, R. C. J. Am. SOC.Moss Spectrom. 1991, 2,45-54.
(5) Blank,M.L.;Robinson,M.;Fitzgerald,V.;Snyder,F.J.Chromotogr. 1984,298,473-483. (6) Rapport, M. M.; Alonzo, N. J. Biol. Chem. 1966,217,19B-205. Q 1992 Amcnlcan chemical Society
2960 ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
Chart I 0 SII-1
II
1,t-diacy lglycerophosphoethanolamine
1-0-alkyl-2-acylglyceropliosphoetlianolamine
Mass Spectrometry. Mass spectra were obtained with a Finnigan TSQPOBtriple-quadrupolemass spectrometer. Xenon gas was used as the particle source for FAB. The FAB gun (Iontech,Ltd.) was operated at 1mA with an acceleration of 5-6 kV. Argon at a pressure of 0.5 mTorr was used as the target gas in the second rf-only quadrupole region. The collision energy offset,&,, was 30eV. Thew conditionsensure multiple collisions between the target gas and the ion exiting the fiit quadrupole mass spectrometer. Approximately 1-2 pL of diethanolamine was used as the matrix for negative-ion FAB. Glycerol was used as the matrix for positive-ion FAB. The phospholipid sample for FAB analysiswas diasolved in CHCbat a typical concentration of 1-2 mM. A 1-pLaliquotwas addedto the probe tip and allowed to evaporate for 1min at room temperature before analysis. Phospholipid Isolation. Human polymorphonuclear leukocytes (neutrophils) were isolated from the blood of normal healthy volunteers as previously described.? Lipids were extraded from 1 X 108 neutrophils using 1:l chloroform:cold methanol according to the method of Bligh and Dyer! Lipid classes were separated using normal-phase high-performance liquid chromatography(25cm X 4.6 mm, 5-pm silica,Phenomenex column) with a solvent system consisting of 53% A (hexane/2propanol; 3/4, v/v) for 6 min followed by a gradient to 100% B (hexane/2-propanol/H& 3/4/0.7, v/v/v) over 20 min. The flow rate was 1mL/min. The elution of the phospholipid classes was monitored by UV absorption at 206 nm as previouslydescribed.' Acid Hydrolysis. Acid-labile GPE molecular species (plasmalogens) were hydrolyzed by addition of 200 pL of 2 N methanolic HC1 to an aliquot of neutrophil GPE purified by normal-phase HPLC after removal of solvent under Na. The mixture was allowed to stand at room temperature for 30 min, and then the solvents were evaporated under nitrogen. The residue was reconstituted in 100pL of CHCb, and 1pL was used for FAB mass spectrometric analysis.
1-0-alk-1 '-enyl-d-acylg~cerophos~hoethanolanli~le (Plasmalogen) t
lW,A
728, 7f7
a7 0
726
1'
C
7f6
1
+ Ve neutralloss 141 u 744
I
II
@ESULTS The large number of different molecular species present in the polymorphonuclear leukocyte GPE fraction is evident from the positive FAB mass spectra shown in Figure 1A. Each of the prominent ions correspond to [M+ HI+derived from individual molecular species. The same molecular species are present by analysisof the negative ions, as shown in Figure 1B. Each of these prominent ions corresponds to [M - H1derived from the individual molecular species. It is our experience that negative ions are more abundantly produced from FAB of GPE than are positive ions, as is also suggested by comparison of the signal-to-background in Figure 1A,B. From either of these molecular ions, one can assign the total number of carbon atoms in the two radyl groups and the total number of double bonds, as shown in Table I. Identification of individual molecular species requires tandem mass spectrometry because isobaric ions are produced from several different molecular species having the same total number of (7)Haelett, C.; Guthrie, L. A.; Kopaniak, M.;Johnston, R. B., Jr.; Hensen, P.M.Am. J. Pathol. 1986,119, 101-110. (8)Bligh, E. G.;Dyer, W . J. J. Biochem. Physiol. 1959,37, 911-917.
