JOURNAL OF MASS SPECTROMETRY, VOL. 31, 1073È1085 (1996)

SPECIAL FEATUREPERSPECTIVE

Characterization of Neutral Fragments in TandemMass Spectrometry : A Unique Route toMechanistic and Structural Information

Michael J. Polce, Beranova� ,¤ Michael J. Nold and Chrys Wesdemiotis°S‹ a� rkaDepartment of Chemistry, University of Akron, Akron, Ohio 44325-3601, USA

The neutral species eliminated upon fragmentation of fast-moving mass-selected ions can be directly identiÐed bycollisional ionization and detection in neutral fragment reionization mass spectra. Establishment of the(N

fR)

identity of neutral fragments yields valuable insight into the decomposition mechanism of a precursor ion, asdemonstrated for fullerene and alkali metal iodide cluster ions as well as metal ion adducts of amino acids. Inaddition, neutral fragment reionization also provides structural information that may not be available from thecomplementary ionic fragments alone ; this is illustrated in the di†erentiation of isomeric mononucleotides. Theparameters inÑuencing the appearance of spectra are discussed and the scope and general applicability of theN

fR

method are brieÑy evaluated.

KEYWORDS: tandem mass spectrometry ; neutral fragment reionization ; fullerenes ; cluster ions ; nucleotides ;metalated amino acids

INTRODUCTION

Tandem mass spectrometry (MS/MS) is widely usedboth in the analysis of molecular structures and in fun-damental gas-phase ion chemistry studies.1,2 In mostcases, structural information in MS/MS experiments isderived from the unimolecular reactions of a mass-selected precursor ion, most commonly induced via col-lisionally activated dissociation (CAD).1,2 Althoughsuch decompositions produce simultaneously ionic andneutral fragments (Scheme 1), only the charged particlescan be identiÐed in conventional MS/MS. The co-produced neutrals and, thus, any mechanistic or struc-tural insight residing in them generally remainaccessible. This deÐciency is alleviated by neutral frag-ment reionization a variant of neutralizationÈ(Nf R),3reionization mass spectrometry (NRMS).4h6 In Nf R,the neutral species released upon CAD of fast (keV)moving ions are collisionally ionized to become directlyanalyzable. The ions emerging upon collisional ioniza-

Scheme 1.

¤ Present address : Charles B. Stout Neuroscience Mass Spectrom-etry Laboratory, University of Tennessee, Memphis, 800 MadisonAvenue, Memphis, TN 38163, USA.

° Author to whom correspondence should be addressed.

tion are separated by their mass-to-charge ratios andrecorded as neutral fragment reionization mass spectra.In this account, we brieÑy describe the procedureNf Rand illustrate its utility primarily in mechanistic butalso in structural problems which are difficult to solveby normal MS/MS.

The prototype experiment was reported byNfRMcLa†erty et al.,7 who detected the neutral fragmentsfrom collisionally activated and metastable acetonecations. Soon thereafter, the groups of Holmes andTerlouw recognized the promise of the method anddemonstrated its usefulness by identifying the neutralfragments from metastable ions with puzzling decompo-sition mechanisms ; the process of ionizing such meta-stably generated neutrals was speciÐcally namedcollisionally induced dissociative ionization (CIDI),Table 1.8h11 The Ðrst problem solved by CIDI was thenature of the neutral (C, H, N) fragment cleaved frommetastable aniline ions, which was shown by Burgers etal.8 to be HNC, not the thermodynamically more stableHCN. Later CIDI studies on metastable methyl acetatecations by the same researchers provided strong evi-dence that cogenerates a substantialCH3C(xO)OCH3`~amount of radicals, together with the expected~CH2OH

during fragmentation to yield~OCH3 , CH3CO`.12Similarly, the McLa†erty rearrangement of metastablen-hexanoic acid ion to was revealed by van BaarC4H8`~et al.13 to release not the enolCH3COOH,

CCC 1076È5174/96/101073È13 Received 7 June 1996( 1996 by John Wiley & Sons, Ltd. Accepted 20 August 1996

1074 M. J. POLCE ET AL .

Table 1. Acronyms and terms used in the study of neutralMS/MS fragments

CAD Collisionally activated dissociation; also calledcollision induced dissociation (CID). It is theprocess of fragmenting mass-selected ions bycollisional activation with gaseous targets.

CIDI Collisionally induced dissociative ionization.Originally defined as the process of ionizing (bycollision) the neutral fragments eliminatedspontaneously from metastable ions. Here, we alsouse the term CIDI to describe the collisionalionization of CAD-generated neutral fragments.

NR Neutralization-reionization. In NR, collisionalneutralization of a mass-selected ion is followed bycollisional reionization of the neutral speciesgenerated in the neutralization step. The neutralintermediate may be unstable, dissociating upon itsformation. In such a case, the neutrals beingreionized were not charged species originally, andthe term reionization rather describes a subsequent,second ionization event (the first having taken placein the ion source to produce the mass-selectedprecursor ion). Note that both CIDI and reionizationinvolve collisional ionization and have, therefore,been used occasionally as synonyms.

Nf R Neutral fragment reionization. In this process, CADof a mass-selected precursor ion is followed by CIDIof the neutral fragments eliminated from theprecursor ion. Reionization describes a secondionization event, as explained above. The ultimatelyobserved spectrum contains the superimposedNf RCIDI spectra of the neutral fragments liberated uponCAD from the precursor ion under study.

expected from a simple c-H rearrange-CH2xC(OH)2ment. These important Ðndings and other CIDI workby Holmes6,14 and Schwarz and Terlouw5 have beenadequately reviewed. More recently, Feng andLifshitz15 utilized CIDI to show conclusively that themetastable ions of proton-bound formic acid clusters,viz. begin to eliminate(HCOOH)

nH`, (HCOOH)2dimers once n P 6.

