Neutral products formed during backbone fragmentations of

ization-reionization mass spectrometry (NRMS). N-terminal acylium ions (b„) arise after the C- terminus is lost as an intact amino acid or peptide; ...
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Anal. Chem. 1993, 65, 1594-1601

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The Neutral Products Formed during Backbone Fragmentations of Protonated Peptides in Tandem Mass Spectrometry Marcela M. Cordero, John J. Houser, and Chrys Wesdemiotis' Department of Chemistry, The University of Akron, Akron, Ohio 44325-3601

Collisionally activated dissociation (CAD) of the protonated polyalanines Ala-Ala,Ala-Ala-Ala, and Ala-Ala-Ala-Ala causes breakup of the peptide bonds leading to sequence-indicative fragment ions. The neutral molecules eliminated during these reactions are identified here using neutralization-reionization mass spectrometry (NRMS). N-terminal acylium ions (b,) arise after the Cterminus is lost as an intact amino acid or peptide; further loss of CO leads to immonium ions (a,). Upon generation of C-terminal sequence ions (y,), a hydrogenatom attachedto a nitrogen rearranges from the N-terminalto the C-terminalside yielding a protonated amino acid (yl) or peptide ( y ~as) the ionic fragment;the complementary neutral fragment is an aziridinone if the N-terminal amino acid is cleaved and a diketopiperazine if two N-terminal amino acid units are eliminated. Detection of neutral dissociationproducts can reveal valuable structure information, as demonstrated with the tetrapeptides Val-Gly-Ser-Glu and ValGly-Asp-Glu. INTRODUCTION Tandem mass spectrometry (MS/MS) is an established technique for the determination of the primary structures of peptides and proteins.14 With MS/MS, these polar, nonvolatile biomolecules are first converted to gaseous ions by desorption ionization: e.g., fast atom bombardment (FAB). Such ionization methods generate abundant protonated or deprotonated molecular ions (MH+ or [M - Hl-) but few, if any, fragments, which are the prerequisite for a reliable sequence determination. The desired fragmentation is achieved in a second step, by mass-selecting an individual precursor ion and inducing its decomposition to structurally diagnostic pieces via collisionally activated dissociation or other means of excitation. Cleavages along the peptide backbone lead to a series of fragments which are indicative of the peptide connectivity. Only the ionic fragments can, however, be mass-analyzed and recorded in (1) Biemann, K.; Scoble, H. A. Science 1987,237,992-998.

(2) Martin, S. A.; Johnson, R. S.; Costello, C. E.; Biemann, K. In The Analysis of Peptides and Proteins by Mass Spectrometry; McNeal, C. J., Ed.; John Wiley & Sons: Chichester, England, 1988; pp 135-150. (3) Biemann, K. In Methods in Enzymology, Mass Spectrometry; McCloskey, J. A., Ed.; Academic Press, Inc.: San Diego, CA, 1990; Vol. 193, Chapter 25, 455-479. (4) Ashcroft, A.; Derrick, P. J. In Mass Spectrometry of Peptides; Desiderio, D. M., Ed.; CRC Press: Boca Raton, FL, 1991; Chapter 7, pp 121-138. (5) Harrison, A. G.; Cotter, R. J. In Methods i n Enzymology, Mass Spectrometry; McCloskey, J. A,, Ed.; Academic Press, Inc.: San Diego, CA, 1990; Vol. 193, Chapter 1, pp 3-36. (6) McLafferty, F. W.; Bente, P. F., III; Kornfeld, R.; Tsai, S.-C.; Howe, I. J. Am. Chem. Soc. 1973,95, 2120-2129. (7) Busch, K. L.; Glish, G. L.; McLuckey, S. A. Mass Spectrometry/ Mass Spectrometry; VCH Publishers, Inc.: New York, 1988. 0003-2700/93/0365-1594$04.00/0