40
20 0
m/z F l p o 1. FAB analysis of human ne@ophil glycarnolamine (WE). (A) Positive bn FAB mess s p e c " using glycerol as matrix. Abundant bns cOcreapOnd to [M H]+ from moh GPE mdeculer s p e c k . The abundant Ion at mlr 737 (*) repreeCNlt0 a matrix duster bn and is not derived tKHn WE. (B) Negative bn FAB mass spectnwn using dlethanolamine as meetrlx. Abundant h m w to [M HI- of varknm molecular species of WE. (C) FAB/MS/MS using neutral kss of 141 unb In the aneryds of human neutrophil WE. ~ h l sneubei kss scan maqxmda to the b~ of phoaphoethenolemlnefrom [M HI+ (eeetext).
+
-
+
carbon atoms and degrees of unsaturation but different distributionsat en-1or 811-2. In addition, different subclasses (diacyl, alkylacyl, or alk-1-enylacyl)can produce isobaric ions despite significant structural differences between these subclasses of phospholipids.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 2967
Table I. Positive [M + H]+and Negative [M- HI-Molecular Ions Corresponding to Selected 1,2-Diacyl,1-0-Alky1-2-acy1,and 1-0-Alk-lf-enyl-2-acyl(Plasmalogen) GlycerophosphoethanolamineMolecular Species. FAB [M+ HI+ FAB [M- HI- diacyl 1-0-alkylacyl plasmalogen FAB [M+ HI+ FAB [M- HI- diacyl 1-0-alkylacyl plaemalogen 676 678 690 692 698 700 702 704 706 714 716 718 720 724 726 728 730 732 734 738 740 742 744 746 748 750
674 676 688 690 696 698 700 702 704 712 714 716 718 722 724 726 728 730 732 736 738 740 742 744 746 748
320 320 321 320 343 34:2 3 41 340
343 34:2 34:l 34:O 343 342 341 340
364 363 3&2 361 360
364 363 362 361 360 365 36:4 36:3 362 361
360
386 385
38:6
752 754 756 758 760 762 764 766 768 770 772 774 776 778 780 782 784 786 788
750 752 754 756 758 760 762 764 766 768 770 772 774 776 778 780 782 784 786
790 792 794 796 798
788 790 792 794 796 898 800
800
802 804 806
386 384 383 382 381 380 386 385 384 383 382 381
38:O
#8
40:7 a 6 a 5 404 403 402 401 400
408 40.7 406 40:5 a 4 403 40.2 #1
384 383 382 381 380
#8 #7 #6 #5 #4 #3 #2
40.1
#O
a0
427 426
428 427 426 425
a The total number of carbon atom in both radyl groups (xz) and the totalnumber of carbon-carbondouble bonds 01)in both radyl group ia given according to the formula m:y. The vinyl ether degree of unsaturation in plasmalogene ie not added to the value of y.
Table 11. Percent Relative Abundance8 of Molecular Ions from a Mixture of 16fip/204 GPE and 18fia/204 OPE scan type
16Op/204
180a/204
362aa
-FAB +neutral loss 141 unite
53 55 3.5
100 100 100
9 8 8
+FAB
Minor contaminant from RP-HPLC purification of GPE species. Corresponds to 18:la/l8:1 and 18:0a/182 GPE.
Neutral Loss of 141 Units. T h e tandem maas spectrometric experiment of a constant neutral loss of 141 units from the (M H)+ ions has been suggested by Cole and Enkee as a more specific means for identification of the positive ions arising from GPE molecular species. This collision-induced decomposition reaction corresponds to loss of the phosphoethanolamine polar head group from the phospholipids. The constant neutral loss scan of 141 units from the neutrophil GPE extract is shown in Figure 1C. It is notable that the relative abundances of the corresponding ions in Figure 1A,B are approximatelythe same, whereas the relative abundances of the ions in the neutral loas scan (Figure 1C)are substantially different from both Figure 1A,B. The [M + HI+ ions which, from Table I, would correspond to ether-linked species appear less abundant in the constant neutral loss of 141 units. To further investigate this apparent discrimination of ether molecular species, an approximate 1:2 mixture of two purified GPE molecular species, 16Opl 20:4 GPE and 1 8 0 4 2 0 4 GPE, was analyzed by positive and negative ion FABIMS and positive ion MSIMS by neutral loss of 141 units. T h e relative abundances of the [M+ HI+ and [M - HI- ions found by each type of mass spectrometric experiment are tabulated in Table 11. The relative abundances of [M + H] and [M - HI- ions were similar for both species in this artificialmixture as well as the minor impurities
+
+
(9) Cole, M.J.; Enke, C. G. Anal. Chem. 1991,63,1032-1038.
