Coenzyme A (CoA) thioesters of long-chain fatty

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1 Analysis f Lng-Chain Fatty Acyl Cenzyme A Thiesters by Negative In Fast-Atm Bmbardment Mass Spectrmetry and Tandem Mass Spectrmetry Jseph A. Zirrlli, Pat Wheelan, and Rbert C. Murphy Natinal Jewish Center fr Immunlgy and Respiratry Medicine, Denver, Clrad, USA Lng-chain acyl Cenzyme A (CA) is essentially cmpsed f three majr chemical grups, fatty acyl-, phsphpantethein-, and 3',5',-adensine diphsph-mieties. The negative in fast-atm bmbardment mass spectrmetry spectra f lng-chain acyl CA thiesters were characterized by the frmatin f abundant [M - H]- and tw distinct classes f fragment ins, ne class which retained the acyl grup and anther class which is related t CA that cntains the phsphpantethene and adenine. The ins which retained the acyl grup in the spectrum f palmityl C.A appeared at m r z 675, 657, 595, and 577 and were fund t decmpse by lss f alkylketene bserved at mrz 357 and 339. Thse ins which retained the adenine grup were bserved at m/z 426 and 408. In cntrast t these ins bserved fllwing fast-atm bmbardment inizatin, tandem mass spectrmetry f the [M - H] -, frm palmityl CA (m/z 4), yielded the adenine-cntaining ins as majr prducts and the acyl-cntaining ins were f lw abundance r nt detected. These results suggested that the frmatin f many characteristic ins bserved in direct FAB analysis ccurred during the desrptin prcess. The unique relatinship between ins which invlved the transitin frm acyl-cntaining ins t nly CA-cntaining ins by the lss f alkylketene allwed the develpment f tandem mass spectrmetry prtcls fr the analysis f acyl CA mixtures. Precursr scans f either m r z 357 r 339 yielded the identificatin f each species in a cmplex mixture. Identificatin f specific species was btained with a neutral lss scan f the mass fr a specific alkylketene. (J Am Sc Mass Spectrm 1994, 5, ) Cenzyme A (CA) thiesters f lng-chain fatty acids (acyl CA) are essential intermediates in all aspects f fatty acid synthesis [1], degradatin [2], and phsphlipid bisynthesis [3]. Acyl CA thiesters are thught t play imprtant rles in many cellular regulatry prcesses including bisynthesis f the lipid mediatr platelet activating factr [4], and intermediates f leuktriene [5, 6] and prstaglandin metablism [7]. In additin, acyl CA thiesters are als frmed with xenbitics in the pathway f cnjugatin t endgenus amin acids [8]. Because f the atmic size f the sulfur atm in the thiester cmpared t the xygen atm in esters, there is less 'IT-electrn verlap and a destabilizatin f the thiester bnd relative t that f an ester. This makes the thilate anin a better leaving grup in nuclephilic displacement reactins that are catalyzed by varius enzymes that use the acyl CA derivative. Address reprint requests t Dr. Rbert C. Murphy, Department f Pediatrics, Natinal Jewish Center, 1400 Jacksn Street, Denver, CO American Sciety fr Mass Spectrmetry /94/$7.00 Lng-chain acyl CA is essentially cmpsed f three majr chemical grups: fatty acyl-, phsphpantethein-, and 3',S'-adensine diphsph-mietics (ADP). Several mass spectrmetric strategies have been used fr the analysis f acyl CAs. Lng-chain fatty alchls frmed frm the reductin f acyl CA esters have been analyzed by electrn capture negative in gas chrmatgraphy mass spectrmetry as the pentaflurbenzyl esters [9]. While this methd is reprted t be quite sensitive, it has the disadvantages inherent with chemical wrkup and derivatizatin. Analysis f intact acyl CAs has invlved direct and cntinuus-flw fast-atm bmbardment mass spectrmetry (FAB/MS) [10-12]. These fast-atm bmbardment (FAB) studies were based upn psitive inizatin mdes. Bth [M + H] + and fragment ins indicative f the CA and acyl grup were bserved, and the base peak f the spectrum was prtnated adenine, m r z 136. A recent reprt using tandem mass spectrmetry f psitive ins frm 3-ket-2-pr pylpentanyl-ca indicated the utility f this apprach t prbe detailed structural features in this Received September 29, 1993 Revised December 20, 1993 Accepted December 21,1993