CIDI studies have focused on metastable ions under-going one (or two) principal dissociation(s). In suchcases the resulting spectra are simple, allowing oneeasily to identify the corresponding neutral fragment(s).Under CAD, which is most widely used in structuralstudies, several neutral fragments are eliminated simul-taneously. When this mixture is reionized, every speciesin it yields ionic products. Hence, the ultimatelyobserved neutral fragment reionization spectrum(NfR)contains the superimposed collisional ionization (i.e.CIDI, cf. Table 1) spectra of all neutral fragments.3,16h19As will be shown here, such convolution does not com-promise the advantage of directly seeing neutral disso-ciation products. Knowledge of their identity is essentialfor learning how larger ions fragment, which in turn(based on the principle of microscopic reversibility)20also reveals how fragments may combine to form largermoieties.

Neutral fragment reionization has been extensivelyused in our laboratory to examine, inter alia, thedecomposition mechanisms of protonated peptides,3,16fatty acid anions and dilithiated cations17 and proto-

nated polyglycols.18 These speciÐc precursor ions wereprimarily selected because of the uncertainty or contro-versy about the nature of their neutral CAD fragments.Concerning protonated peptides, it has been shown thatbreak-up of the amide bond releases amino acids orsmaller peptides from the C-terminus and aziridinonesor diketopiperazines from the N-terminus.3,16 Regard-ing fatty acid ions, strong evidence has been providedthat their charge-remote fragmentations at keV energiescleave alkenes, not alkanes or alkyl radicals.17 And forprotonated polyglycols, it was found that linear precur-sors decompose by elimination of smaller oligomerswhile cyclic precursors (crown ethers) favor the loss of1,4-dioxane units.18 This paper presents more recentapplications of the method, pertaining to theNf Rdecomposition mechanisms of fullerene ions,19 alkalimetal iodide cluster ions21 and metalated aminoacids.22

Few studies have so far explored the implica-Nf Rtions for molecular structure elucidation of the structur-al information residing in neutral CAD fragments. Wuand Fenselau23 and Cordero and co-workers3,16b reion-ized peptides cleaved from proton-bound dimers andfound that collisional ionization produces sequenceindicative N-terminal ions. Additionally, Squire et al.16cshowed that distinguishes leucine from isoleucineNf Rresidues when they are eliminated from a protonatedpeptideÏs C-terminus. Here, we also discuss how Nf Rcan help characterize protonated nucleotide isomers.24

NEUTRAL FRAGMENT REIONIZATION

involves two sequential collisions. The Ðrst colli-Nf Rsion promotes CAD, giving rise to ionic and neutralfragments (Scheme 2). After removal of the ionic frag-ments and any una†ected precursor ions, the neutralfragments are subsequently ionized in the second colli-sion. This procedure is very similar to that encounteredin neutralizationÈreionization (NR),4h6 where the Ðrstcollision neutralizes the precursor ion and the secondreionizes the intermediate neutral (after deÑection ofnon-neutralized ions), viz. Scheme 2. Whether the Ðrstcollision causes CAD or charge exchange depends, interalia, on the target (see below). Helium has been the pre-ferred CAD gas in studies, because its high ioniza-Nf Rtion energy and negative electron affinity25 minimizethe degree of concomitant charge exchange.26 In con-trast, metallic targets,26 xenon,26 dimethyl disulÐde27and trimethylamine19,28,29 have been found primarilyto e†ect neutralization (i.e. electron transfer) and shouldbe avoided in measurements.Nf R

Scheme 2.

CHARACTERIZATION OF NEUTRAL FRAGMENTS IN MS/MS 1075

Regarding the second, reionizing collision, is theO2most efficient collision gas for the production of cations,causing the least fragmentation.30 We have found tri-methylamine (TMA) or Xe to be adequate choices forgenerating anions.19,31 The collisional ionization yieldsare O2% for cations and O0.1% for anions. Thecharges of the precursor ion and of the ultimate reioni-zation products may be indicated by superscripts to the

acronym. For example, the He/Xe spec-Nf R `NfR~trum of is acquired by dissociating(CsI)3Cs` (CsI)3Cs`cations (with He) and subsequently ionizing the col-lisionally generated neutral fragments to anions (withXe).

in competition with NRNfR

As mentioned above and illustrated in Scheme 2, colli-sion of a mass-selected precursor ion with a gaseoustarget can cause CAD and/or charge exchange ; both ofthese processes create neutral species. In addition,neutral fragments may be formed by the spontaneousdecomposition of remaining metastable ions (i.e. of pre-cursor ions that did not collide but still possess suffi-cient internal energy to undergo a unimolecularreaction). In the Akron tandem mass spectrometer (seeExperimental), collision-induced fragmentation at gaspressures corresponding to P20% beam attenuationgenerally yields [20 times more fragments than doesmetastable ion fragmentation. Consequently, in thepresence of a collision gas, the proportion of metastablygenerated neutral fragments is negligible compared withthose arising by CAD.