conventional MSIMS. This study concerns the detection and direct characterization of the complementary neutral fragments, using neutralization-reionization mass spectrometry (NRMS).a12 In NRMS, beams of neutral species are produced in the gas phase by charge exchange or dissociation of mass-selected ions. After removal of any remaining ions the neutrals are identified by the mass spectra obtained after their reionization. So far, this technique has mainly been used for the investigation of reactive intermediates, such as hypervalent radicals, carbenes, and diradicals,'3 accessed by neutralization14of the corresponding readily available cations or anions. The neutral products eliminated upon ion decompositions have been studied to a much lesser e ~ t e n t ; ~nevertheless, l~J~ the few reported cases have demonstrated that detection of such neutral MS/MS fragments can reveal structural and mechanistic insight which is impossible to gain from the ionic fragments only. For example, the elucidation of the structure and precise dissociation pathways of ionized methyl acetate (CH3COOCH3'+) relied on establishing that besides the expected CH30*a sizable amount of 'CHZOH (-30%) is liberated during dissociation to CH3C0+.16J7Combined with desorption ionization, the NRMS methodology can also be applied to biomolecules. First experiments with oligopeptides18 indicate that disclosure of the complementary neutral fragments can be particularly valuable for clarifyingproposed fragmentation mechanisms as well as for providing supplementary sequence information. The most commonly observed fragment ions from protonated peptides arise by backbone cleavagesat the amide bond and contain the N- (b, and a,, series) and C-terminus (y,, series), respectively. It is currently accepted that in the absence of basic side chains these reactions are initiated by migration of the added proton to the amide as rationalized in eq 1for the tripeptide Ala-Ala-Ala. Whether (8) Wesdemiotis, C.; McLafferty, F. W. Chem. Reo. 1987,87,485-500. (9) Terlouw, J. K.; Schwarz, H. Angew. Chem., Int. Ed. Engl. 1987,26, 808-815. (10) Holmes, J. L. Mass Spectrom. Reo. 1989, 8, 513-539. (11) Polce, M. J.; Wesdemiotis, C. In Mass Spectrometry i n the Biological Sciences: A Tutorial; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992; pp 303-310. (12) References 9-11 concern studies of kiloelectronvolt beams. The following references involve neutralization and reionization of slow beams (eV range) by proton transfer: (a) Orlando, R.; Fenselau, C.; Cotter, R. J. Org. Mass Spectrom 1989,24, 1033-1042. (b) Orlando, R.; Murphy, C.; Fenselau, C.; Hansen, G.; Cotter, R. J. Anal. Chem. 1990,62,125-129. (13) McLafferty, F. W. Science 1990,247,925-929. (14) Gellene, G. I.; Porter, R. F. Acc. Chem. Res. 1983, 16, 200-207. (15) (a) Burgers, P. C.; Holmes J. L.; Mommers, A. A.; Szulejko, J. E.; Terlouw, J. K. Org. Mass Spectrom. 1984, 19, 442-447. (b) Clair, R.; Holmes, J. L.; Mommers, A. A.; Burgers, P. C. Org. Mass Spectrom. 1985, 20, 207-212. (16) (a) Holmes, J. L.; Hop, C. E. C. A.; Terlouw, J. K. Org. Mass Spectrom. 1986, 21, 689-695. (b) Wesdemiotis, C.; Feng, R.; Danis, P. 0.;Williams, E. R.; McLafferty, F. W. Org. Mass Spectrom. 1986, 21, 776-778. (17) Heinrich, N.; Schmidt, J.; Schwarz, H.; Apeloig, Y. J. Am. Chem. SOC.1987, 109, 1317-1322. (18) Cordero, M. M.; Selby, T. L.; Boyle, S. V.; Wesdemiotis, C. Proceedings of the 40th A S M S Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31-June 5, 1992; pp 41-42. 0 1993 American Chemlcal Society

ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993

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the C-terminus is indeed eliminated as a complete unit or as an isobaric mixture of small neutrals (e.g., H3N + CzH4 + Con) is not known. Concerning the y fragment, recent D-labeling e ~ p e r i m e n t s 'have ~ , ~ ~revealed that the hydrogen transferred from the N- to the C-terminal side originates from a nearby nitrogen atom, not from the a-carbon. However, it has not been clear yet whether this rearrangement involves the immediately adjacent amide hydrogen releasing an aziridinone or whether H-transfer from a more remote functionality can also take place leading to elimination of a more stable diketopiperazine (eq IC).These mechanistic questions are addressed here by identifying the neutral fragments generated with the a,,, b,,, and y, sequence ions from the protonated oligopeptides (Ala),, (n = 2-4). The present study also evaluates the structural information attained from the detection of such neutral dissociation products.

EXPERIMENTAL SECTION The experimentswere performed with a modifiedVG AutoSpec tandem mass spectrometer that has been described in detail elsewhere.21 In this EBE instrument the first two sectors serve as MS-1 and the second electric sector as MS-2. One collision cell (Cls-1)is located after the ion source and two (Cls-2,Cls-3) are located in the region followingthe magnet. Any of these cells can be used for acquiring conventional MS/MS spectra via CAD; Cls-2 was employed for the spectra shown in this article. MS/ MS/MS experiments were performed by utilizing both Cls-1and Cls-2. NRMS studies made use of Cls-2 and Cls-3,which are the places of precursor ion fragmentation (or neutralization) and neutral product reionization, respectively. The peptides were ionized by FAB using a Cs+ion gun as the source of -20-keV primary particles. The MH+ cations were mass-selected by MS-1 and subjected to dissociating collisions with He in Cls-2. After electrostatic deflection of the generated fragment ions and the residual precursor ions, the remaining neutral fragments were reionized with O2 in Cls-3. Finally, the newly formed cations were mass-analyzed through MS-2 and recorded in the respective +NfR+He/Ozspectrum; the acronym NfR stands for neutral fragment reionization while the superscripts indicate the charges of the precursor ion and the ultimate product ions, respectively. Referencecollision-induceddissociativeionization (CIDI)mass spectral5of individual neutral molecules were acquired similarly by producing them from the corresponding proton-bound dimers.22 For smaller, volatile neutrals, reference spectra were obtained by neutralization-reionization (NR) of the respective (19)Mueller, D. R.; Eckersley, M.; Richter, W. J. Org.Mass Spectrom. 1988,23, 217-221. (20)Kenny, P. T.M.; Nomoto, K.; Orlando, R. Rapid Commun. Mass Spectrom. 1992,6, 95-97. (21)Poke, M. J.; Cordero. M. M.; Wesdemiotis, C.; Bott, P. A. Int. J. Mass Spectrom. Ion Processes 1992, 113, 35-58. (22)Biemann, K. In Biochemical Applications of Mass Spectrometry; Waller, G. A., Dermer, 0. C., Eds.; Wiley: New York, 1983;Chapter I11 15,p 469.