2 J Am Sc Mass Spectrm 1994, 5, NEGATIVE ION MASS SPECIROMEffiY OF FAlTY ACYL CA nnoesters 417 unique CA ester [13]. Little wrk has been perfrmed t evaluate the efficacy f negative inizatin f intact acyl CAs. One previus study did reprt that abundant [M - H]- ins were prduced frm LTB 4-CA [6]; hwever, this study did nt describe any ther in frmatin. Fr these reasns we have investigated the negative in FAB/MS and fast-atm bmbardment tandem mass spectrmetry (FAB/MS/MS) behavir f lng-chain acyl CAs. Methds Standard lng-chain acyl CA species, in their free acid frm, were btained frm Sigma Chemical Cmpany (St. Luis, MO). Individual and mixture f standards were disslved in methylene chlride/methanl (2/1) t give a final cncentratin f 1 ug/ul per each cmpund. Aliquts (1 ul) were applied t the FAB prbe using a variety f matrices (reagent grade) as described in the Results. F H]IO-(N,O)-Palmityl-CA was prepared by disslving 10 ug f palmityl-ca in ul f ethanl-od (99.5% D, Aldrich, Milwaukee, WI), evapratin f the slvent in vacu, and repeating the prcedure three times. [2H]1O-(N,O)-Palmityl-CA was analyzed using [2Hh-glycerl (98% D, Aldrich, Milwaukee, WI) as the FAB matrix. The mst abundant istpic species bserved was FH]10-(N,O) palmityl-ca which was selected fr cllisin-induced dissciatin (CID) and analysis by tandem mass spectrmetry. Other species were bserved at lwer abundances cntaining eight and nine deuterium atms indicating incmplete deuterium exchange, but these species did nt interfere with the CID experiments. Analyses were perfrmed n a Finnigan TSQ 70 mass spectrmeter (Finnigan Crpratin, San Jse, CA) equipped with a FAB gun frm In Tech (Teddingtn, England). Scans were btained ver the range m r» 200 t 1200 u at the rate f 2 s/scan. The FAB gun was perated with xenn as the particle surce at 6 key. Tandem mass spectrmetry emplyed a cllisin energy f 30 ev (labratry frame f reference) with argn as the cllisin gas at a pressure f 0.5 m'trr, Results FAB I MS (Surce-frmed ins) With negative in FAB/MS desrptin and inizatin each grup, fatty acyl-, phsphpantethein-, and 3',5' -adensine diphsph-mieties (Scheme I) exerted an influence t yield fragment ins characteristic f that grup. Indeed, lng-chain acyl CA thiesters prduced abundant [M - H] - ins and tw distinct classes f fragment ins, ne class retaining the acyl grup (designated [-V) and the ther related t cenzyme A and/r adensine diphsphate (designated A-E). The class f ins (A-E) related t the CA r ADP grups are cmmn t all acyl CAs while acylcntaining ins ct-v) shift in mass accrding t the mass f the acyl grup. This is illustrated in Figure 1 fatty acyl Palmityl - CA phsphpantetheine Schemel a Pslmltyl- CA mw 5 I Cenzyme A 3'.5'-diphsphadensine with the negative in spectra f palmityl- and arachidnyl-ca which displayed [M - H]- at mrz 4 and 1052, respectively. (Even thugh the number f hydrgen atms in acyl CA species is relatively high (66-74 prtns), the exact masses bserved fr mlecular in species is nly abve the nminal mass because f the number f mass deficient heteratms als present. The exact mlecular masses fr palmityl and arachidnyl CA are 5.3 and , respectively. In this publicatin we will reprt the nminal masses fr bserved ins rather than the exact masses.) The majr fragment ins f each class are described belw. The assignment f in structures is supprted by apprpriate mass shifts between the spectra f palmityl- and [2H]1O-(N,O)-palmityl-CA as depicted belw in specific in structures. a e V 271 m III IV 595 t. 577 J s 575.Q 0 "C 357 m :;:: E(..,;.l!! 339 C B II ]srl JI 6&7,I.1 I b Arschldnyl- CA mw 1053 m E V '{ D 357 m IUJS C IV 5 52 A 766.I '24 fm-h]" 4 MI 0 1UJ0 600 BOO 0 [M-H]" 1052 miz Figure 1. Negative in FABjMS f (a) palmityl CA and (b) arachidnyl CA btained in glycerl matrix; letter m designates matrix ins.