(1814181 GPE and 1 8 0 4 1 8 2 GPE). However, a striking reduction in the abundance of the ion from 16:Op/204 GPE measured by neutral loss of 141 units was observed relative to the loss of 141 units from the diacyl GPE species. Radyl Species Identification. Identification of individual molecular species within the neutrophil GPE extract can be made on the basis of product ion spectra following collisionalactivation of [M - HI- ions. Example product ion mass spectra from collision-induced dissociation of mlz 766 and 750 in a tandem quadrupole instrument are shown in Figure 2A,B,respectively. Collision-induced dissociation of m/z 766 results in abundant carboxylate anions at mlz 283 and 303 from the acyl groups esterified at en-1 and 811-2, respectively, on the glycerol backbone. For these fatty acyl groups, the abundance of the 811-2carboxylate anion is 2-3 times the abundance of the carboxylate anion from the an-1 position, providing a meam for identifying the position of acyl substituent on the glycerol backb0ne.l' In the caw shown here, the collision-induceddissociation of [M - HI- provides information to identify the molecular Species shown in Figure 2A as 1 8 0 4 2 0 4 GPE. Other carboxylate anions present at mlz 279,281,306, and 307 are from decomposition of the [M - HI- at mlz 766 corresponding to isobaric molecular species such as 2 0 2 4 1 8 2 GPE and 2 0 3 4 1 8 1 GPE in this extract of the human polymorphonuclear leukocyte. The product ion mass spectrum resulting from collisioninduced dissociation of the ion at mlz 750, shown in Figure 2B, consists of abundant ions a t mlz 303,436, and 418. The anion at mlz 303 corresponds to the arachidonoyl carboxylate anion. The tons at mlz 436 and 418 correspond to the loes of arachidonic acid and the arachidonoylketene, as previously described for GPC molecular specie^.^*^ The less abundant ions appearing at m/z 279 and 331 are carboxylate anions arising from isobaric molecular species that contain linoleic acid (182, mlz 279) or docomtetraenoic acid (mlz 331,224). Given the mass of the [M - HI- ion (mlz 7601, the presence
2968
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
Table 111. Percent Relative Abundance of [M - HI- Ions from a Synthetic Mixture of 16*p/20:4 GPE and l&Oa/20:4 GPE before and after Hydrolysis with Methanolic HCl
766
m/z 722 160p1204 GPE
-FAJ3 before hydrolysis after hydrolysis a
98.3 0.75'
Signal at the noise level of the FAE! experiment. 750
A
I
Precursors of m/z 303 748 100
200
300
400 m/z
500
600
700
800
mlz 766 1801204 GPE 100 100
1
I
9)
a
752
722
*-u
s
Neutrophil GPE
/
B
776
750
800
700
900
m/Z 724
B
752
750 100
200
300
400
500
600
700
800
m/z F w e 2. Product kin scans for two maJorprecursor ions of mlz 303 found in the GPE fractionfrom human neutrophils. (A) Coilislonlnduced dissociation of mlz 766 corresponding to 18:0a1204 GPE revealing abundant carboxylate anions at mlz 303 and 283. Other carboxylate anions are also present, revealingaddMona1isobaric molecular spcies (carboxylate anion regkin expanded in inset). (B) Collision-induced dissociation of mlz 750 corresponding to 18:0p1204 W E . Other minor molecular species are present as indicated by the carboxylate anions at mlz 279 and 331 (see text).