In an experiment, the extent of concomitant NRNfRcan be minimized by proper target choice (see above) ;however, it may be impossible to eliminate it com-pletely. Alternatively, some precursor ions may ratherdissociate than undergo charge exchange, even withsuperior neutralization targets. The relative cross-section for neutralization vs. dissociation depends onthe nature of the precursor ion, the thermochemistryand FranckÈCondon factor of charge exchange,26,27 aswell as on other, not completely understood variables.Which product ions in the ultimately observed spec-trum originate from neutral fragment reionization (i.e.from dissociative collisions) and which fromneutralizationÈreionization (i.e. from neutralizingcollisions) can nevertheless be assessed by contrastingspectra taken with He (which primarily dissociates) vis-

trimethylamine (TMA) or Xe (which primarilya-visneutralize). This is illustrated here for two precursorions that exhibit di†erent behavior.

Figure 1 compares the and `NR``Nf R` He/O2spectra of ionized glycine.32 The majorTMA/O2CAD process of this radical cation [[70% of totalfragments, Fig. 2(a)] is the decomposition

(m/z`~H2NCH2COOH]`H2NxCH2 30) ] ~COOH(45 u), which eliminates the carboxyl radical. Since onlyone major neutral fragment is released, the `Nf R`spectrum [Fig. 1(a)] basically represents the CIDI spec-trum of ConÐrmatory evidence that Fig. 1(a)~COOH.contains reionized is provided by the similarity~COOHof the fragmentation patterns in this Ðgure and theCAD spectrum of authentic `COOH [Fig. 2(b)].

Figure 1. (a) and (b) ½NR½ spectra of½NfR½ He/O

2TMA/O

2ionized glycine, (m /z 75), formed by EI.½~H

2NCH

2COOH

TMA ¼trimethylamine. Part (a) essentially represents the CIDIspectrum of (see text).~COOH

Finally, it is pointed out that the spectrum of`Nf R`[Fig. 1(a)] also includes a minor`~H2NCH2COOH

peak at m/z 30 that cannot be formed from reionizedThis ion presumably originates from the minor~COOH.

CAD channel [m/z 45`~H2NCH2COOH] `COOHin Fig. 2(a)], which liberates the radical (30H2NCH2~u).

The `NR` spectrum of ionized glycineTMA/O2[Fig. 1(b)] duplicates all features of the spec-`Nf R`trum, indicating that a substantial portion of the pre-cursor ion undergoes CAD, even with TMA. Inaddition, a recovered molecular ion (survivor ion or

Figure 2. CAD spectra of (a) ionized glycine, ½~H2NCH

2COOH

(m /z 75) and (b) the carboxyl cation, ½COOH (m /z 45), formed byEI of formic acid. served as the collision gas.O

2

1076 M. J. POLCE ET AL .

recovery peak at m/z 75) and additional fragments (e.g.m/z 74, 57 and 30) appear and can readily be explainedas arising from the fraction of glycine ions that wereneutralizedÈreionized ;32 overall, CAD prevails witheither target. For this reason, the `NR` spectrum ofionized glycine is not an adequate reference spectrum ofneutral glycine (see below).

The situation changes with precursor ions.19C60`Now, the and `NR` spectra`NfR` N2/O2 TMA/O2di†er dramatically, cf. Fig. 3 (in this case, was usedN2for CAD because He can also be entrapped in the ful-lerene cage33). The large survivor ion in the `NR`spectrum [Fig. 3(b)] can only result from neutralization.Moreover, the fragment ion pattern in this spectrumclosely resembles that in the CAD spectrum of C60`[Fig. 3(c)],19 indicating that they are formed from disso-ciations of the survivor ion. The small relativeC60`abundance of recovered in the spectrumC60` `Nf R`[Fig. 3(a)] reveals, on the other hand, that targetsN2e†ect little neutralization and that the abundant iongroup extending up to must mainly originateDC28`from the neutral fragments eliminated from byC60`CAD (see below for interpretation). In this case, chang-ing the target has a dramatic e†ect on the competitionbetween CAD and neutralization.

Figure 3. (a) spectrum of The region up to½NfR½ N

2/O

2C

60½ .

m /z B 400 in this spectrum arises from the neutral fragments elimi-nated from upon CAD with The ions above m /z 400 areC

60½ N

2.

due to the small fraction of that underwent neutralization,C60½

instead of CAD, with the targets (see text). (b) ½NR½N2

TMA/O2

spectrum of (c) CAD spectrum of using as the colli-C60½ . C

60½ O

2sion gas. This spectrum contains the ionic fragments generatedfrom was formed by EI in all spectra. Reprinted from Ref.C

60½ , C

60½

19.

Mixtures of neutral fragments

As mentioned above, CAD gives rise to a mixture ofneutral fragments, all of which are reionized concurrent-ly (see entry in Table 1). The interpretation ofNfR Nf Rspectra is, therefore, facilitated if reference reionization(CIDI) spectra of individual neutral fragments areobtained independently. This can be achieved by Ðndingprecursor ions that dissociate to yield just the desiredneutral fragment. SpeciÐc neutrals can be generated bydissociation of metastable ions,5,6,8h14 however, theÑux of metastably generated neutral fragments is notlarge enough in our instrument34 to a†ord usable CIDIspectra. All reference CIDI spectra from our laboratoryhave been measured on CAD-generated neutral losses,from precursors whose CAD liberates mainly oneneutral species. The latter condition is fulÐlled byseveral classes of ions,3,11,16,23,35h38 especially amineradical cations35,36 and proton-bound dimers,3,11,16,23which can be used to prepare individual radicals andmolecules, respectively. For example, we showed abovethat CAD of ionized glycine (i.e. an amine radical ion)supplies radicals of adequate purity [see Fig.~COOH1(a) for the CIDI spectrum of Proton-bound~COOH].dimers can be used analogously, as exempliÐed in Fig.4, which shows the CIDI spectra of glycine (Gly) andalanine (Ala) molecules formed from the dimers GlyÈH`ÈGly and AlaÈH`ÈAla, respectively. The ionspresent in these CIDI spectra also appear in the elec-tron impact (EI) spectra of Gly and Ala39 (insets) andthe CAD spectra of ionized Gly [Fig. 2(a)] and Ala (notshown), as expected. Note, however, that the relativepeak intensities in EI, CAD and CIDI spectra vary,probably because of the di†erent internal energiesdeposited in these processes.40