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molecular cations produced by electron impact at 70 eV. For example, the +NR+Xe/02spectrum of 3-methylmiridinone (M) resulted after charge-exchange neutralization of M*+with Xe and reionization of M with 0 2 . Since the abundances in +NfR+ and +NR+spectra were found to be dependent on the kinetic energy of the mass-selected precursor ion, the reference neutrals were produced with the same kinetic energy as the neutral MS/ MS fragments under investigation. He, Xe, and 02 targets were used for dissociating, neutralizing, and reionizing collisions, respectively. The pressure of each gas was adjusted to achieve a 30% reduction of the precursor ion intensity. Glycerol, thioglycerol, a 5:l mixture of dithiothreitol and dithioerythritol, and a 1:l mixture of thioglycerol and 2-hydroxyethyl disulfide served as matrices for FAB ionization. For each of the various peptides studied, the matrix maximizing the abundance of MH+and minimizing contamination by isobaric peaks was chosen; to enhance protonation, the sample was acidified by trifluoroacetic acid. The spectra shown are multiscan averages (10-50 scans for CAD and 50-1000 scans for NfR and NR data depending on the primary beam abundance). In some cases it was necessary to suspend data acquisition in order to load more sample before continuing with more scans. An average of 5-20 wmol of oligopeptide was used to acquire its CAD and NfR spectra. The reproducibility of relative abundances is better than *20 % . a,a'-d2-Ala-Alawas synthesized from a-deuterated alanine by following standard procedure^.^^ The deuterated starting material, all other samples,and the FAB matrices were commercially available and introduced into the mass spectrometer without further purification. The heats of formation of some ionic and neutral fragments were estimated by semiempirical MO calculations at the AM1 carried out on an IBM 3090 computer using the MOPAC 5.0 program.25

RESULTS CAD and +NrR+Spectra of Protonated (Ala),. Figures 1 and 2 display the CAD and +NfR+spectra, respectively, of

the MH+ cations from the oligopeptides Ala-Ala, Ala-AlaAla, and Ala-Ala-Ala-Ala. Figure 1contains all ionic products generated upon CAD and Figure 2 all reionized complementary neutral losses. The major CAD product ions observed originate from ruptures at the peptide bonds, generating N-terminal b,, and a, as well as C-terminal y,, fragments. The neutral losses postulated to accompany these a,,, b,,, and y, cations are summarized in Table I. With increasing size of the MH+ precursor ion, bigger neutral moieties are eliminated. This is indeed reflected in the corresponding +NfR+spectra, in which the number and intensities of higher mass ions grow in the order Ala-Ala < Ala-Ala-Ala < Ala-Ala-Ala-Ala. Nonetheless, all +NfR+spectra in Figure 2 are dominated by low-massions and for most proposed polyatomic neutral losses no molecular ions are observed. The extensive fragmentation of the reionized neutrals results from the fact that collisional reionization is a hard ionization technique.26 Furthermore, since several neutrals are liberated simultaneously, their ions from reionization may overlap. Because of these obstacles the determination of the correct composition and structures of the neutral CAD products necessitates proper reference spectra of the individual neutral components. Reference Spectra of C-Terminal Losses. According to Table I the C-terminus of protonated oligoalanines is eliminated as an amino acid or as a peptide. The ions resulting (23)Bodanszky, M.; Bodanszky, A. The Practice of Peptide Synthesis: Springer Verlag: Berlin, 1986. (24)Dewar, M. J. S.; Zoebisch, E. G.; Eamonn, F.; Stewart, J. J. P. J . Am. Chem. SOC. 1985, 107, 3902-3909. (25)QCPE,No.581,Quantum ChemietryProgramExchange,Creative Arts Building 181,Indiana University, Bloomington, IN 47405. (26)Danis, P.0.; Feng, R.; McLafferty, F. W. Anal. Chem. 1986,58, 355-358.

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993

-

I I

1 80

50

lL--L20

40

-f

60

M h

MH+

70

60

I

IWZ

Flgure 1. CAD mass spectra of (a) [Ala-Ala]H+ (7.2 keV), (b) [AlaAla-Ala]H+ (5.8 keV), and (c) [Ala-Ala-Ala-Ala]H+ (5.2 keV). (d) A partial CAD spectrum of a,a'-dr[Ala-Ala]H+ Is inset In Figure la.