of the arachidonoyl acyl substituent, the confirmation of phosphoethanolamine as the polar head group by NP-HPLC retention time, and fairly weak neutral loss of 141unita from the corresponding M H+(mlz 752 in Figure lC),the radyl group present at the other glycerol backbone position must either be an odd chain fatty acid containing one double bond (17:la) or an ether substituent (18le or 18Opat sn-1). Since no ion at mlz 267 for the 17:l carboxylate anion is observed, we conclude that the ion at mlz 750 correspondsto either the 181e/20:4 GPE ether-linked molecular species or the 18Opl 20:4 GPE plasmalogen species. From this tandem mass spectrometric experiment it is not possible to ascertain the position of the double bond in the 181chain at sn-1. Plasmalogen Identification. Since plasmalogen molecular species are known to be quite unstable to mild acidic conditions, a method to distinguish plasmalogen molecular species from 1-0-alkylacyl species based upon rapid and specific hydrolysis of plasmalogens was tested.' A mixture of two purified molecular species, 16Opl204 GPE and 180al 204 GPE was analyzed by negative ion FABIMS before and after mild acid hydrolysis. For this mixture, approximately equal abundances of the plasmalogen and acyl molecular species were present before hydrolysis; however, after 30 min the signal at mlz 722 (16:Op/204 GPE)W ~ Ereduced I to the
+
Precursors of m/z 303
766
After ..,ldrolysis
722
v
800
700
900
m/Z Flgum 9. Negative ion FABIMSIMS analysis of human neutrophil OPE. (A) Precursor ion scan for mlz 303 indicating arachidonic acid containing W E species in an aliquot of human neutrophil WE. (B) Precursor ion scan of mlz 303 following miid acid hydrolyslsof human neutrophil GPE (seeMethod and Experiments).
noise level of the FAB experiment (Table 111). Considering the extensive studies of acid hydrolysis of plasmalogens, the reaction conditions employed were sufficient to destroy plasmalogens without effecting 1,a-diacylspecies, as shown, or the even more stable 1-0-alkylacylGPE species (data not shown). Polymorphonuclear Leukocyte Arachidonate GPE. Identification of allarachidonat.8-containingmolecular species in the neutrophil GPE was carried out generating parent ion scansfor the arachidonoylcarboxylateanion (mlz 303) shown in Figure 3A as previously described for glycerophosphocholine analysis.* The ions observed at mlz 808,780, 766, 764, 752, 736, and 724 can be identified as 22:0e/20:4, 20:08/204,180a/204,181a/204,180e/204,161aI204, and 16:Oe/204 GPE,on the basis of the mass of their [M - HIions and the fact that they contain arachidonic acid and the phosphoethanolaminepolar head group. The abundant ions
ANALYTICAL CHEMISTRY, VOL. 64. NO. 23, DECEMBER 1, 1992
2909
Table IV. Arachidonate-ContainingMolecular Species of GPE Found in the Human Neutrophil As Measured by Parent Ion Abundance for m / z 303 before and after Acid Hydrolysis parents mlz 303
re1 ion abundance'
720 722 724 736 738 746 748 750 752 764 766 776 778 780 790 792 794 804 806 808 832 834
3.9 f 0.3 44.7 f 2.2 10.2 & 1.3 6.5 f 0.8 2.5 f 0.4 4.8 f 0.5 58.4 & 4.3 100 10.9 f 1.3 7.8 f 0.4 12.0 f 0.5 16.7 f 0.9 11.5 f 0.4 2.5 f 0.4 3.4 0.5 5.5 f 0.4 2.2 & 0.2 6.3 f 0.2 2.4 f 0.1 1.4 f 0.1 3.8 & 0.7 2.7 f 0.5
1,2-diacyl
molecular species (mol % ) 1-0-alkylb 162e (0.12 f 0.01) 161e (0.35 t 0.02) 160e (2.68 & 2.0)
plasmalogenb 161p (1.1t 0.09) 16Op (13.6 t 0.8) 16Op (0.5 t 0.4)c
161a (2.02 f 0.27) 160a (0.78 f 0.13) l 8 3 e (0.14 f 0.02) 182e (0.34 t 0.03) 1 8 l e (2.78 f 0.08) 18Oe (3.42 f 0.22)
182p (1.35 t 0.16) 1 8 l p (17.9 1.42) 18Op (28.4 0.78)
202e (0.21 f 0.01) 201e (0.53 f 0.03) 20Oe (0.69 f 0.04)
2 0 l p (5.00 f 0.31) 20Op (3.06 0.15) 20Op (0.19 t O.15)C
222e (0.11 f 0.01) 221e (0.31 t 0.02) 22:Oe (0.31 f 0.07) 242e (0.07 f 0.01) 241e (0.18 f 0.04)
2 2 l p (1.86 f 0.16) 22Op (0.45 t 0.04) 22Op (0.14 & 0.04)e 241p (1.12 t 0.22) 24Op (0.65 0.17)
*
1 8 l a (24.3 f 0.14) 180a (3.75 f 0.19)
*
202a (1.07 f 0.16) 201a (1.71 t 0.13) 200a (0.69 f 0.08)
*
a Values expressed as the average &E, n = 4. See text for description of calculation for 1-0-alkylacyl and plasmalogendistribution. Stable isotope containing plasmalogen, e.g. two 13C atoms at positions other than arachidonoyl substituent.
