Here we describe methodology based on tandem mass

Transcription

1 Proc. Nat. Acad. Sc. USA Vol. 86, pp , February 1989 Chemstry Structural characterzaton of toxc cyclc peptdes from blue-green algae by tandem mass spectrometry (lqud secondary-on mass spectrometry/mcrocysts aerugnosa) THAIYA KRISHNAMURTHY*t, LINDA SZAFRANIEC*, DONALD F. HUNTt, JEFFREY SHABANOWITZt, JOHN R. YATES 1t1, CHARLES R. HAUERt, WAYNE W. CARMICHAEL, OLAV SKULBERG, GEOFFREY A. CODDII, AND STEPHEN MISSLER** *Research Drectorate, U.S. Army Chemcal Research, Development, and Engneerng Center, Aberdeen Provng Ground, Aberdeen, MD ; tdepartment of Chemstry, Unversty of Vrgna, Charlottesvlle, VA 2291; 2Wrght State Unversty, Dayton, OH 45435; I'Department of Bologcal Scences, The Unversty, Dundee, Unted Kngdom; Norwegan Insttute for Water Research, N Oslo 3, Norway; and **U.S. Army Medcal Research Insttute of Infectous Dseases, Fort Detrck, Frederck, MD Communcated by Fred W. McLafferty, November 14, 1988 (receved for revew September 6, 1988) ABSTRACT Combned use of chemcal degradaton, dervatzaton, and tandem mass spectrometry for rapd structural characterzaton of toxc cyclc peptdes from blue-green algae at the nanomole level s descrbed. Prevously, all blue-green algal toxns were thought to belong to a famly of seven-resdue cyclc peptdes, havng the general structure cyclo-d-ala-l-xaa-erythro-,b-methyl-d-soaspartc acd-l-yaa- Adda-D-soglutamc acd-n-methyldehydroalanne, where Xaa and Yaa represent varable amno acds of the L confguraton and Adda s 3-amno-9-methoxy-2,6,8-trmethyl-1-phenyldeca-4,6-denoc acd. Structural characterzaton of two addtonal toxns ndcates that further varablty can exst wthn ths famly of naturally occurrng toxc cyclc peptdes. Isoaspartc acd and dehydroalanne can substtute for (3-methylsoaspartc acd and N-methyldehydroalanne, respectvely. Producton of a famly of toxc cyclc peptdes n freshwater supples by the cyanobacterum Mcrocysts aerugnosa has been lnked to the death of lvestock n both Australa and South Afrca (1, 2). Growth of toxc blooms from the above blue-green algae s enhanced n stagnant water enrched n mnerals and organc nutrents and depleted of dssolved oxygen (1). Snce the toxns are unaffected by normal chlornaton, flocculaton, and fltraton procedures used by water treatment facltes, growth of the cyanobacterum n reservors represents a potentally serous threat to the human populaton. Structural studes nvolvng extensve use of both NMR and mass spectrometry on fve of the toxns ndcated that they all belonged to a famly of seven-resdue cyclc peptdes havng the general structure cyclo-d-ala-l-xaa-erythro-,3-methyl-d-soaspartc acd-l-yaa-adda-d-soglutamc acd- N-methyldehydroalanne, where Xaa and Yaa represent varable amno acds of the L confguraton and Adda s 3-amno-9-methoxy-2,6,8-trmethyl-1-phenyldeca-4,6-denoc acd (3-5). Cyclc peptdes n whch the Xaa and Yaa resdues were dentfed as Leu and Ala and Tyr and Met, respectvely, were characterzed fully n earler papers (4, 5). For three other toxns, only four of seven resdues were placed unambguously. Compostonal analyss, however, confrmed the presence of Adda and Glu n each of the samples. The combned data suggested strongly that the three addtonal toxns all belonged to the above famly of cyclc peptdes. The two L-amno resdues, Xaa and Yaa, n each of these toxns were assgned tentatvely as Leu and Arg, Tyr and Arg, and Tyr and Ala, respectvely (5). The publcaton costs of ths artcle were defrayed n part by page charge payment. Ths artcle must therefore be hereby marked "advertsement" n accordance wth 