3 r 357 rz ZIRROLLI ET AL. J Am Sc Mass Spectrm 1994, 5, Frmatin f mst negative ins can be ratinalized by an initial hydrgen abstractin frm and charge lcalizatin n ne f the phsphate hydrxyl grups. Fragmentatin f the 5'-phsphate ester (adensine 3' -mnphsphate(amp)) bnd f palmityl-ca with charge retentin n the acyl diphsphpantetheine grup yields the in bserved at mjz 675 (1), while m r z 657 (II) nminally represents an additinal lss f H 2 0. In the spectrum f [ZHllO-(N,O)-palmityl-CA these ins were shifted t m/z 680 and 660, respectively, crrespnding t a lss f DzO. This lss f water invlving exclusively exchangeable prtns is cnsistent with the frmatin f a cyclic phsphate ester bnd. (0) (D) (0) If If n f! f?,<"""'n)('-ny'x-w : f.! en, 0 0 (D70(D10 In I miz 675 (680) In II m/z 657 (660) The ins bserved at m r z 595 and 426 (III and B, respectively) may be frmed by fragmentatin n either side f the central xygen in the diphsph ester bridge between AMP and acyl pantetheine. Thus III, an acyl diagnstic in, may be frmed with charge retentin n the acylphsphpantetheineand B, a CA diagnstic in, may be frmed with charge retentin n the 5'-phsph-AMP. In III miz 595 (599) In IV miz 577 (579) El,,-:'0""6' (;N-r I, NllNJ N (V)HO 0 OH(D) (D) HO' 'OH(D) In B mlz 426 (432) NH,(D,) Ins C (mjz 408) and IV (mjz 577) are mst likely dehydrated analgs f B and III, respectively. In the spectrum f [ZH]lO-(N,O)-palmityl-CA these ins were shifted t mr : 599 (III), 579 (IV), 432 (B), and 412 (C), again cnsistent with the frmatin f cyclic phsphate esters in IV and C. z tn-tn NU, (D,) N N J 0,; f ---() (D) YO 0 0 'p' (f" 'H(D) tn t: miz 408 (412) The ins bserved at m (D) and m (E) in the mass spectrum f palmityl-ca were als bserved in the mass spectra f all ther acyl CAs studied (Figure 1 and Table 1). One reasnable pathway fr the frmatin f these ins (D) and (E) is by lss f alkylketene frm the acyl phsphpantetheine ins, III and IV. The significance f this transitin is Table 1. Abundance f surce-frmed fragment ins frm fatty acyl C.As Abundance f CA Diagnstic Fragment Ins frm Acyl CA" Fragment In Fatty Acyl Grup'' A 8 C D E m Iz 766 m Iz 426 m Iz408 m /z 357 m/z : : : : : : : Abundance f Fatty Acyl Diagnstic Fragment Ins frm Acyl CA" Fragment In" [M- H]- II III IV V 16:0 4 (42' 675 (33) 657 (12) 595(116) 577 (106) 271 () 17: (23' 689 (32) 671 (12) 609 (68) 591 (79) 285 () 18:1 1030(40' 701 (28) 683 (16) 621 (48) 603 (149) 297 () 18: (40' 703 (38) 685 (14) 623 (52) 605 (90) 299 () 19: (28) 717(25) 699 (10) 637 (58) 619 (47) 313() 20: (54) 723 (46) 705 (24) 643 (95) 625 (73) 319 () 20: (20) 731 (31) 713 (14) 651 ( (55) 327 () "Spectra were btained in glycerl and abundances listed are nrmalized t in V (). ba Cy l nmenclature cc:b, where cc is the carbn chain length and b is the 101al number f duble bnds. cdata are listed as me se-t-charqe rati values with relative abundances in parentheses.