Figure 4. CIDI mass spectra of (a) 4.0 keV Gly and (b) 4.0 keVAla, eliminated from the proton-bound dimers Gly–H½–Gly andAla–H½–Ala, respectively (formed by FAB). The amino acids wereliberated by CAD with He and reionized by collisions with TheO

2.

insets show EI spectra of Gly and Ala. Reprinted from Ref. 16a.

CHARACTERIZATION OF NEUTRAL FRAGMENTS IN MS/MS 1077

An alternative route to reference collisional ioniza-tion spectra of an individual neutral species is byneutralizationÈreionization (NR) of the correspondingion.3,17 When using this approach, care should be takenthat the NR spectrum is not contaminated by asNf R,was the case for ionized glycine [cf. Figs 1 and 4(a)].Further, the neutral accessed in the neutralization stepmust not decompose before reionization otherwise theNR spectrum represents a neutral mixture, not thedesired species alone. NR can provide reference spectrafor stable molecules, such as alkenes17 or enols,4,32 butis not suitable for weakly bonded species. For example,the `NR` spectrum of `COOH (Fig. 5) is aTMA/O2poor reference spectrum of because, as docu-~COOH,mented by Holmes et al.,11 the neutralization stepdeposits enough energy in the incipient radical to causeextensive dissociation to andCO2] ~H CO] ~OHbefore reionization (Fig. 5). Fortunately, can be~COOHgenerated by a di†erent pathway, namely via CAD ofthe glycine ion, as outlined above [Fig. 1(a)]. If thelatter choice is not available, the EI spectrum of aneutral or the CAD spectrum of its ionized form (oreven similar data for a chemically related species)41 mayprovide some information on what types of productions are to be expected upon collisional ionization ofthis neutral.

How selected reference spectra help in the identiÐca-tion of speciÐc neutral fragments is illustrated for theprotonated dipeptides AlaÈGly and GlyÈAla, whose

spectra are shown in Fig. 6.16a Note`NfR` He/O2that only C-terminal amino acids are eliminated asintact units ;3 thus, [AlaÈGly]H` is expected to loseGly, whereas [GlyÈAla]H` should lose Ala (Scheme 3).Indeed, the spectrum of [AlaÈGly]H` is strikinglyNf Rsimilar to the CIDI spectrum of authentic Gly [cf. Fig.6(a) vs. Fig. 4(a)]. Both spectra display abundant m/z 30

and m/z 45 (`COOH), which are dignos-(`H2NxCH2)tic for the glycine connectivity. In contrast, these frag-ments are insigniÐcant in the spectrum ofNf R[GlyÈAla]H` [Fig. 6(b)] ; this instead includes all pro-ducts present in the CIDI spectrum of genuine Ala [Fig.4(b)], inter alia the structurally indicative immoniumions of m/z 44 and 88(`H2NxCHCH3)Overall, the availability of(`H2NxC(COOH)CH3).reference CIDI spectra (Fig. 4) makes it easier to detectGly and Ala among the neutral CAD fragments from[AlaÈGly]H` and [GlyÈAla]H`, respectively (Fig. 6).Of course, reference CIDI and spectra do notNfRmatch completely, because the latter also contain

Figure 5. ½NR½ spectrum of ½COOH (m /z 45), formedTMA/O2

by EI of formic acid. It resembles qualitatively the ½NR½ Xe/Hespectrum of ½COOH published by Holmes et al .11

spectra of (a) ÍAla–GlyËH½ and (b)Figure 6. ½NfR½ He/O

2ÍGly–AlaËH½, formed by FAB at 8.0 keV. Gly and Ala cleaved fromthese peptide ions have kinetic energies of 4.1 and 4.8 keV,respectively. Reprinted from Ref. 16a.

signals due to all other neutral fragments liberated fromthe protonated peptides.16a

Several variables a†ect the contribution of a speciÐcneutral to an spectrum, including the kinetic andNf Rinternal energy of the species, its structure and thereionization efficiency and internal energy depositionassociated with the target used. Owing to this com-plexity, it is virtually impossible to identify quantitat-ively every neutral fragment generated from a given pre-cursor ion. The power of the method rather lies inNfRthe qualitative detection of certain speciesin neutral CAD mixtures to thereby decipher puzzlingmechanistic (or structural) problems. For compositionalassignments of these mixtures, it is important to recog-nize the following limitations.