acids and peptides can be acquired by producing them independently from their proton-bound dimers, via M2H+ MH+ + M.22 The CAD spectra of MzH+ (M = Ala, Ala-Ala, or Ala-Ala-Ala) indeed contain major fragments stemming from loss of M; this is illustrated for [Ala-Ala]zH+in Figure 3. In all three cases MH+is the base peak and, thus, M (MH+'s complementary neutral) the most abundant neutral loss. Based on the [MH+I/[total fragments+] ratio in the CAD spectra, the fractionof M in the totalneutral mixture liberated upon CAD of MzH+ is 90% for M = Ala, 80% for M = AlaAla, and 70% for M = Ala-Ala-Ala. Evidently the neutral beams from CAD of MzH+ are primarily composed of M, so that the resulting +NrR+mass spectra (Figure4) closely match the CIDI spectra of pure Ala, Ala-Ala,and Ala-Ala-Ala. These molecules are the neutral losses expected from the C-termini of the studied peptides (Table I). The CIDI spectrum of Ala (Figure 4a) looks strikingly similar to the published E1 mass ~pectrum.~'Both spectra include intense low-mass fragments and insignificant molecular cations; these characteristics are also true for Ala-Ala and Ala-Ala-Ala. The resemblance between E1 and CIDI spectra of alanine confirmsthat collisionalionizationparallels electron impact in causing hard ionization.26 It should be noted here that the pure CIDI spectrum of a neutral species is obtained only if the precursor ion dissociates to just one ionic and one neutral fragment.*5 This is usually true for metastable precursor ions; for the studied protonbound dimers, however, the sensitivity is so low without any form of activationthat the acquisition of a useable reionization spectrum is not possible. With CAD, the extent of the decomposition MzH+ MH+ + M increases by a factor of >lO"lO3, leading to spectra with acceptable signalhoise ratios, viz. Figure 4. At the same time, the primary dissociation product, namely MH+,can be generated with sufficient internal energy for sequential cleavages (Figure 3). In spite of such consecutive reactions, M remains the predominant neutral eliminated (70-90%,vide supra);therefore,the +NfR+ data of Figure 4 are assumed to represent adequate reference mass spectra for Ala, Ala-Ala, and Ala-Ala-Ala. Reference Spectra of N-Terminal Losses. Before the N-terminus is cleaved, one H-atom migrates from the N- to the C-terminal side to form the ynfragment ion (eq 1). Recent labeling experiments with the peptides Phe-Phe-Phelg and Gly-Gly-Gly-Ala-AlaZ0have shown that the transferred hydrogen is originally attached to a nitrogen atom, not the a-carbon, as often postulated in the ~ a s t . ~This . ~ is corroborated here for a-perdeuterated Ala-Ala (eq 2). The CAD spectrum of ita MH+ cation is consistent with rearrangement of an amino hydrogen Figure Id). The labeling result is also incompatiblewith elimination of the N-terminus as an amino ketene.

-

+xi0 10 0

/\ ,I ,\,a ,n,r \ , ,-

20

410

6'0

810

160

1hO

,^_ ,

,

1)O

,

,

,

,

h/Z

WZ

Flguro 2. Neutral-fragment rebnlzatbn (+N,R+) mass spectra of (a) [Ala-Ala]H+ (7.2 keV), (b) [Ala-Ala-Ala]H+ (5.8 keV), and (c) [AlaAla-Ala-Ala]H+ (5.2 keV). The largest proposed neutral losses from the Gtermlnus are (a) Ala, (b) Ala-Ala, and (c) Ala-Ala-Ala, respectively, all three of which are formed with 4.0 keV. (d) The Inset of Figure 2a shows a partlal +N,W spectrum of a,a'-dr[Ala-Ala]H+.

upon the collisional ionization of such a neutral fragment can be found from ita collision-induced dissociative ionization mass spectrum.15 Reference CIDI spectra of specific amino

Migration of an amino hydrogen in [Ala-Ala]H+ upon formation of the y1 fragment ion would lead to loss of 3-methylaziridinone, a C3HdO molecule (eq 2a). Such aziridinones are not easily available, thus preventing the acquisition of reference CIDI mass spectra from the corre~~

(27) McLafferty, F. W.; Stauffer, D. B. WileylNBS Registry of Mass Spectral Data; Wiley: New York, 1989.

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Table I. Postulated Neutral CAD Products. from Protonated Oligoalanines sequence ion (mlz)

[Ala-Ala]H+

y3 (232) b3 (214) a3 (186) YZ (161) bz (143) a2 (115) Y1

(W

C3H5NOb (71) Ala (89) Ala (89) CO (28)

bi (72) a1 (33)

[Ala-Ala-Ala]H+

[Ala-Ala-Ala-AlalH+

C3H5NOb (71) Ala (89) Ala (89) + CO (28) C6H10%@2C(142) Ala-Ala (160) Ala-Ala (160) CO (28)

C3H5NOb (71) Ala (89) Ala (89) + CO (28) CsHioNzOzC(142) Ala-Ala (160) Ala-Ala (160) CO (28) C9Hi&Kkd (213) Ala-Ala-Ala (231) Ala-Ala-Ala (231) + CO (28)

+

+

+

* T h e mass of the neutrals is given in parenthesis. 3-Methylaziridinone, H2NC(CHs)=C=O, or H N 4 H C H 3 structures. Possible structures analogous to those postulated for C6HloNzOz.

+ CO.

See eq I Cfor proposed

x3,. 00

1007 90

BO

M2H+

MHf

70 60 50 40

30 20 10 0

510

ibo

11

,

140

,

,

zbo

,

,

,

,

,

,

260

,

,

H/k

d

WZ

Flgwo 9. CAD mass spectrum of the 8.0 keV) from Ala-Ala.

proton-bound dimer (M2H+of

i "

I

25

50

75

100

m'Z x6.40

Flguro 5. CAD mass spectra of the C3HsNO'+ radical catlons (3.2 keV) from (a) 3,&dlmethyC2,eplperaln~~ne and (b) acrylamlde.

3-methylaziridinone (videinfra). From this cation, a suitable reference mass spectrum of the aziridinone itself can be obtained via neutralization-reionization.

115

m'z Flguro 4. +Np+ mass spectra of the proton-bound dimers (M2H+of 8.0 keV) from (a)Ala, (b) Ala-Ala, and (c)Ala-Ala-Ala. They represent colllsloninduced dlssoclathre bnlzatlon mass spectra of (a) 4." Ala, (b) 4.O-keV Ala-Ala, and (c) 4.0keV Ala-Ala-Ala, respecthrely. Inset In Figure 4a Is the E1 spectrum of alanine.27

sponding proton-bound dimers. In contrast, aziridinone radical cations have been generated in the gas phase by dissociative ionizationof cyclic compounds.% Electron impact ionization of the diketopiperazine shown in eq 3 leads to an abundant C3H5NO*+product at mfz 71 27 whose CAD mass spectrum is most compatible with the structure of ionized (28) Moskal, J.; Nagraba, K.; Moskal, A. Org. Mass Spectrom. 1980, 15,251-262.