at mlz 722, 748, and 750, which arise from ether-linked molecular species (based on the mass of [M - HI- and the product ion spectra data, represented for mlz 750 in Figure 2B),have additionaldegreesof unsaturation in the alkylchains present in the radyl group at sn-1. Therefore, these species could be plasmalogenmolecular species, 1-0-alkylacylspecies, or a mixture of both. Mild acid hydrolysis was used to differentiate the plasmalogen arachidonate-containing molecular species from isobaric 1-0-alkylacyl species that contain double bonds within the alkyl chain. Following the hydrolysis reaction, FABIMSIMSwas repeated to determine the parents for mlz 303 (Figure3B). This spectrum correspondsto residualdiacyl and 1-0-alkylacyl arachidonate-containing GPE molecular specieswhich are not hydrolyzed by HC1. The plasmalogens were converted to 1-lyso species and fatty aldehydes which do not have [M - HI-ions within the mass range scanned. After the hydrolysis reaction, the largest peaks in the precursor ion scan for mlz 303 (Figure 3B) appear at mlz 724 and 752 correspondingto16Oe/204GPE and 180e/204 GPE, respectively. These were only minor molecular species in the precursor ion scan prior to hydrolysis(Figure3A). Calculation of the percentage of plasmalogen and 1-0-alkylacylfor each isobaric species was carried out first by normalizing the relative abundances of those ions appearing in Figure 3A to mlz 752 and those appearing in Figure 3B to mlz 752. Under the assumption that the relative amount of mlz 752 is the same in hydrolyzed and nonhydrolyzed GPE, the normalized abundances for each molecular species following hydrolysis were substracted from the relative abundance prior to hydrolysis. At each mass these differences were divided by the relative abundances prior to hydrolysis to obtain the percentage GPE hydrolyzed, which corresponds to the percentage of plasmalogen. The assumption is based on the fact that there is no unsaturation in the sn-1 radyl moiety and, therefore, the substituent producing the ion at mlz 752 cannot be a plasmalogen that would undergo hydrolysis. Identification and relative abundances of the major arachidonate-containing neutrophil GPE molecular species, includingthe plasmalogenand alkylether species,are tabulated in Table IV. These results represent the average of four
different neutrophil sample analyses by FABIMSIMS and acid hydrolysis. Table V summarizes these data as the percentageof arachidonate-containingGPE molecular species within the specific GPE subclasses in order to comparethese results to values obtained from the literature in which conventionaltechniques were used to determine the subclass distribution of arachidonate-containingGPE molecular s p a cies.1° The secondpart of Table V compares the mole percent of diacyl, alk-1-enylacyl and 1-0-alkylacyl for all GPE molecularspecies in the neutrophil GPE as found by negative ion FABIMS and in the 1iterature.l' The percentage of plasmalogen was calculated from the difference in the total ion current between FABJMSscans (normalized to mlt 744, [M - HI- ion corresponding to 18Oal181 GPE) before and after hydrolysis. By comparison of the means1*there was no statistically significant differences @ < 0.01) found between the two methods for mole percent GPE molecular species subclassesor arachidonate-containii GPE subclaseesin spite of expected biological variability between normal human subjects with differing diets of unsaturated fatty acids.