4 J Am Sc Mass Spectrm 1994, 5, NEGATIVE ION MASS SPECTROMETRY OF FATIY ACYL CA THIOESTERS 419 imprtant because the fatty acyl cntaining ins (III and IV) decmpse t CA diagnstic ins (D and E, respectively) which lack the fatty acyl grup. ('i/ (If! ) e HS"-NY'-N-.!x:O-':-,fi) 0 (ljo In D miz 357 (361) The ther tw ins, A and V, bserved at m/z 766 and 271 in the spectrum f palmityl-ca (Figure 1) crrespnd t fragmentatin n either side f the thiester bnd. Lss f alkylketene frm [M - H]- in yields A, m/z 766, an in analgus t the [M - H] frm free CA. Interestingly, this in shifted by 9 u t mrz 775 in the spectrum f the deuterated analg indicating the lss f ne deutern. Scissin f the sulfur bnd between thiester and pantetheine yields anther imprtant acyl diagnstic in V, the acyl thilate anin. sej CH, (n V miz 271 (271) (D) (0) \ 0(3 '! 0' () HS"-N..,;--N-r;'>( (n A miz 766 (775) In E miz 339 (341) A series f seven saturated and unsaturated lng chain-acyl CAs 06:0, 17:0, 18:0, 18:1, 19:0, 20:0, and 20:4) were studied under negative in FAB/MS using a variety f matrices. The negative in FAB mass spectra f this series f lng-chain acyl CAs are summarized in Table 1 (glycerl matrix). The mst abundant fragment ins are the acyl-specific ins III, IV, and V, and the CA diagnstic ins D and E. The least abundant fragment ins frmed were the CA related A, B, and C ins. The matrices emplyed included thiglycerl, glycerl, diethanlamine, and triethanlamine. In general, spectra cntaining abundant [M - H]- and fragment ins, in particular abundant type V ins, were btained with the mre basic matrices, diethanlamine and triethanlamine. Spectra generated in the presence f the relatively mre acidic matrix, thiglycerl, als yielded abundant [M - H]- ins and mre abundant acyl-cntaining ins III and IV, but less abundant thilate type anins V (Table 2). Frm the perspective f identifying specific acylcntaining CA thiesters, negative in FAB/MS demnstrated gd perfrmance and prmpted further study f their CID behavir t evaluate specific decmpsitins fr the analysis f cmplex mixtures. FAB/MS/MS: Tandem mass spectrmetry included prduct in, precursr in, and neutral lss scans fllwing CID studies f the lng-chain acyl CAs t investigate the frmatin f the fragment ins bserved in FAB/MS spectra. Prduct ins frmed frm CID f the majr surce-frmed ins frm palmityl CA are summarized in Table 3 and listed in cmparisn with the FAB/MS spectrum. Prduct in Spectra frm the ther acyl CA species were qualitatively similar. [M - ut: (m/z 4). As shwn in Figure 2, the mst abundant prduct ins frmed frm CID f the [M - H]- f palmityl-ca are the CA class ins, B and C (m/z 426 and 408, respectively). The acyl cntaining ins were either f lw abundance, I (m/z 675), II (m/z 657), and IV (m/z 577) r cmpletely absent, III (m/z 595) and V (m/z 271). Als absent were the CA ins D and E (m/z 357 and 339, respectively), which are derived frm acyl-cntaining ins by the lss f alkylketene. The prduct in spectrum is in cntrast t the FAB/MS analysis in which the mst abundant ins bserved were acyl-cntaining fragments and the least abundant ins were CA related (Figure Ia). Other minr prduct ins were bserved at m rz 924, lss f 80 u (HP0 3)frm [M - HI-, and tw fragments nt seen in the FAB/MS spectrum, mrz 328 and 273. These tw ins, m/z 328 and 273, mst likely derive frm ins B (m/z 426) and C (m/z 408) by lsses f 98 u (H 2P04 ) and 135 u (adenine), respectively. In I (m/z 675). The prduct in spectrum f I frm palmityl CA is shwn in Figure 3. Tw abundant ins m rz 577 and 339 rrv and E, respectively) are frmed by an initial lss f H 3P04 (98 u) and the lss f H 3P04 fllwed by the lss f hexadecylketene (238 u), respectively. These lsses are supprted by the CID f this in (m/z 680) frm [2H]l--(N,O).-palmityl-CA in which bth prduct ins lst three deuterium atms (as 2H 3 P0 4 ) and the bserved prduct ins, retaining Table 2- Matrix effects n bserved abundances f surce-frmed fragment ins frm pahnityl CA a Matrix [M - HI A 8 C D E I 11 (If IV V m/z TEA DEA 26 7 B Glycerl Thiglycerl aln abundances are frm an averaged spectrum f five scans, nrmalized t the mst abundant i...