The structure of C3H5NO*+from eq 3 can be determined both by interpretation of its CAD spectrum and by comparison of this spectrum to that of ionized acrylamide (Figure 5 ) . Acrylamideand its molecular cation are the most stable known C3H5NO molecule and C3HsNO*+radical ion, respectively.m CAD of the C3H5NO*+cation from eq 3 produces fragments distinctively different from those formed upon CAD of the other isomer. The presence of a methyl group in the former is indicated by the intense peak at mfz 56 in Figure 5a, which is absent in the other spectrum. A further characteristic differenceis observed in the abundancesof the CAD fragments arising by H2 loss from M + and M2+. The C3H5NO0+ precursor from eq 3 shows significantly more abundant products at mfz 69 (C3H3NO0+)and 34.5 (C3H3N02+)than the acrylamidecation. H2 elimination should be favored with an ionized aziridinone, since an aromatic three-membered ring can emerge through this reaction. These features are most consistent with generation of the 3-methylaziridinone ion in eq 3. Its +NR+ spectrum (Figure 6a) represents a (29) Lias, S. G.; Bartmess, J. E.; Liebman, J. F.; Holmes, J. L.; Levin, R. D.; Mallard, W. G . J. Chem. Phys. Ref. Data 1988, 17, Suppl. 1.

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993 loo$

g

e

Q

100%

ii

miz

Fburr 8. Neutrallzatlon-reionization (+NR+)mass spectra of the C3H5NO" radlcel cations (3.2 keV)from (a)3,~imethyCS,epIperazinedlone and (b) acrylamide.

mi2

Flgure 8. CAD mass spectra of (a) the sequence Ion b2 from CAD of 8.0-keV [Ala-Ala-Ala]H+ and (b) the MH+ cation (4.9 keV) from

3,6-dlmethyC2,4-piperazlnedione.

collision cell situated in the first field-free region of the instrument (Cls-l), mass-selected through the magnetic sector, and subjected to CAD in the collision cell immediately following the magnet (Cls-2). The resulting spectra were compared to spectra of reference ions independentlygenerated in the ion source by FAB. The incipient bz fragment from [Ala-Ala-AlalH+has the acylium cation structure depicted in eq 4a. Interaction of

100%

miz

Flgure 7. +Np+ spectrum of the proton-bwnd dimer (M2H+ of 7.4 keV) of 3,&dlme~yC2,4.plperazln~~ne. It represents a C I D I mass spectrum of 3.7keV 3,~lmethyC2,~plperazln~lone (CeHloN202). The peak of mlz 133(Cs+) orlglnates from contamination with Cs2F+. whlch is isobarlc with the mass-selected M2H+.

reference mass spectrum for the neutral 3-methylaziridinone molecule. Again, this +NR+spectrum is substantially distinct from the correspondingspectrum of acrylamide (Figure 6b). Differences are observed between the CAD and +NR+ spectra of the same C3H5NO0+cation. These variations can arise if the internal energy distribution resulting after CAD is different from that resulting after reionization; further, some neutral dissociation can take place before reionization, enhancing the abundances of the lower mass products in the +NR+spectra.a'0121 Discussion of these effects is beyond the scope of the present paper. The important fact is that collisional ionization of the C~HSNO isomers produces distinguishable spectra which can be used for the characterization of the neutral MSIMS fragments from the N-termini of the studied peptides. As illustrated in eq IC,the yn fragment formation may proceed with expulsion of a diketopiperazine moiety. With polyalanines, this molecule is the commercially available 3,6dimethyl-2,5-piperazinedione.The CAD spectrum of the corresponding proton-bound dimer, MZH+, is dominated by MH+ (89% of total fragment ion current), resulting from M loss. Reionization of the neutral losses leads to the +NfR+ spectrum of Figure 7, which is assumed to constitute an acceptable reference CIDI spectrum for the aforementioned diketopiperazine. MSIMSIMS Spectra. Before proceeding with the characterization of the neutral fragments coproduced with the b, and y, series, the postulated structures of the bp and y2 sequence ions from [Ala-Ala-AlalH+were examined by MS1 MSIMS experiments. These ions were produced in the

$3

the acylium group with the N-terminus could lead to a cyclic cation with the connectivityof a protonated diketopiperazine (eq 4a). MO calculations predict higher thermodynamic stability for the cyclic isomer; the corresponding AHof values (kJ mol-') are 516 for the linear bp cation, 407 for N-protonated, and 365 for 0-protonated diketopiperazine. The MSIMSI MS spectrum of the bz ion (Figure 8a) contains prominent products of mlz 44 (N -terminal immonium ion) and 115 (a2 ion, CO loss) which agree well with the proposed acylium structure. The reference CAD spectrum of protonated 3,6dimethyl-2,5-piperazinedione(Figure 8b) is significantly different, thus clearly showing that cyclization of bz has not taken place. Ring closure is most likely hindered by stabilizing hydrogen bonding in the linear ion. According to eq 4a, extra stabilization can be achieved in linear bz by an H-bond between the amino nitrogen and the amido proton and by ion-dipole attraction between the carbonyl and acylium groups.30 The yz fragment ion generated by CAD of [Ala-Ala-Ala]H+is presumed to be the protonated dipeptide [Ala-AlalH+ (30)O-C bond formation betweenthe carbonyloxygenand the acylium carbon of linear bz would lead to a substituted N-protonated oxazolone. The AM1 estimated heat of formation of this isomer is 423 kJ mol-I.The CAD spectra of such oxazolone ions must first be studied before it can be determinedwhether they are formed upon the decompositionof peptide ions. These experiments will be performed after suitable oxazolone precursors have been found.