18 U.S.C solely to ndcate ths fact. Here we descrbe methodology based on tandem mass spectrometry that facltates rapd characterzaton of toxc cyclc peptdes from blue-green algae at the nanomole level. Toxn contanng the two L-amno acds Leu and Arg (Fg. 1) has been solated from freshwater lakes n fve countres and characterzed by tandem mass spectrometry. Structures for two addtonal peptde hepatotoxns from blue-green algae blooms are also reported. MATERIALS AND METHODS Isolaton and purfcaton of toxc peptdes from blue-green algae have been descrbed (6). Fnal HPLC separatons were performed on an Appled Bosystems model 13A system. Sample dssolved n.1% trfluoroacetc acd was njected onto a mcrobore RP3 Aquapore column (2.1 mm X 3 cm) and eluted wth a 4-mn lnear gradent of-6% acetontrle (.85% trfluoroacetc acd) n.1% trfluoroacetc acd. Column effluent was montored at 214 nm and fractons were collected by hand. The Waters Pco-Tag system was used for amno acd analyss of the purfed toxns. Amno acd chralty was determned by HPLC analyss of dastereosomers formed on reacton of the amno acds wth 1-fluoro- 2,4-dntrophenyl-5-(L-alannamde) (Perce) (7). Standard solutons of peptde n ether 5% acetc acd or methanol (1 nmol/,p) were stored at C. Mass Spectrometry. Mass spectra were recorded on both a trple quadrupole mass spectrometer (8) and a Fourer transform mass spectrometer (9). Operaton of these nstruments has been descrbed (8, 9). Samples for mass analyss were prepared by addng 1 tul of a methanol soluton contanng peptde at the.5-1. nmol/ptl level to a matrx contanng.5 pkl of monothoglycerol and.5,ul ofa 1: 1 mxture ofdmethyl sulfoxde/6 M HCl on a gold-plated stanless steel probe tp 2 mm n dameter. The resultng mxture was then subjected to bombardment by a beam of 6- to 8-keV Cs+ ons generated from a cesum on gun (Antek, Palo Alto, CA). Collsonactvated dssocaton mass spectra were recorded on the trple quadrupole nstrument wth argon as the collson gas ( mlltorr; 1 torr = Pa) and wth collson energes between 2 and 3 ev. Hydrolyss of the Cyclc Peptde Toxns wth Trfluoroacetc Acd. An alquot of the standard peptde soluton (1 1.l) was lyophlzed, treated wth trfluoroacetc acd ( tal), and Abbrevatons: Adda, 3-amno-9-methoxy-2,6,8-trmethyl-1- phenyldeca4,6-denoc acd; MAsp, f3-methylaspartc acd; Mdha, N-methyldehydroalanne. tto whom reprnt requests should be addressed. 77

2 FIG. 1. Hepatotoxn from blooms of blue-green algae M. aerugnosa. Masp MC Mdha NH NH CO2H NH C-NH12 NH Arg MeO C6H5 H3C ~~Adda Glu allowed to stand at room temperature overnght. The soluton A.l Chemstry: Krshnamurthy et al. was then lyophlzed and the resdue was dssolved n 2-4 of methanol. Esterfcaton. A standard soluton of 2 M HCl n methanol was prepared by addng acetyl chlorde (16 41.) dropwse wth strrng to methanol (1 ml) and by allowng the resultng soluton to stand for 1 mn at room temperature. Peptdes at the 1-2 nmol level were esterfed by treatng lyophlzed sample wth 2 M methanolc HCl ( 1.d) and by allowng the reacton mxture to stand at room temperature for 2 hr. Samples were then lyophlzed and dssolved n ether methanol or 5% acetc acd. Deuterum Labelng of Amno Acd Resdues Contanng Free a-carboxylc Acd Groups (1, 11). Peptde (1-2 nmol) was treated wth 6,p1 of a 2:1 mxture of acetc anhydrde and % (vol/vol) formc acd. After 1 mn at room temperature, the mxture was lyophlzed and the resdue was allowed to stand for 3-6 hr at room temperature n a soluton contanng,ul of deuterum oxde, 2,u1 of pyrdne, and 5,ul of acetc anhydrde. Reagents were removed by lyophlzaton and deuterum ncorporated