DISCUSSION The analysis of complex glycerophospholipids has been significantlyimproved by application of newer techniques in mass spectrometry, including fast atom bombardment ionization and tandem instruments. It is possible to obtain specificinformationconcerningnot onlythe polar head group but also the radyl substituents at en-1 and sn-2 based upon characteristic decompositions of ions corresponding to the intact molecule. For glycerophosphoethanolamine,fast atom bombardment ionization leads to the production of [M HI+ as well as [M - HI- ions representative of each species in the complex mixture from a biological extract. Unfortunately, there has been no exhaustive study comparing the FAB ionization efficienciesof different phospholipid molec-
+
(10) Mueller, H.W.; O'Flaherty, J. T.; Green, D. G.; Samuel, M. P.; Wykle, R. L. J. Lipid Res. 1984,26,383-389. (11)Tence, M.; Jouvin-Marche,E.; Beeaou, G.; Record,M.;Benvenista, J. Thromb. Res. 1985,38, 207-214. (12) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry; Halatad Press: New York, 1984, pp 53-66.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 23,DECEMBER 1, lQQ2
Table V. Subclass Distribution of Arachidonate-ContainingGPE Molecular Species and Total GPE Subclass Species in Human Neutrophils
2-arachidonoylspecies lit) all molecular speciesc 1it.d
% l,2-diacyla
% 1-0-alkyl
% plasmalogen
12.5 f 2.7 6.3 f 3.2 29.0 28.1 f 6
12.1 f 2.6 9.2 f 2.7 18.5 23.9 f 5.3
75.4 f 1.9 84.5 & 11.4 52.5 47.9 f 4
Scheme I
i a : w z o : 4 CPE mh 768
"Data expressed as the average from four separation human subjects, *SE. Reference 10. Average from two human subjects. Variation of 5 % or less observedfor s u b c h distribution. Reference 11.
ular speciesand such informationwould be necessary in order to accurately determine the relative concentrations of molecular species directly from the relative abundances of their [M-HI-or [M+ H]+ions byFAB1MS. Reasonsforthelack of such studies include the difficulty in obtaining completely purified molecular species from natural sources to serve as primary standards and the difficulty in determining the concentrationsof such isolated phospholipids. Perhaps more importantly, naturally occurring phospholipids exist as mixtures of hundreds of individual molecular species, each of which would need to be synthesized, quantitated, and analyzed to test FAB ionization efficiencies and surface activities. However, Chilton and Murphy13 observed that the relative concentrations of glycerophosphocholine molecular speciescould be estimated from the relative abundances of their [M + HI+ions as long as the contributions of isobaric molecular species and background noise were taken into account. Precursor ion scanning (mlz 303)was used to directly estimate the relative concentrations of arachidonic acid containingGPE molecular speciesin human neutrophils.The accuracy of this estimation was assessed by comparison with quantitative results in the literature (Table V). Collision-induceddissociation tandem maas spectrometry was used to c o n f i i identification of the arachidonatecontaining GPE molecular species found in human neutrophils. Decomposition of [M + HI+and scanning at a constant neutral loss of 141units was attempted to identify those ions in the FAB mass spectrum arising from glycerophosphoethan~lamine.~ However, as noted above, comparison of relative abundances in Figure 1A to those in 1C indicates that the neutral loss scan does not accurately reflect the relative concentrationof all GPE molecular species. The data in Table I1 reveal that plasmalogen species do not undergo neutral loss of phosphoethanolamine(141 units) to the aame extent as diacyl molecular species. The reaction for the loss of phosphoethanolamine likely proceeds by one or both of the mechanisms shown in Scheme I. In either a five- for six-membered transition state, the nonbonded electrons on an ester carbonyl oxygen atom attack at the sn-3 methylene carbon atom with loss of the electronically neutral, but zwitterionic, phosphoethanolamine moiety. These mechanisms would predict an unequal abundance of product ions for diacyl and ether-linked species due to the presence of only one ester carbonyl in the ether-linked species but two such moieties in diacyl species. For diacyl species, both mechanisms could operate whereas only mechanism B can operate in fragmentation of sn-1 ether-linked species. The overallreaction is much more abundant for the diacyl species, suggestingthat mechanism A, in which a six-membered ring product is formed, may be more facile than mechanism B. In light of these results, a constant neutral loss of 141 units cannot be used to defiie the complete profile of GPE (13) Chilton, F. H.; Murphy, R. C. Biomed. Mass Spectrom. 1986,13, 71-76.