Equivalent amounts of di†erent neutral fragmentsmay not contribute similarly to the spectrum. TheNf Rlarger neutral species, which have higher kinetic ener-gies and hence superior transmission and collection effi-ciencies,2,42 usually dominate the ultimately observedspectra.17 A higher translational energy also appears toraise the collisional ionization yield,43 favoring thelarger fragments, but it also increases the internalenergy deposited upon CIDI,40,43 causing more exten-sive fragmentation and product ion overlap. Finally,even if the above parameters are very similar, the ener-getics of dissociation may inÑuence which neutral willdominate after A dissociation with considerableNf R.

Scheme 3.

1078 M. J. POLCE ET AL .

reverse activation energy could produce internallyexcited neutral fragments which, according to studies byHarnish and Holmes,43 have a reduced reionizationcross-section ; this will in turn decrease their contribu-tion to an spectrum.16b Consideration of theseNf Rproblems is essential for the correct interpretation of

data.Nf R

EXPERIMENTAL

involves high-energy collisions. The experimentsNfRdescribed in this study were conducted on a modiÐedVG AutoSpec tandem mass spectrometer,E1BE2housing two collision cells and an intermediate deÑectorin the Ðeld-free region between (MS-1) andE1B E2(MS-2).34 Precursor ions were formed by EI at 70 eV orby fast atom bombardment (FAB) with a Cs` ion gunoperated at D20 keV. They were accelerated to 8 keV,mass selected by MS-1 and subjected to CAD with Heor in the collision cell following the magnet. All ionsN2exiting this cell were removed by electrostatic deÑectionand the remaining beam of neutral fragments was reion-ized in the collision cell preceding The newlyE2 .formed ions were mass analyzed by MS-2 and recordedas the respective spectrum. NR spectra wereNf Racquired similarly by replacing the Ðrst collision gaswith trimethylamine or Xe.

All samples used are commercially available and wereintroduced into the mass spectrometer as received fromthe manufacturer. The spectra shown are multi-scansummations and their relative abundances are repro-ducible to within ^15%. Absolute abundances (i.e. Nf Ryields) were calculated by dividing the abundance of aspeciÐc ion or of the total ions exiting the reionizationcell by the abundance of the unattenuated precursor ionsubjected to CAD. Reionization yields were calculatedby dividing the total ion current emerging from reioni-zation by the Ñux of the neutral fragments (assumed tobe identical with the total fragment ion current in thecorresponding CAD spectrum). Total and reioni-Nf Rzation yields range between D10~6 and 10~4 andbetween D10~3 and 10~2, respectively (^50%).3,16h19The pressure of each collision gas was adjusted toachieve 20% (50% for reduction of the precursorC60Z`)ion abundance. Glycerol, thioglycerol, a 5 : 1 mixture ofdithiothreitol and dithioerythritol and a 1 : 1 mixture ofthioglycerol and 2-hydroxyethyl disulÐde served asmatrices for FAB ionization. To protonate the sample,it was acidiÐed with hydrochloric or triÑuoroacetic acid.To form metal ion adducts, the sample was mixed witha solution of a suitable metal salt in one of the afore-mentioned matrices. The alkali metal iodide clusterswere ionized by FAB without any matrix. Buck-minsterfullerene was thermally desorbed at 375 ¡C fromthe direct insertion probe.

LOSSES FROM FULLERENE CATIONSC2n C60z`

Singly and multiply charged fullerene cations that havebeen supplied with sufficient internal energy for disso-ciation (e.g., upon EI or CAD) primarily undergo

Scheme 4.

nominal eliminations (Scheme 4). These reactionsC2ncould involve n sequential cleavages or the loss ofC2larger, intact units.33,44 Appearance energy mea-C2nsurements cannot help determine the true mechanismowing to the large kinetic shifts associated with C60z`decompositions.44c The problem can, however, be reli-ably solved by neutral fragment reionization, whichascertains directly the identity of the neutral fragments.

CAD of gives rise to an abundant fragment ionC60`series that ranges between and [Fig. 3(c)] andC32` C58`formally arises by the above-mentioned losses.C2nAnother group of fragment ions stretches from toC7`and results, based on studies by Doyle and Ross45C29`and us,19 from consecutive catastrophic dissociations of

which release very small neutrals[C60[ C2n]`, (Cx3).The neutral CAD fragments from lead to theC60`spectrum in Fig. 3(a), which contains`NfR` N2/O2abundant ions extending up to (The higher massC28` .

ions arise, at least in part, from the competitive NRprocess.) Clearly, CAD of with does liberateC60` N2large neutral moieties, which must originate from theprimary cleavages (see above). The dominantC2nproduct ions from reionization of are odd carbonC2nclusters similar to the magic clusters(C11` , C15` , C19` ),found in the laser ablation of graphite.46 This can beexplained as follows : (1) collisional ionization depositshigh average internal energies, resulting in extensivefragmentation of the reionized (2) TheC2n .40 C2nmixture consists of small, even-numbered carbon clus-ters (2n \ 30). Such clusters are known to dissociatepreferably by and losses,47 which would produceC1 C3odd cluster ions from the even-numbered as indeedC2n ,is observed.

The negligible abundance of in the spec-C2` `NfR`trum in Fig. 3(a) does not necessarily rule out the possi-bility of sequential losses. from 8 keV hasC2 C2 C60`only 267 eV of kinetic energy and would therefore besubject to serious transmission and scattering losses (seeabove). In addition, its reionization efficiency can beexpected to be low. In our instrument, mass discrimi-nation e†ects become severe when the translationalenergy drops well below 1 keV.40 To minimize theseproblems, multiply charged (z\ 2È4) were alsoC60z`studied.