on oxygen, and ntrogen was then back-exchanged by treatng the resdue wth 2 x 2,ul of water and by lyophlzng the resultng solutons. Treatment of the resdue wth 2 M methanolc HCI for 2 hr converted the labeled peptde sample to the correspondng methyl ester. Dervatzaton wth N-t-Butyloxycarbonylprolylhydroxysuccnmde. Lnear peptde (5-1 nmol) was dssolved n 75,ul of 5% aqueous pyrdne and then treated wth a 5-fold molar excess of reagent n pyrdne. After 1 hr at 75 C, the mxture was treated wth a second 5-fold molar excess of reagent, allowed to stand for an addtonal 1 hr at 75 C, and then lyophlzed. The resdue was taken up n 5,ul of water and the resultng mxture was lyophlzed. To remove the t- butyloxycarbonyl group, the dred resdue was treated wth 5,ul of neat trfluoroacetc acd. The resultng soluton was heated at 37 C for 1 mn and then lyophlzed. Traces of acd were removed by dssolvng the sample n 5 p.1 of water and by lyophlzng the mxture. Dervatzed peptdes were separated from excess reagent by HPLC. RESULTS AND DISCUSSION Characterzaton of Toxn I Isolated from Akersvatn, Norway. Isolaton and purfcaton of a hepatotoxc peptde produced by blooms of the blue-green algae M. aerugnosa n Akersvatn, Norway, has been descrbed (6). Amno acd Proc. Natl. Acad. Sc. USA 86 (1989) 771 analyss on the purfed peptde ndcated the presence of Ala, Arg, Glu, Leu, and B-methylaspartc acd (,6-MAsp) n equmolar quanttes. Only Leu and Arg were found to have the L-confguraton by HPLC analyss of dastereomerc dervatves (7). The summed masses of the above resdues total 598 Da. Analyss of the peptde by lqud secondary on mass spectrometry afforded a spectrum contanng a strong sgnal for an (M + H)+ on at m/z 995. Prevous work ndcated that the toxc peptdes from blue-green algae all belonged to a famly of seven-resdue cyclc peptdes, termed cyanognosn-xy, that contan two unusual amno acd resdues, Adda and Mdha (N-methyldehydroalanne) (4, 5), not detected n the normal amno acd analyss scheme. Incluson of masses for Adda (313 Da) and Mdha (83 Da) n the above calculaton affords a mol mass of 994 that s n agreement wth that determned by mass spectrometry. Thus, t seemed lkely that the solated toxn was a member of the above famly of cyclc peptdes. The varable amno acds could be ether Leu and Arg or Arg and Leu. Collson-actvated dssocaton mass spectra recorded on the toxn (M + H)+ on contaned a large number of fragment ons that could not be assgned unambguously to a sngle sequence of amno acds. Results of ths type are not atypcal for complex cyclc peptdes, although successful characterzaton of several cyclc peptdes by ths approach has been acheved (12). To convert the cyclc peptde to a lnear structure, toxn was treated drectly wth neat trfluoroacetc acd. Progress of the reacton was montored by mass spectrometry. Two products formed n about equal abundance. The (M + H)+ on for product I occurred at m/z 113, a number consstent wth that expected for a lnear structure resultng from the addton of water to the cyclc peptde of mol mass 994. The mass spectrum of product II showed an (M + H)+ on at m/z 93. Ths s the expected m/z value for a peptde formed by loss of the Mdha resdue (83 Da) from the lnear peptde. Shown n Fg. 2A s the collson-actvated dssocaton mass spectrum recorded on the on at m/z 93. Nomenclature (13) and mechansms of formaton (14) for the fragment ons observed n ths type of spectrum have been dscussed. Daughter ons present n Fg. 2A are consstent wth the partal sequence Ala-Leu-MAsp-Arg-Adda-Glu. Masses for fragments of type B and Y" expected for ths sequence