5 420 ZIRROLLI ET AL. J Am ScMass Spectrm 1994,5, C 41t II 657 IV ,J l.l Palmityl-CA Prduct ins CID mjz 4 Cllisin Energy 30 ev I 675 I l,j, [M-H], 4 [A-80)" 924 Mit t1t BOlt 0 m/z Figure 2. Prduct ins btained fllwing cllisin induced dissciatin (30 ev cllisin energy, 0.5 mtrr argn) f [M H]- ins f palmityl C A. tw deuterium atms, were shifted by 2 u t m r z 579 and 341, respectively (Table 3). Als the frmatin f in E by lss f alkylketene was supprted by the bservatin f this in in the FAB/MS spectra f all ther acyl CAs studied (Table 1) as well as in their CID spectra f the respective I type ins (data nt shwn). Other prducts ins, II, III, D, and V (m/z 657,595,357, and 271, respectively) were als bserved in the FAB/MS spectrum. Of these ins, 1II and D were minr ins in cntrast t the FAB/MS analysis in which they were very abundant. Prduct ins nt bserved in the FAB/MS spectrum included m/z 305, IItIt Palmityl-CA Prduct Ins CID mlz 675 Cllisin Energy 30 ev B IV t5 /I V III I,J E 339 D I.1 I t 501t 600 mjz Figure 3. Prduct ins btained fllwing cllisin induced dissciatin (30 ev cllisin energy, 0.5 mtrr argn) f rnjz 675, Type I ins frm palmityl ea. 208, and 159. These prduct ins were unchanged in bserved mass in the CID f I frm the ther lngchain acyl CAs studied and therefre did nt retain the acyl grup. The in at mr z 305 was als bserved in the cm spectra f II (m/z 657) and IV (m/z 577) as listed in Table 3, and the frmatin f this prduct in mst likely invlves lss f the thiacid (RCOSH, 272 u) frm IV (m/z 577). That this in is shifted by nly 1 u in the CID spectrum f the deuterated analg Table 3. FABjMS and FABjMSjMSins frm palmityl ea Cllisin Induced Dissciatin ln",b FABjMS"'C [M - H]- I II III IV 4(1013) (775) 675 (680) (660) (599) 577 (579) (432) (412) (361) (341) 271 (271) (680) 657 (660) 657 (660) 595 (595) 595 (59S 577 (579) 577 (579) 577 (579) 357 ( (361) 357 (361) 339 (341) 339 (341) 339 (341) 339 (341) 323 (326) 305 (306) 305 (306) 305 (306) 271 (271) 271 (271) 271 (271) 271 (271) 208 (20S) 208 (209) 208 (209) ( (160) 159 (l60) IIMass-t-charge rati values in parentheses are frm [<-H]lO-(N.O)-palmityl CA. bmass-t-charge rati values f precursr ins selected fr CID are underlined. csurce-frmed ins bserved in FAB /MS.