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sional activation N-terminal sequence ions via elimination of C-terminal moieties. For the fragment ions containing at least two amino acid residues, the abundance of b, (n = 2, 3) is substantially larger than that of a,,. In sharp contrast, [bll l, the adjacent N-lone pair is delocalized in the peptide bond and, hence, less capable of assisting a CO loss. An additional reason of the smaller tendency of b>l to lose CO could be the presence of stabilizing H-bonds in these larger acylium cations (vide supra). According to Figure la, the principal ionic fragment from protonated Ala-Ala is the a1 backbone ion at rnlz 44 (58% of the total product ion current), formally arising by loss of CO plus alanine from MH+(Table I). If alanine is eliminated 88 an intact molecule,the corresponding+NfR+spectrum (Figure 2a) should be similar to the reference CIDI spectrum of authentic alanine (Figure 4a). Indeed the former contains all peaks appearing in the latter, including the structurally significantm l z 44 (a-cleavageof COOH from ionized alanine), 74 (a-cleavage of *CH3),and 88 (a-cleavage of OH). With a,a'-dz-[Ala-AlalH+ (partial spectrum in Figure 2d), the analogous cleavages produce ions of rnlz 45, 75, and 88, respectively, as expected if now a-deuterated alanine is reionized. These facts confirm that the C-terminal amino acid is eliminatedasa completealanine molecule. The relative abundance8 of the ions below rnlz 72 are higher in the +NfR+ spectrum of [Ala-AlalH+ than in the CIDI spectrum of alanine, because the former also incorporates the reionization products of additional neutral fragments. The differences between Figures 2a and 4a are mainly accounted for by the presence of C~HBNO (N-terminalloss) in the neutral fragment beam from [Ala-Ala]H+ (vide infra). It is noteworthy, that the centroid value of rnlz 28 in Figure 2a shifts to rnlz 29 in the deuterated sample. Thus, this ion is primarily composed in CH2N+ (CHDN+ for m l z 29), not COO+.CH2N+can be formed from alanine (see Figure 4a). Although CO and alanine are liberated in equivalent amounta upon formation of a1from [Ala-AlalH+(Table I) and CO has the larger reionization yield (Table II), the much lighter molecule does not contribute appreciably to the observed +NfR+products. As mentioned earlier, the discrimination of CO is most likely caused by the increased scattering losses and decreasedtransmittance-collection efficiencies for neutral fragments with low mass.32 An a1sequence ion is also formed upon CAD of protonated Ala-Ala-Ala (Figure lb, 5% of total fragment current). Generation of a1from this tripeptide involves elimination of CO and Ala-Ala. The reference CIDI spectrum of Ala-Ala (Figure 4b) is dominated by low-mass ions but also contains fragments of m l z 99,113,and 115which are characteristic for the dipeptide as they are absent in the reference mass spectrum of alanine itself (Figure 4a). These weak yet diagnostic peaks are present in the +NfR+spectrum of [AlaAla-Ala]H+ illustrated in Figure 2b, thus establishing that, during a1 ion formation from the latter, the C-terminus is liberated as an intact dipeptide unit. Other N-terminal

-

n lW2

Fl@urr CAD mass spectra of (a) the sequence Ion y2 from CAD of 8.0 keV [Ala-Ala-Ala]H+ and (b) authentic (sourcegenerated)5.6keV [Ala-AlaIH'.

Table 11. Reionization Efficiencies of Neutral Backbone Fragments fragment

kinetic energy, keV reioniz efficc

AlaR Ala-Ala" Ala-Ala-Alao

CsHioNzOzo

4.0 3.0 4.0 4.0 4.0

8x 9x 8x 3x 8x

3.7

10-3 10-3 10-3 10-2 10-3

(3,6-dimethyl-2,4-piperazinedione)

C3H5NOb

3.2

8 x 10-3 1x 10-2 9 x 10-2

(3-methylaziridinone) COb

3.0

3 X 1k2

2.5

-

"Molecules (M) produced via MzH+ MH+ + M. Their reionization efficiencieswere obtainedby dividing the total ion current in the Corresponding CIDI mass spectra (Figures 4a-c and 7) by [MH+]. Molecules produced by Xe neutralizationof the respective Ma+. Here, the given value represents [totalions in NR spectrum]/ [total neutral flux upon Xe neutralizationz1]. *50%.

*

(eq 4b). This structure is indeed confirmed by the respective

MSIMSIMS spectrum,which is identical within experimental error to the M S I M S spectrum of authentic [Ala-Ala]H+ (Figure 9). Similar results were found by Richter et al.l9 for y2 ions formed by chemical ionization in the ion source.