The spectra of (z\ 2È4) are`Nf R` N2/O2 C60z`shown in Fig. 7. They all include larger cluster ions,verifying that larger neutral clusters are lost from disso-ciating precursors. As the charge of the fullereneC60z`precursor increases from 2 to 3 to 4, the largest detect-able neutral fragment expectedly decreases (to avoidCoulombic explosion in the remaining fragment ion)from to to [to for z\ 1, cf. Fig.C24 C18 C12 C283(a)].19 It is worth noting that is minuscule in allC2`spectra in Fig. 7, even for which would formC604`, C2with 1.1 keV kinetic energy. Thus, successive losses of

are a minor channel ; their occurrence cannot beC2totally excluded because the detection of such light frag-

CHARACTERIZATION OF NEUTRAL FRAGMENTS IN MS/MS 1079

spectra of (a) (b) and (c)Figure 7. ½NfR½ N

2/O

2C

602½, C

603½ C

604½,

formed by EI. Reprinted from Ref. 19.

ments is problematic (see above). On the other hand,the data provide conclusive evidence that`Nf R` C60z`decomposition to liberates whole units. ThisC

x56z` C2nresult fully supports the fullerene formation mechanismproposed by Bowers,48 according to which fullerenestructures are produced by association of larger carbonclusters. Such an analogy between dissociation andassociation mechanisms is expected, based on the prin-ciple of microscopic reversibility.

EVAPORATION OF OLIGOMERS FROMCLUSTERS(CsI)

xCs‘

A question that is closely related to the one discussedabove concerns the dissociation of cations.(CsI)

xCs`

Upon CAD, these species decompose by nominal CsnIn(n O x) losses,49 which could be intact oligomers(CsI)

nor a mixture of n CsI monomers (Scheme 5). The kineticenergy release distributions for the cleavage of 2 CsIunits from metastable measured by El-Sayed(CsI)

xCs`,

and co-workers,50 were consistent with the Ðssion of a

Scheme 5.

whole dimer rather than the consecutive loss of(CsI)2two CsI molecules. Reaction intermediate scans byDrewello et al.51 supported this supposition. Morerecently, Ñoated collision cell experiments by Luda� nyiand Ve� key52 demonstrated that the losses of andCs2I2from collisionally activated proceed inCs3I3 (CsI)6Cs`one step as well as sequentially ; whether the one-stepprocess involves ejection of n CsI molecules or intact

multimers could not be answered. As will be(CsI)nshown here, neutral fragment reionization provides

strong evidence for the evaporation of complete largeroligomers from energetically excited cluster(CsI)

xCs`

ions.21CAD of with He leads to the elimination of(CsI)3Cs`

up to three CsI units (Fig. 8). Reionization of the so-liberated neutrals to cations (with or anions (withO2)Xe) gives rise to the spectra in Fig. 9. Both of`NfR`,~these spectra contain ions heavier than CsI`~/CsI~~,indicating that oligomers (i.e. complete moieties)(CsI)

w2

Figure 8. CAD spectrum of formed by FAB. served(Csl)3l½, O

2as the collision gas.

Figure 9. (a) and (b) He/Xe spectra of½NfR½ He/O

2½N

fRÉ

formed by FAB. The neutral losses from this precursor(Csl)3Cs½,

ion have been reionized to (a) cations with or (b) anions withO2

Xe.

1080 M. J. POLCE ET AL .

are evaporated from the collisionally activatedprecursors. Notice that the largest cation(CsI)3Cs`

observed in the spectrum [Fig. 9(a)] has three`Nf R`cesium atoms, whereas the largest anion observed in the

spectrum [Fig. 9(b)] has three iodine atoms.`NfR~Positive and negative products emerge from the sameneutral mixture. Consequently, the biggest neutral mol-ecule in this mixture possesses three cesium and threeiodine atoms and must be an intact trimer. Appli-(CsI)3cation of to precursors (spectra notNfR (CsI)2I`shown) similarly reveals that they lose complete (CsI)2dimers during CAD. It is therefore plausible that alsothe loss of two CsI molecules from which gives(CsI)3I`,rise to the CAD base peak in Fig. 8, involves the Ðssionof a dimer.(CsI)2The only radical ions present in Fig. 9 are andCsI`~

Apparently, collisional ionization of andCsI~~. (CsI)3favors the formation of closed-shell fragments(CsI)2 (in the positive mode) or (in the(CsI)yCs` (CsI)

yI~

negative mode). The radical ions observed presumablydo not originate from reionization of the and(CsI)3oligomers but from reionization of CsI, which is(CsI)2eliminated upon CAD, too (Fig. 8). In addition, anystepwise elimination of or generates CsICs2I2 Cs3I3monomers,52 thereby contributing to the andCsI`~

peaks. The small relative abundances of the latterCsI~~ions in Fig. 9 suggest, however, that the sequentiallosses are of lesser importance compared with the con-certed evaporation of whole dimers and trimers.

Even larger oligomers are cleaved from larger clusterions. and are found to lose intact(CsI)4Cs` (NaI)4Na`tetramers, i.e. and respectively, and the(Csl)4 (Nal)4 ,heaviest oligomer detected so far is the pentamer (Csl)5from precursor ions.21 The preference for(Csl)6Cs`elimination of intact clusters instead of a mixture ofmonomers could be the result of a lower energy require-ment for the former process which produces thermody-namically more stable neutral molecules (see below).