6 J Am Sc Mass Spectrm 1994, 5, NEGATNE ION MASS SPECTROMETRY OF FATIY ACYL CA THIOESTERS 421 m/z 577 (579) )l.h(d) R S + nth. 305 (306) Scheme II implies a deuterium transfer frm the amide nitrgen, perhaps t frm an xazle ring (Scheme II), The fragment in at m/z 159 is cnsistent with [HP2061 and this in als shifted by 1 u in the CIO spectrum f the deuterated analg (Table 3). The ther in (m/z 208) is a cmmn prduct in in the CID spectra f I, II, and IV, and the frmatin f this in mst likely invlves the lss f RCOSCH 2CH2NHCOCH=CH2 frm mr z 577 (IV) perhaps via a McLafferty-like y-prtn rearrangement (Scheme III). The resultant structure f this in retains nly ne exchangeable hydrgen and is in agreement with a 1 u shift t mrz 209 in the CID spectrum f the deuterated analg (Table 3). In II (mit 657). The CID spectrum f this in frm palmityl CA was quite similar t that f I, except that the abundance f IV, m/z 577, was very lw in this case and n type III, mrz 595, was bserved. All ther prduct ins listed in Table 3 are as described abve in the CIO spectrum f I, m/z 675. In III (m/z 595). A Single abundant in, m/z 357 (D) dminated the prduct in spectrum f III as shwn in Figure 4. This is a cmmn in bserved in CID f III frm all ther acyl CAs and is frmed by the neutral lss f alkylketene, in this case hexadecylketene (238 u), Lw abundance ins bserved in the spectrum included m/z 339, 323, and 271. Of these, mrz 339 (E) and 271 (V) have been discussed abve. The in at m/z 323, which is bserved in the cm f III frm all ther acyl CnAs, is derived by lss f the acylthiacid (RCOSH, 272 u) as in the frmatin f m/z 305 in the CID spectrum f I (Scheme II). This in shifted by 3 u in the cm f the deuterated analg, which again implied the transfer f a deuterium atm frm an amide t frm the neutral thiacid. In IV (m rz 577). The CIO behavir f IV was similar t that bserved with III in that a single abundant prduct in was frmed by the lss f the neutral alkylketene, resulting in the in at mrz 339 (E). Other ins bserved in the spectrum (Table 3) were f very lw abundance. In B (m/z 426). This in, which des nt cntain the acyl grup, prduced fur ins with cllisinal activatin. The ins bserved were m/z 408 (lss f water), 328 (lss f H 2P04 ), 159 ([HP 2P06 ] - ), and 134 ([adenine - Hl-) (data nt shwn). In C (m jz 408). The prduct in spectrum f C cntained nly ne in nt bserved in decmpsitin f B. This in, mr z 273, which was als bserved in the prduct in spectrum f the [M - Hl-, is mst likely due t the lss f adenine, 135 u (data nt shwn). In V (m rz 271). The acyl thilate anin, [RCOS1-, is unique fr each acyl CA studied and is analgus t the carbxylate anin derived frm negative in FAB/MS f fatty acids and phsphlipids [14-16]. The CID f V did nt yield many prduct ins. Hwever, R s H y 'Y \.OCH'en, Q H (D) N ;, 0 0 (D) 'p' In IV " '0_ Palmityl - CA Prduct Ins miz 595 CID Energy 30 ev D 3!l III 595 V E Scheme III m/z. 208 (209) miz Figure 4. Prduct ins btained fllwing cllisin induced dissciatin (30 ev cllisin energy, 0.5 mtrr argn) f mjz 595, Type III ins frm palmityl CA. 400!l00 600

7 422 ZlRROLLI ET AL. J Am Sc Mass Spectrm 1994, 5, increasing the cllisin ffset (Q2) ptential t V did prduce a lw abundance series f prduct ins differing in 14 u, beginning at mrz 60, [SCOj-. The abundance f this in series was nt sufficient t determine duble bnd psitins in unsaturated acyl CAs, as has been demnstrated in the high energy CID f carbxylate anins frm fatty acids [14-16]. Precursr in spectra in the frmatin f this in (m/z 271) cntained nly ne abundant in, IV, and t a much lesser extent, III (m/z 577 and 595, respectively, fr palmityl CA). 