DISCUSSION With reference spectra of the postulated neutral losses on hand, the structures of the actual neutral M S I M S products from the studied oligopeptidescan be elucidated. Here, only a qualitative analysis is attempted due to the complexity of the +NfR+spectra arising upon reionization of the neutral mixtures. Reionization efficienciesare not affected greatly by kinetic energy (Table II).26 The transmission efficienciesof a neutral fragment and ita reionization products and the collection efficiency of the ultimately analyzed ions increase, however, with rising kinetic energy.26 The heavier neutral losses are expected, therefore, to contribute the major product ions in the +NfR+spectra. Due to this +NfR+dependence on kinetic energy, the reference spectrum of a specific neutral was obtained at exactly the kinetic energy with which it is eliminated from the protonated peptide. N-Terminal Sequence Ions/C-Terminal Neutral Fragments. All three oligoalanines studied produce upon colli-

(31) Basedonthereported29heatsofformation(kJmol-1) ofCO (-111) and a1 (657),and the theoretically (AM1) estimated values for bl (711), bp (516),and a2 (520),the reaction enthalpies for b, an+ CO are -165 and -107 kJ mol-' for n = 1 and n = 2, respectively. (32) Other small neutral losses from [Ala-AlalH+ include H20and HCOOH, which accompany the produgion of the weak fragment ions at rnlz 143 and 115, respectively (Figure la). ?'%e contributions of these species to the +NfR+spectrum should also be insignificant.

-

1600

ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE I,1993

m'Z

Flgure 10. +Nfi+ mass spectra of (a) [Ala-Ala-Ala]H+ (5.8 keV) and (b) [Ala-Ala-Ala-Ala]H+ 8.0 keV). Cleavage of two amino acMs from the N-termini to form (a)y1 and (b) y2, respectively, liberates a -3.7keV CBHION20P moiety in both cases.

sequence ions from [Ala-Ala-Ala]H+ include the larger bz fragment ion (34% of total product ions) and, after consecutive CO loss, the az cation (9%). These dissociations proceed by elimination of the C-terminal alanine, whose reionization products overlap in part with those of Ala-Ala. From the tetrapeptide precursor [Ala-Ala-Ala-Ala1H+, N-terminal ionic fragments at rnlz 44 (al, 2% of total fragments),115(az,8%),143(b2,17%),186(a3,2%),and214 (b3,23%) are observed upon CAD (Figure IC).The smallest (al) corresponds to elimination of CO plus the tripeptide AlaAla-Ala,whose reference CIDI mass spectrum is displayed in Figure 4c. In line with the behavior of its homologs Ala and Ala-Ala, collisional reionization of Ala-Ala-Ala gives rise to relatively small higher mass ions. The heaviest among them, extending between rnlz 139 and 145, are unique to the CIDI spectrum of Ala-Ala-Ala and, therefore, indicative for its structure. The appearance of the same products with similar peak shapes in the +NfR+spectrum of protonated Ala-AlaAla-Ala (Figure 2c) provides evidence that a1 is formed by elimination of a complete tripeptide molecule. The relative intensity of the rnlz 143 ion is much lower in the +NfR+than in the CIDI spectrum, in agreement with Ala-Ala-Ala being a minor component of the neutral MSIMS fragments from the tetrapeptide. The products of lower rnlz values in Figure 2c must, therefore, originate mainly from the C-terminal neutrals coproduced with bz (and a2)and b3 (and ad, namely, Ala-Ala and Ala, respectively, as well as from the reionized N-terminal neutrals (vide infra). The contributions of these lighter neutral fragments are enhanced if the kinetic energy of the [Ala-Ala-Ala-Ala]H+precursor is raised from 5.2 (Figure 2c) to 8.0 keV (Figure lob), due to the above-mentioned better transmission and collection of the lighter products at higher kinetic energies. C-Terminal Sequence IonsIN-Terminal Neutral Fragments, TheCADspectrumof [Ala-Ala]H+inFigurelashows a weak y1 fragment ion at rnlz 90 (7% of total product ion current) corresponding to loss of C3HsNO from the N-terminus. As verified by deuterium labeling, the C3HsNO expulsion is accompanied by migration of an amino hydrogen to the C-terminus (Figure la,d, eq 2a). The simplest neutral resulting from such a rearrangement would be 3-methylaziridinone. The reference +NR+ spectrum of this molecule contains C&NO+ ions between rnlz 68 and 71 (Figure 6a) which are diagnostic for an intact C3H5NO unit. Such ions cannot originate from reionization of alanine, the major