mh 724
molecular species in all biological extracta. If GPE etherlinked speciesexist, their quantitative contributionwould be misrepresented by the constant neutral loss scanning for 141 units. Unfortunately, the separation of ether-linked and diacylGPE subclasseshasnever been demonstratedby HPLC or TLC without prior phospholipase C treatment to remove the polar head group. Collision-induceddissociation of [M - HI- ions resulta in the formation of abundant carboxylate anions from those radyl groups which are esterified to the glycerophosphoethanolamine backbone, allowing identification of isobaric molecular species. The ratio of the carboxylate anions can provide information as to the site of esterification,either at an-1 or 811-2, for the most abundant fatty acyl groups that are found in a eukaryotic cell, such as the neutrophil.'" The sn-1 and 811-2 substitutions of stearic and arachidonic acid for the molecular species shown in Figure 2A were assigned by applying this generalization to the product ion spectrum. The relationship is not valid for phospholipids containing either very short-chain or long-chainhighly unsaturated fatty acids, such as docosahexanoic acid,14 or oxygenated fatty acids.16 When these types of phospholipids have been analyzed, it has been suggested that assignment of the sn-1 and sn-2 positions can be obtained through collision-induced dissociation of the phosphatidic acid anion produced during FAB of the phospholipid (for GPE this ion corresponds to [M - 441-1. The relative abundances of the ions produced by neutral low of the sn-1 and sn-2 fatty acids reveal the acyl group position on the glycerol backbone.14 Preliminary identificationof the arachidonate-containing GPE specieswas made on the basis of the precursor ion scan for mlz 303 (the arachidonate carboxylate anion). Identifications of the sn-1 substituents as ether-linked rather than acyl-linkedodd chain fatty acids were confirmed by product ion scans such as those shown in Figure 2. However, differentiation of 1-0-alkyland 1-0-alk-1'-enyl substituenta in glycerophospholipidsis not poeaible by direct FAB/MS/ MS techniques. Major chemicaland biochemicaldifferences exist between isobaric compounds having a degree of unaab uration at the vinyl position (plasmalogen)comparedto those containing one degree of unsaturation at any other position in the alkyl chain of the ether substituent. The lability of the alk-1-enyl ether group to mineral acids has been known for sometime.16 Acid-catalyzed hydrolysis of plasmalogens is known to be a rapid reaction initiated by protonation of the vinyl ether double bond with formation of a carbonium ion. The carboniumionthen rapidly reacts with water or methanol (14) Huang, Z.-H.; Gage, D. A.; Sweeley, C. C. J. Am. Soc. Mass Spectrom. 1991,.9,71-78. (15)Bernatzom, K.; Kayganich, K.; Murphy, R. C. AM^. Biochem. 1991,198,203-211. (16)Siggia, S. Quantitative Orgunic A ~ l y ~via i sFunctional Groupe; Wdey New York, 1949.
ANALYTICAL CHEMISTRY,VOL. 64,NO. 23, DECEMBER 1, 1002 2971
to yield an aldehyde or acetal, re~pectively.~~ This reaction is the basis for several quantitative assays for plasmalogena in complex phospholipid mixtures.'* The rapidity of this reaction makes it quite suitable for selective decomposition of plasmalogensprior to the FABIMSIMSexperiment. Since the change in molecular weight following hydrolysis by elimination of the sn-1 radyl group is quite substantial, the remaining [M - HI- ions correspond only to the acid-stable 1-0-alkylacyl GPE or 1,a-diacylGPE molecular species. The relative abundances of percursors for mlz 303 before and after hydrolysis were wed to calculate the percentage of arachidonate-containing plasmalogen and alkylacyl GPE molecular species. The calculation assumes that the surface activities of each arachidonate-containing GPE molecular species are not significantly different and that all species (17) Frosolono, M. F.; Rapport, M. M. J. Lipid Res. 1969, 10, 504. (18) Snyder,F.EtherLipids: ChemistryandBiology;Academic Preas, New York, 1972; pp 44-48. (19) Zirrolli, J. A.;Clay,K. L.; Murphy, R. C. Lipid 1991,26, 11121116.
have similar CID behavior. Minor differences in surface activity would have to result from the different sn-1 substituents as the remainder of the moleculesare identical. The CID efficiencies for the production of mlz 303 is not likely to differ greatly from species to species,as the mechanism for the decomposition does not involve the different sn-1 substituents.l@In spite of these assumptions,the resulta obtained by FABIMSIMS were not statistically different from the published results obtained using more conventionalmethods to measure subclasses distributions. There have been no reports published to date identifying in detail the molecular species of glycerophosphoethanolamine in the human polymorphonuclear leukocyte.
ACKNOWLEDGMENT
This work was supported, in part, by a grant from the National Institutes of Health (HL34303).
RECEIVED for review May 14, 1992.
20, 1992. Accepted September