200 a Acyl. CA Mixture HI" IV V '31""1' E , - 51' "C 65' , D s.q '" -; Q: Analysis f Acyl ea Mixtures by FAB / MS/ MS The prduct in spectra f III and IV frm all acyl CA species studied cntained abundant ins frm the lss f alkylketene at m/z 357 and 339 (0 and E, respectively). The precursr scans f these ins, D and E, als cntained nly abundant ins crrespnding t III and IV, respectively (data nt shwn). As D and E are fragment ins cmmn t all acyl CA species, precursr scans f these ins during FAB/CID f mixtures f acyl CA thiesters cntained ins diagnstic f each acyl CA species in the mixture. Figure 5a shws the negative in FAB/MS f a mixture f seven standard acyl CA thiesters with the inset expanding the mass-t-charge rati regin cntaining the III and / V type ins frm each f the CA species present. A much simpler spectrum results frm CID and precursr scans f m r z 339, E (Figure 5b). In this spectrum, nly IV ins are detected and the expanded regin in Figure 5b reveals ins clearly indicating each acyl CA species present in the mixture. An alternate strategy wuld be t use CID and tandem mass spectrmetric analysis t search a mixture f acyl CA esters fr the presence f ne r mre specific mlecular species. Neutral lss scans f a specific alkylketene wuld detect nly the transitin f the III and IV type ins t E (m/z 357) and D (m/z 339) ins assciated with that specific mlecular species. This is demnstrated in Figure 5c with the same acyl CA mixture emplying neutral lss scans f 238 u (lss f hexadecylketene), a specific lss fr palmityl CA. As shwn nly m/z 595 and 577 (Ill and lv, respectively frm palmityl CA) are bserved (Figure 5c). Discussin The negative in FAB/MS spectra f lng-chain acyl CA thiestcrs are characterized by the frmatin f abundant [M - H]- and tw distinct classes f fragment ins, ne class which retains the acyl grup and anther class which is related t CA. Fr the identificatin f specific acyl CA species the first series f fragment ins (I-V) are bviusly mre imprtant as these ins retain the lng-chain fatty acyl grup. With t c:: {'l s.q "C :;:,.!!! II! '00 Ii c c::.q "C " j II! b 40' C IV Ins, , 6O' III Precursr Ins miz S39 Cllisin Energy 30..V :0 'S: D3 19:0 18:0 6' :0 :20:4 591 "", r51.r33 0 " ; t10(j 900 f} 1 rnfz IV E 571_ :);3'9 Neutral Lss Scan f238 u Cllisin En"rgy 30 "v miz Figure 5. (a) FAB/MS f a mixture f acyl CAs cntaining C16:0, C17:0, C18:1, C18:0, C19:0, C20:0, and C20:4 acyl species. The spectrum was btained frm a glycerl matrix and the letter m designates matrix ins; (b) precursr ins f m/z 339 btained fllwing cllisin induced dissciatin (30 ev cllisin energy, 0.5 mtrr argn) f surce frmed ins, m/z frm the same mixture f acyl CA species; and (c) precursr ins f the neutral lss f hexadecylketene, 238 u. btained fllwing cllisin induced dissciatin (30 ev cllisin energy, 0.5 mtrr argn) f surce frmed ins, mrz , frm the same mixture f acyl C A species. negative inizatin the class f fragment ins retaining the acyl grup are much mre abundant than the C.A related class f ins (Table 1). The mst prbable site f the initial negative inizatin is ne f the three phsphate grups and many f the fragment ins are derived frm fragmentatin f a phsph anhydride r ester bnd and subsequent lsses f either water r alkylketene. As has been described abve, the ins which retain the acyl grup in the spectrum f palmityl CA appear at m/z 675, 657, 595, and 577. Ins frmed frm these by lss f alkylketene are m rz 357 and 339. Fragment ins which retain the adenine grup are bserved at m r z 426 and 408 (Figure n, In cntrast t these ins bserved fllwing FAB inizatin, tandem mass spectrmetry f

8 J Am Sc Mass Spectrm 1994, 5, NEGATIVE ION MASS SPECTROMETRY OF FATTY ACYL CA THIOESTERS 423 the [M - H]-, frm palmityl CA (m/z 4), yielded the adenine-cntaining ins as majr prducts and the acyl-cntaining ins were f lw abundance r nt detected. In additin, the mst abundant fragment in bserved in the FAB/MS spectrum, the thilate anin (m/z 271), was nt bserved at all (as well as the lesser abundant in at m/z 766). These results suggested that the frmatin f many characteristic ins bserved in direct FAB analysis ccurred during the desrptin prcess. Althugh the excitatin and decmpsitin f surce-frmed ins can be quite different frm cllisinal activatin with respect t energy and time cnsideratins, ther bservatins argue that these are nt the nly parameters affecting fragmentatin. The bserved abundances f fragment ins in the FAB/MS spectra were dependent upn the matrix emplyed. With relatively basic matrices such as diethanlamine and triethanlamine, the mst abundant fragment ins frmed were the thilate anins (V). In cntrast, with less basic matrices such as glycerl and thiglycerl, the mst abundant ins frmed