Theoretical calculations by Welch et al.53 indicatethat the most stable conÐgurations of and(Csl)2 (Csl)3have the connectivities of a rhombus and a hexagon,respectively, as depicted in Scheme 6. In these geome-tries, the binding energy relative to the separated mono-mers is 1.59 eV for and 2.69 eV for(Csl)2 (Csl)3 .53Parallel characteristics are predicted for sodium iodidedimers and trimers.53 Scheme 6 includes two likelystructures of cations, as proposed by Morgan(Csl)

xCs`

et al.54 and resembling a distorted cube and an elon-gated dodecahedron, in which alternating Cs` and l~form closed lattice-like surfaces. Since the faces of thelatter structures have the shapes of rhombuses and/orhexagons, the evaporation of intact dimers or(Csl)2trimers (or even larger oligomers) could progress(Csl)3by the rupture of complete faces from the (Csl)

xCs`

cluster. This should be true for and other(Nal)xNa`

Scheme 6.

alkali metal halide cluster ions, too, all of which havebeen postulated to possess equivalent structures.54,55 Inreverse, ionized alkali metal iodide clusters may beformed by the addition of complete faces, not just con-secutive attachment of monomers.

Na‘- vs. Cu‘-CATALYZED FRAGMENTATIONOF GLYCINE AND ALANINE

Collisional activation of the Na` adduct of glycine,[Gly] Na]` (m/z 98), predominantly leads toGly] Na` (m/z 23 ; [50% of total fragment ionÑux).22 The corresponding spectrum (Fig. 10) is`NfR`very similar to the ClDl spectrum of authentic glycine[Fig. 4(a)], conÐrming that an intact H2NCH2COOHmolecule (75 u) is eliminated from the activated[Gly] Na]` precursor ion. The peak resolution isnoticeably better in the than in the reference`Nf R`ClDl spectrum [cf. Figs 10 and 4(a)]. This, and thesmall di†erences in the relative abundances in the twospectra, can be accounted for by the higher kineticenergy of Gly generated from [Gly] Na]` vs. GlyÈH`ÈGly (6.1 vs. 4.0 keV, respectively).

The copper(l) attachment ion of glycine, [Gly] Cu]`(m/z 138 for 63Cu), also yields upon CAD the metal ion,viz. Cu` (m/z 63), albeit to a smaller extent [ca. D30%of the total fragment ion Ñux ; Fig. 11(a)].22 However,now, the corresponding spectrum [Fig. 12(a)]`Nf R`indicates that the accompanying neutral loss is not anintact Gly molecule ; this is attested to by the absence ofa recognizable (m/z 75) in Fig.[H2NCH2COOH]`~12(a) and the overall incompatibility of the `Nf R`spectrum with the reference ClDl spectrum of pure Gly[Fig. 12(a) vs. 4(a)]. Apparently, Cu` catalyzes reactionsthat destroy the glycine frame, in sharp contrast to Na`(see below).

Another unique fragmentation of [Gly ] Cu]` is thegeneration of at m/z 92 [Fig. 11(a)], for-HNCH2Cu`mally emerging by cleavage of a neutral with mass 46 u

Expectedly, the perdeuterated isotopomer(H2CO2). loses 48 u to produce m/z 95[d5-Gly ] Cu]` (D2CO2)cf. Fig. 11(b). Studies by Bouchonnet et(DNCD2Cu`),al.,56 Wen et al.57 and Lei and Amster58 have shownthat the elimination of (“elements of formicH2CO2acidÏ) is a common feature of the Cu` adducts of most

spectrum of ÍGly ½Na˽, formed byFigure 10. ½NfR½ He/O

2FAB at 8.0 keV. This spectrum is strikingly similar to the CIDIspectrum of 4.0 keV Gly depicted in Fig. 4(a). Note that Gly rel-eased from ÍGly ½Na˽ has 6.1 keV kinetic energy.

CHARACTERIZATION OF NEUTRAL FRAGMENTS IN MS/MS 1081

Figure 11. CAD spectra of (a) ÍGly ½63Cu˽ (m /z 138) and (b)(m /z 143), formed by FAB. was used as theÍd

5-Gly ½63Cu˽ O

2collision gas. The narrow peak at m /z 46 (which disappears in the

or the 65Cu adducts) is probably a matrix-related iso-d5-sample

baric impurity dissociating to m /z 46 plus glycerol.

a-amino acids. Several di†erent molecules are possibleas the neutral fragment(s) in this process, includingHCOOH (formic acid), (dihydroxycarbene),C(OH)2 H2or a combination thereof. Here,] CO2 , H2O ] COthis uncertainty is partially resolved by Nf R.

The recently reported neutralizationÈreionization(`NR`) mass spectra of andHCOOH`~ `~C(OH)2 ,59show that collisional ionization of HCOOH and

gives rise to abundant survivor ions (m/z 46).C(OH)2

spectra of (a) ÍGly ½63Cu˽ and (b)Figure 12. ½NfR½ He/O

2formed by FAB. These spectra arise from theÍd

5-Gly ½63Cu˽,

neutral fragments co-produced with the ionic fragments shown inFig. 11.

However, the spectrum of [Gly ] Cu]` does`Nf R`not contain any measurable ions at m/z 46 [Fig. 12(a)].Similarly, there is no detectable m/z 48 in the `Nf R`spectrum of cf. Fig. 12(b). Hence[d5-Gly] Cu]`,neither formic acid nor dihydroxycarbene is eliminated.