are shown above and below the structure at the top of Fg. 2A. Those observed n the spectrum are underlned. The on at m/z 783 corresponds to B5 and results from loss of water and the C-termnal resdue (129 Da) from the (M + H)+ on. Glu and MAsp both have a mol mass of 129, so ether could be located at the C termnus. Assgnment of Glu to ths poston s based on the observaton that ths resdue nvarably occupes a ste on the C-termnal sde of Adda wthn the famly of toxc cyclc peptdes from blue-green algae. That Adda s adjacent to Glu s ndcated by the characterstc mass dfference of 313 Da between the B5 and B4 fragment ons at masses 783 and 47, respectvely. The appearance of the Y2" on at m/z 461 s also consstent wth the above assgnment. Placement of Arg on the N-termnal sde of Adda s dctated by the fndng that Y3" appears at m/z 617, 156 Da hgher than Y2". MAsp s assgned to the next poston n the chan because the Y4" on appears 129 Da hgher than Y3' and because ths locaton s nvarably occuped by MAsp n all blue-green algal peptdes shown to contan ths resdue. Amno acd analyss on the parent cyclc peptde and the mass dfference between Y4' and the (M + H)+ on (184 Da) both support assgnment of Ala and Leu to the remanng two postons n the peptde although the fragment on Y5' requred to prove ths s mssng n the daughter on spectrum of m/z 93.

3 'Prelyl 772 Chemstry: Krshnamurthy et al. 7n n 7P3 93 A Ale Lou Moep Arg Ade Glu C-d Cd * N- hib -@L -. N~t N -LAL,&1 I I< a.4 T 76 1 fiso 'Il l 1 A J4 757 B Me Inj Ester Ale Lee Keep Arg Ades Gv V I U a ye so 1 9- m a, I. F. L ~L~I..L ALA.-I. t-. I ISO t3 n to 3 ~ Cd Cd~~d 1vI '1I 5o I It d d d - --T I N 1, N I I,, m 5 5o 6*o 75 G o C Sc Z ' SC la IL I. L 11 D 54 :: loc 55 LII3. ~ 17559L.. Dervetwe Pro Ale Lee "eep Arf Adde 1le M #I? 4±1 145 Yoe A / 65 5oo Me* Tuter Om AU s* MA-4-CO-Ale Lou Meep Arg Addse IV-NNCH OLe A; a~ a - L4 L I, J lis 2oo ISO 45 2I 's t- - II n NO a 3 Cd 6 I I.1I I 1, ( 55 6 */ f5 oo.1to eel. -4 FIG. 2. Collson-actvated dssocaton mass spectra recorded on toxn I. Spectra generated from the (M + H)+ on of product II (A), product II methyl ester (B), prolylated dervatve of product II (C), and product I methyl ester (D). To confrm the fragment on assgnments made n Fg. 2A, products I and II obtaned from acd hydrolyss of the cyclc peptde were converted to the correspondng methyl esters. Product II was also dervatzed by addng a prolyl moety to Proc. Natl. Acad. Sc. USA 86 (1989) the unblocked N termnus. Collson-actvated dssocaton mass spectra of the (M + H)+ ons correspondng to the methyl ester and prolyl dervatve of the lnear peptde of mol mass 929 are shown n Fg. 2 B and C, respectvely. Note that all fragment ons predcted to contan one or more free carboxylc acd groups n Fg. 2A do ndeed shft to hgher mass n the methyl ester spectrum by the expected multple of 14 Da per COOH group. In Fg. 2C, daughter ons desgnated as beng of type B shft by 97 Da as a result of addng a prolyl moety to the N termnus of the peptde sample. Note also that Fg. 2C contans a complete seres of ons oftype B. Introducton ofthe strongly basc prolyl group at the N termnus of the peptde promotes formaton of fragment ons contanng the amno termnus and allows the complete sequence of the peptde to be determned. The abundant on at m/z 169 dctates that the order of the frst two amno acds n undervatzed product II be Ala-Leu rather than Leu-Ala. Collson-actvated dssocaton of m/z 113, the (M + H)+ on observed for product I n the acd catalyzed hydrolyss of toxn I, affords a seres of ons of type Y", all of whch occur at m/z values