were the type III and IV (Table 2) which invlve fragmentatin f the phsph anhydride bnd. These bservatins argue fr an active rle f either matrix mlecules r matrix-derived anins in the frmatin f fragment ins rather than inizatin slely at a phsphate residue. Such interactins ccurring in the selvedge regin during desrptin and inizatin f bth psitive and negative ins have been reviewed [17] and previusly bserved in the negative in FAB/MS f anther phsphate-cntaining lipid, glycerphsphchline [18]. Fragmentatin f the central phsph anhydride bnd can frm either an acylpantetheinphsphate anin r a diphsphadensine anin. The abundant type III ins (m/z 595 in palmityl CA) bserved in the FAB/MS spectrum suggest that the frmatin f the acylpantetheinphsphate anin was greatly favred ver the frmatin f the diphsphadensine anin, m/z 426. Fragmentatin n either side f the anhydride bnd may be equally favred, but the strngly basic amin sites in adenine may als abstract a prtn frm ther matrix mlecules t frm a neutral zwitterinic diphsphadensine species. This wuld result in the bserved greater abundance f the acyl-cntaining species. In the absence f matrix mlecules, fragmentatin f this anhydride bnd als ccurred, as in the cm spectrum f the [M - H]- frm palmityl CA (Figure 2). In this decmpsitin mde charge retentin by the diphsphadensine was favred. Withut the presence f matrix mlecules during CID, frmatin f the diphsphadensine anin may be favred with charge lcalized ver tw phsphate grups, while the acylpantetheinphsphate cntains nly ne such grup t stabilize the charge. Psitive inizatin FAB/MS f acyl CAs als yielded abundant [M + H]+ and acyl retaining fragment ins [10, 11]. Tw majr differences were bserved between psitive and negative in spectra: the base peak bserved with psitive inizatin was prtnated adenine, m/z 136, while the base peak with negative inizatin was typically the acyl thilate anin, V. The acyl thilate anin was nt bserved in the psitive in FAB spectrum f acyl CAs, and the frmatin f this in in the negative inizatin mde, especially when enhanced by the presence f a basic matrix, may be particularly useful in the identificatin f ismeric lng-chain acyl CAs. Clearly, tandem mass spectrmetric analysis f bth psitive and negative ins generated frm lng-chain fatty acyl CA esters prvide structurally useful and cmplementary infrmatin. The use f CID and tandem mass spectrmetric techniques with negative in frmatin will undubtedly be useful in the analysis f cmplex mixtures f acyl CAs. The unique relatinship between type IV and E r III and D ins which invlve the transitin frm acyl-cntaining ins t nly CA-cntaining ins allws the develpment f tandem mass spectrmetry prtcls similar t thse develped fr the analysis f phsphlipid mixtures. Such techniques have been used with gd success with phsphchline mixtures t determine bth mlecular species [19, 20] and t identify nvel individual species [21). These techniques are directly adaptable t mixtures f acyl C.As, using either precursr r neutral lss scans. Acknwledgments This wrk was supprted, :in part, by grants frm the Natinal Institutes f Health (HL34303) and Amgen Inc. References 1. Black, K; Vance, D. Ann. Rev. Bichem. 1977, 46, Waku, K Bichim. Biphys. Acta 1992,1124, Vance, D. In Bichemistry f Lipid» and Membranes, Vance, D. E., and Vance, J. E., Eds. Benjamin Cummings: Menl Park, CA 1985; Chapter 8, pp, Venable, M. E.; Zimmerman, G. A.; McIntyre, T. M.; Presctt, S. M. t. Lipid Res. 1993, 34, Shirley, M. A; Murphy, R.. C. J. BiI. Chem. 1990, 265, Yamaka, A; Sumimt, H.; Isbe, R.; Minakami, S. Bichem. Biphys. Res. Cmmun. 1988, 154, Klis, S. T.; Rstma, E. J.; Williams, T. H.; Sass, G. J. Drug Metab. Disp.1986, 14, Gregus, F.; Fekete, T.; Varga, F.; Klaasen, C. D. Drug Metab. Disp. 1992, 20, Wlf, B. A; Cnrad-Kessel, W.; Turk, J. t. Chrmaigr, 1990, 509, Taylr, D. c; Weber, N.; Hgge, L. R.; Underhill, E. W. Anal. Bichem.1990, 184, Nrwd, D. L.; Bus, C. A; Millingtn, D. S. l- Chrmatgr, 1990, 527, Millingtn, D. S.; Nrwd, D. L.; Kd, N.; Mre, R.; Green, M. D.; Berman, J. [. Chrmatgr. 1991, 562, Li, J.; Nrwd, D. L.; Ma, L.-F.; Schultz, H. Blchem. 1991, 30,

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