C-terminal neutral loss, as they are insignificant in the CIDI spectrum of this species (Figure 4a). The clear appearance of peaks at m/z 68-71 in the +NfR+spectrum of [Ala-AlalH+ (Figure 2a) must therefore mean that the N-terminus of protonated Ala-Ala is cleaved as a complete CsHbNO moiety. This conclusion is substantiated by the higher intensities (relative to rnlz 74 or 88)in Figure 2a, as compared to Figure 4a, for the ions of rnlz 52-56,38-42, and 26-28, i.e., for cations expected from reionization of 3-methylaziridinone(seeFigure 6a). From the minuscule [NHz+l(mlz 16) in Figure 2a, it can also be seen that acrylamide (Figure 6b) is not present among the neutral MSIMS fragments. N-terminal units containing more than one amino acid residue can be eliminated from the larger [Ala-Ala-Ala]H+ or [Ala-Ala-Ala-Ala]H+. In such cases, the rearranging H can be supplied by more than one nitrogen site. As illustrated in eq IC for the tripeptide, migration of the H atom immediately adjacent to the cleaved bond leads to loss of a substituted aziridinone, while H migration from the more remote N atom produces a diketopiperazine. The heats of formation of these two isomers are not known. Semiempirical MO calculations predict AHofvalues of -160 and -331 kJ mol-', respectively, justifying an incentive for elimination of the latter because of its substantially higher thermodynamic stability. According to Figure 7, a unique characteristic of the CIDI mass spectrum of the diketopiperazine moiety (C6Hd202) is the relatively abundant fragments of rnlz 71 and 99. For the isomeric aziridinone (see eq IC)no reference spectrum is available. This molecule has the same N-terminus as the polyalanines for which CIDI spectra have been measured. Because CIDI of such species mainly generates ions from the N-terminus, the substituted aziridinone of eq ICshould also yield a reionization spectrum similar to those of Figure 4, namely, one primarily containing rnlz 44 and much weaker ions of rnlz 71 and 99. Elimination of C6H1~N2O2 from the N-terminus of [AlaAla-Ala]H+ to form y1 is a minor dissociation channel (4% of total product ions); the same process from the N-terminus of [Ala-Ala-Ala-AlalH+yields a strong y2 ion, accounting for 21 % of the total fragment ions (Figures 1,spectrum b vs ~1.33 If these reactions involve release of the thermodynamically more stable diketopiperazine, a substantially larger amount of this molecule should be present in the neutral fragments from [Ala-Ala-Ala-AlalH+than in the neutral mixture from [Ala-Ala-Ala]H+. The amount of diketopiperazine (CeH10N202) in the respective neutral beams can be monitored based on its distinctive reionization products qf rnlz 71 and 99 (vide supra). The abundances of these fragments indeed rise in the +NfR+spectrum of protonated Ala-Ala-Ala-Alacompared to the respective spectrum of the tripeptide (Figure lo), consistent with elimination of the N-terminus as a sixmembered diketopiperazine. Novel Structural Information. In addition to providing mechanistic information, identification of the neutral sequence fragments can also be of analytical value. This is demonstrated here for the tetrapepides Val-Gly-Ser-Glu and Val-Gly-Asp-Glu. Both show the same abundant bz ion in their CAD spectra, arising by cleavages of Ser-Glu and AspGlu, respectively. The corresponding +NfR+spectra (Figure 11)readily distinguish these dipeptides on the basis of their al fragments: reionization of Ser-Glu yields the immonium ion +HzN=CH(CHzOH) of rnlz 60 while Asp-Glu gives rise to +HZN=CH(CHzCOOH) of rnlz 88. Although the tetra(33)An abundant yz fragment is formed from [Ala-Ala-Ala-AlaIH+ J. M.; Bursey, M. M. Org. Mass Spectrorn. 1991,20, 443-450. The result has been interpreted as indicative for the elimination of a diketopiperazine moiety. ala0 upon low-energy collision conditions: Yeh, R. W.; Grimley,

ANALYTICAL CHEMISTRY, VOL. 65, NO. 11, JUNE 1, 1993

a

Ir/z

Figwo 11. +Np+ mass spectra of the MH+ catlons (8.0 keV) from the tetrapeptides (a) VaW-Ser-Glu and (b) VaW-Asp-Glu.

peptide precursors can also be differentiated from the yz sequence ions in the corresponding CAD spectra, redundant structural information is particularly helpful in order to establish and/or confirm the correct connectivity.2~3

CONCLUSIONS The +NfR+data of the polyalanines studied indicate that the b, and y, sequence ions are produced by elimination of the residual part of the peptide as a whole unit. Such a process involves the breakup of fewer bonds and is expected to be less energy demanding than elimination of an isobaric mixture of smaller neutral species.29 Upon formation of the N-terminal acylium (b,) and immonium (a,) ions, intact peptide molecules are cleaved from the C-terminus except for the largest b, and a, cation (34)Polce, M. J.; Saigusa, H.; Wiedmann, F. A.; Nold, M. J.; Wesdemiotis, C.; Bott, P. A. Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington, DC, May 31June 5, 1992;pp 850-851.

1601

whose complementary neutral is the C-terminal amino acid. The mass spectra arising by collisional reionization of such amino acids and peptides include abundant low-mass fragments, among them the structurally diagnostic immonium ion a1 (viz. Figures 2 and 11). In contrast, the higher mass ions are weak and molecular cations are absent. Because of this shortcoming, reference mass spectra are invaluable for unequivocally identifying a C-terminal neutral fragment. Fortunately, beams of individual amino acids and small peptides can be accessed by dissociationof the corresponding proton-bound dimers, thus enabling the acquisition of the needed reference spectra. Before the C-terminal y, sequence ions are formed, a hydrogen originally attached to a nitrogen atom rearranges to the charge site. The largest y, fragment containsan H-atom from the N-terminal amino group;its complementaryneutral fragment is an aziridinone. With the smaller y, fragments, H-rearrangement from the more remote N-atom is possible, so that a more stable six-membered diketopiperazine can be eliminated. NfR spectra can become particularly helpful to real problems if softer reionization methods are developed. A technique forming only one ion per neutral loss (preferably the molecular ion) would enable identification of the complementary neutral fragmentsfrom their mass to chargeratios. Chemical ionization could be used to reionize the neutral fragments.12 Photoionization is another alternative;34 with pulsed lasers, this technique would require phase-sensitive or array detection.

ACKNOWLEDGMENT We thank &rka Beranovb, Jinnan Cai, Francesco Giorgianni, Michael J. Polce, Thomas L. Selby, and Fred A. Wiedmann for experimental assistance and helpful discussions. We are grateful for financialsupport from the National Institutes of Health. Acknowledgement is also made to the University of Akron for partial support of this work. RECEIVEDfor review November 3, 1992. Accepted February 15,1993.