that are 13 Da hgher than those seen n Fg. 2A. Converson of product I to the correspondng methyl ester shfted the (M + H)+ from m/z 113 to 187, an ncrease of 74 Da. Incorporaton of three methyl groups and one molecule of methanol nto the lnear peptde accounts for the observed mass shft. The collson-actvated dssocaton mass spectrum of m/z 187 s shown n Fg. 2D. Partcularly noteworthy s the observaton of a seres of fragment ons of type Y" that occur at m/z values that are 1 Da lower than those n Fg. 2B. We conclude that product I s formed by protonaton of the enamne moety n the Mdha resdue, as shown n Fg. 3. Ths s then followed by acd-catalyzed hydrolyss of the resultng Schff base to afford a lnear peptde blocked at the N termnus and C termnus wth a-ketoacyl and N-methyl amde groups, respectvely. Treatment of ths molecule wth 2 M methanolc HCl would be expected to esterfy the two carboxylc acd sde chans n MAsp and Glu and also to convert the keto group to the correspondng dmethyl ketal. The result of these transformatons would be to ncrease the mass of the molecule by the observed 74 Da. Note that the presence of a complete seres of Y" ons n Fg. 2D allows all resdues except MAsp and Glu to be placed n the proposed structure unambguously. Once the above sequence nformaton had been obtaned, addtonal experments were performed to determne whch of the two carboxyl groups n MAsp and Glu were nvolved n the amde lnkages along the backbone of the cyclc peptde. Base-catalyzed ncorporaton of deuterum selectvely onto carbons adjacent to all free a-carboxyl groups n Mdha 1I I " CHI NH "1 f OH2 TFA -k NH., % HI I %".,'. I H+ CHE3 H --ANN NH" N " J _ FIG. 3. Acd-catalyzed cleavage of cycloheptapeptde toxns from blue-green algae. TFA, trfluoroacetc acd.

4 Chemstry: Krshnamurthy et A the peptde was used to obtan ths nformaton (4, 9, 1). Labelng of MAsp and Glu s expected even when they are n non-c-termnal postons, provded that these resdues are connected by an so-lnkage and thus have a free a-carboxyl group. When the above experment was performed on the mxture of two products obtaned n the acd-catalyzed hydrolyss of the toxn I and the labeled peptdes were then converted to the correspondng methyl esters, the m/z values for the (M + H)+ ons of products I and II shfted from m/z 187 and 972 to and 974, respectvely. Product I ncorporates up to fve deuterum atoms, and product II ncorporates two deuterum atoms. Fg. 2B (Inset) shows a porton of the collson-actvated dssocaton mass spectrum recorded on the (M + H)+ at m/z 974 for product II. Note that m/z values for the major fragment ons contanng ether MAsp or Glu n Fg. 2C all shft by 1 Da to hgher mass n the spectrum of the labeled peptde. Ths result requres that MAsp be connected to Arg by an so-lnkage. Incorporaton of deuterum nto Glu results because ths resdue s C termnal n the sx-resdue fragment. That ths resdue s also part of an so-lnkage n the ntact cyclc peptde s strongly suggested by the observaton that the methyl ester of the lnear peptde n Fg. 2D ncorporates up to fve deuterum atoms, three on the carbon a to the ketoacyl moety and one each a to the free carboxyl groups of MAsp and Glu. Further support for the presence of soglu was obtaned from a lqud secondary on mass spectrum of the deuterum-labeled methyl ester of product I recorded on the tandem quadrupole Fourer transform nstrument. Fragment ons correspondng to Y1'-Y3" all showed deuterum ncorporaton to an extent greater than 25%. We conclude that the toxc heptapeptde solated from blooms of the blue-green algae M. aerognosa n Akersvatn, Norway, has the structure cyclo-d-ala-l-leu-d-masp- L-Arg-Adda-D-soGlu-Mdha. Further work n our laboratory has dentfed ths same toxn n blooms of blue-green algae taken from waters n South Afrca, Scotland, Canada, and the Unted States. Characterzaton of Toxn II from Canada. Mass spectra recorded on toxn II from Canada showed (M + H)+ ons at both m/z 981 and 995. HPLC was used to separate the mxture and the sample of mol mass 994 was then hydrolyzed wth neat trfluoroacetc acd. Products of ths reacton were converted to methyl esters and prolylated as descrbed n Materals and Methods. Collson-actvated dssocaton spectra recorded on the resultng dervatves confrmed that the materal of mol mass 994 was dentcal n structure to toxn I descrbed above. Amno acd analyss on the sample of mol mass 98 ndcated that Ala, Arg, Glu, Leu, and Asp were present n equmolar quanttes. Only Leu and Arg were found to have the L-confguraton by HPLC analyss of dastereosomer dervatves. Mass spectra recorded on the sample after treatment wth neat trfluoroacetc acd showed two (M + H)+ ons at m/z 916 and 999, respectvely. Converson of the two products to the correspondng methyl esters shfted the observed (M + H)+ ons to m/z 958 and 173, respectvely. Shown n Fg. 4A s the collson-actvated dssocaton spectrum recorded on the (M + H)+ on of m/z 173. Comparson of these data wth that obtaned on the same dervatve of toxn I (Fg. 2D) shows that all fragment ons assumed to contan the MAsp resdue n Fg. 2D are now shfted to lower mass by 14 unts n Fg. 4A. Thus, sample of mol mass 98 n toxn II contans Asp n place of the MAsp resdue n toxn I. Fragment ons of type B and Y" predcted for open-chan dervatve of the Asp-contanng peptde are shown above and below the structure n Fg. 4A. Those observed n the collson-actvated dssocaton spectra are underlned. Deuterum labelng experments conducted on the lnear peptde dervatves confrm that both Asp and Glu A B so I" a c Proc. Natl. Acad. Sc. USA 86 (1989) 773 Fo: P ta n t : A IShIL e 6L P L L- omf La on MesC[CO-AIs Los Asp AreAdds 6Is-NH OM* Y * t 4to : ISO ISo. *A a 2 oa eie * e - P- l :I 1. A ;OAP e.l.i 21 1 L j J L WA A.AMn s5 oo 15 Me Ester OM 1I on lo~~~~~~~~~~~~~~~~~~y M"-C-CO-Als Arg Asp Arg Adds 19-NH2 Mf DoI 'a1. h S 1l, :,e z _ r rn/z FIG. 4. Collson-actvated dssocaton mass spectrum generated from the (M + H)+ ons of the lnear peptdes derved from toxn II (A) and toxn III (B). resdues are attached to the adjacent amno acds by solnkages. We conclude that the sample of mol mass 98 n toxn II has the structure, cyclo-d-ala-l-leu-d-soasp-l- Arg-Adda-D-soGlu-N-Mdha. Characterzaton of Toxn mii from Norway. A mass spectrum recorded on the toxn III from Norway showed an (M + H)+ on at m/z 11. Amno acd analyss confrmed the presence of Ala, Asp, Glu, and Arg n the molar rato 1:1:1:2. Only Arg was found to have the L confguraton by HPLC analyss of dastereomerc dervatves (7). Treatment of the sample wth neat trfluoroacetc acd produced a sngle product of mol mass 127. Unlke toxns I and II above, toxn III does not elmnate Mdha to form a second product n the acd hydrolyss reacton. Mass spectra recorded on the sngle hydrolyss product and the correspondng methyl ester showed (M + H)+ ons at m/z 128 and 112, respectvely. The observed shft of 74 mass unts s analogous to that observed for toxns I and II and suggests that toxn III also suffers hydrolyss n trfluoroacetc acd to a lnear peptde blocked at the N termnus and C termnus wth a-ketoacyl and amde groups, respectvely. Shown n Fg. 4B s the collson-actvated dssocaton spectrum recorded on the (M + H)+ on of m/z 112. Comparson of ths spectrum to that obtaned for the same dervatve of toxn II (Fg. 4A) shows that the frst four fragment ons of type Y" are all shfted to lower mass by 14 unts. Combned wth the results of amno acd analyss, these data ndcate that the C termnus of the lnear peptde s blocked wth an unsubsttuted amde group. We conclude that toxn III contans dehydroalanne n place of the usual Mdha resdue. Placement of Asp and two Arg resdues wthn the peptde s facltated by the presence of a complete seres of Y" type fragment ons n Fg. 4B. The proposed sequence for the lnear hydrolyss product s shown at the top of Fg. 4B. Predcted fragment ons of types B and Y" appear above 1

5 774 Chemstry: Krshnamurthy et al. and below the structure, respectvely. Those observed n the collson-actvated dssocaton spectrum are underlned. Deuterum ncorporaton nto the product of acd hydrolyss confrms that the Asp and Glu resdues contan free a- carboxylc acd groups and are therefore attached to adjacent resdues by so lnkages. We conclude that toxn III has the structure, cyclo-d-ala-l-arg-d-soasp-l-arg-adda-dsoglu-dehydroalanne. The results presented n ths paper ndcate that tandem mass spectrometry, n combnaton wth specfc chemcal degradaton and dervatzaton technques, s deally suted for the drect characterzaton of toxc cyclc peptdes solated as mxtures at the nanomole level from blue-green algae. Samples are treated wth neat trfluoroacetc acd to convert the cyclc peptdes to lnear structures, whch are then ether prolylated or converted to the correspondng methyl esters. Tandem mass spectrometry provdes the necessary structural nformaton to deduce the sequence of amno acds n the dervatzed lnear peptdes. Deuterum labelng of the peptdes facltates detecton of amno acd resdues lnked through sde-chan carboxyl groups. In the present work, the above methodology was used to determne structures for two toxc cyclc peptdes solated from bluegreen algae. A contract awarded to D.F.H. from the U.S. Army and nstrument development funds awarded to D.F.H. from the Monsanto Co., Center for Innovatve Technology (Rchmond, VA) (BIO-876), and the Natonal Scence Foundaton (CHE ) are gratefully acknowledged. Proc. Natl. Acad. Sc. USA 86 (1989) 1. Carmchael, W. W. & Mahmood, N. A. (1984) n Seafood Toxns, Amercan Chemcal Socety Symposum Seres No. 262, ed. Ragels, E. (Am. Chem. Soc., Washngton, DC), p Botes, D. P., Kruger, H. & Vljoen, C. C. (1982) Toxcon 2, Botes, D. P., Vljoen, C. C., Kruger, H., Wessels, P. L. & Wllams, D. H. (1982) Toxcon 2, Botes, D. P., Tunman, A. A., Wessels, P. L., Vljoen, C. C., Kruger, H., Wllams, D. H., Santkarn, S., Smth, R. J. & Hammond, S. J. (1984) J. Chem. Soc. Perkn Trans. 1, Botes, D. P., Wessels, P., Kruger, H., Runnegar, M. T. C., Santkarn, S., Smth, R. J., Barna, J. C. J. & Wllams, D. H. (1985) J. Chem. Soc. Perkn Trans. 1, Krshnamurthy, T., Carmchael, W. W. & Sarver, E. W. (1986) Toxcon 24, Marfey, P. (1984) Carlsberg Res. Commun. 49, 591-5%. 8. Hunt, D. F., Gordan, A. B., Rhodes, J. & Herold, D. A. (1982) Cln. Chem. (Wnston-Salem, N.C.) 28, Hunt, D. F., Shabanowtz, J., Yates, J. R., III, Zhu, N. Z., Russell, D. H. & Castro, M. E. (1987) Proc. Natl. Acad. Sc. USA 84, Holcomb, G. N., James, S. A. & Ward, D. N. (1968) Bochemstry 7, %. 11. Steffens, J. C., Hunt, D. F. & Wllams, B. G. (1986) J. Bol. Chem. 261, Tomer, K. B., Crow, F. W., Gross, M. L. & Kopple, K. D. (1984) Anal. Chem. 56, Roepstorff, P. (1984) Bomed. Mass Spectrom. 11, Hunt, D. F., Yates, J. R., Shabanowtz, J., Wnston, S. & Hauer, C. R. (1986) Proc. Nat. Acad. Sc. USA 83,