Regarding the alternative neutral losses, viz.H2CO2and characteristic ionsH2] CO2 H2O] CO, `Nf R`are observed only for their heavier components ; theseappear in Fig. 12(a) at m/z 44 and 28(CO2`~) (CO`~).Their presence is substantiated by the spectrum`NfR`of [Fig. 12(b)] which includes clearly[d5-Gly] Cu]`resolved peaks for (m/z 44) and (m/z 28).CO2`~ CO`~The lighter constituents of the above H2CO2 (D2CO2)neutral products, viz. and are dis-H2(D2) H2O (D2O),criminated against by more severe scattering losses andpoorer reionization and transmission efficiencies (seeabove), which obstruct their detection and can thusexplain their negligible contribution to the `Nf R`spectra (water gives just a trace signal). With consider-ation of this limitation, it is concluded that the neutralfragment of mass 46 u is eliminated in the form of H2and/or] CO2 H2O] CO.

Being a relatively soft Lewis acid, Cu` presumablyattaches close to the soft amino group of glycine (a inScheme 7) ;60 additional coordination by the carboxylsubstituent is plausible.56

Scheme 7.

A hint as to what happens to this structure when col-lisionally activated is given by the neutral fragmentsthat can be identiÐed by The heaviest organic`Nf R`.(i.e. non-copper containing) neutral lost from[Gly] Cu]` is the carboxyl radical, (45 u),~COOHwhich after reionization gives rise to a recognizablesignal at m/z 45 [Fig. 12(a)] ; similarly, [d5-Gly ] Cu]`cleaves [46 u, cf. Fig. 12(b)]. The residual frag-~COODment of glycine, viz. of 30 u (or ofH2NCH2~ D2NCD2~34 u) is also present in the neutral loss mixture, asattested by the observation of m/z 30 (or 34) in Fig.12(a) (or 12(b)). These results strongly suggest thatcopper(I) catalyzes the cleavage of the CaÈCOOHbond, possibly by insertion,61 as shown in Scheme7.56h58 In the resulting intermediate b, the glycine con-nectivity is destroyed, consistent with the absence ofintact Gly molecules in the neutral fragment mixturefrom [Gly] Cu]` (see above). SigniÐcant CAD frag-ment ions that can be rationalized by direct cleavagesfrom b are (i) [m/z 30 in Fig. 11(a)], (ii) Cu``H2NCH2(m/z 63) and (iii) `CuCOOH (m/z 108). The complemen-tary neutral losses are the aforementioned radicals (i.e.

and and Cu (63 u), all of which are~COOH H2NCH2 Æ)detected in the spectrum [Fig. 12(a)]. Further,`NfR`as has been proposed by many other researchers,56h58simple hydrogen or hydroxyl rearrangements in ion bcan account for the other products observed in CADand spectra, inter alia (m/z`Nf R` HNxCH2 É É É Cu`92 ; loss of or andH2O ] CO H2] CO2) Cu(OH2)`(m/z 81 ; loss of The heaviest neutralHNxCH2] CO).fragment observed is CuOH (80 u)/CuOD (81 u), cf. Fig.12. Such a molecule can be envisioned as arising from b

1082 M. J. POLCE ET AL .

by OH~ transfer to Cu` and cogeneration ofReionization of CuOH/CuOD`H2NxCH2] CO.

gives rise to and CuO` (m/z 79) andCuOH`~/CuOD`~presumably also is a partial source of Cu` (m/z 63) inthe spectra.`Nf R`

Competitive pathways that disrupt the glycine struc-ture are conceivable. For example, transition metal ionsare known to increase the acidity of amines in solu-tion.62 Proton transfer from the amino to the carboxylgroup of Gly could then be facile, yielding the ionÈmolecule complex c as depicted in Scheme 8. Someimportant CAD fragments that can be formed from c byligand evaporation are (m/z 92)HNxCH2 É É É Cu`

and (m/z] CO] OH2 Cu(OH2)` 81)] COother products, such (m/z] HNxCH2 ; `H2NxCH230) ] CO] CuOH, could emerge via proton transfer

reactions within c.

Scheme 8.

Finally, the m/z 42 ion in the spectrum of`Nf R`[Gly] Cu]` is probably (ionizedCH2xCxO`~ketene). With this ion shifts to m/z 44(d5-Gly ] Cu]`,and overlaps with (Fig. 12). Ketene loss suggestsCO2`~that not all fragmentations of [Gly ] Cu]` cleave theCaÈCOOH bond. could be eliminated,CH2xCxOtogether with or upon generation ofH2N~ ~OH,

(m/z 80) or (m/z 79), respectively.CuOH`~ CuNH2`~Alanine completely parallels glycine in its reactivity

towards Na` vs. Cu`. Thus, CAD of [Ala] Na]`mainly yields Na` by liberation of a whole alanine mol-ecule ; this is conÐrmed by the striking similarity of the

spectrum of [Ala ] Na]` (Fig. 13) with the ref-`NfR`erence ClDl spectrum of authentic Ala [Fig. 4(b)]. Incontrast, collisionally activated [Ala] Cu]` does noteliminate an intact alanine unit upon dissociation togive Cu`. In analogy with [Gly ] Cu]`, the copper(I)adduct of Ala shows an abundant loss of (46 u)H2CO2in its CAD spectrum and signiÐcant CO, asH2O, CO2well as CuOH losses in its spectrum. Taking`NfR`the dissociation behavior of [Gly] Cu]` (or[Ala] Cu]`) in reverse, copper(I) complexed withappropriate ligands (e.g. an imine, water and carbonmonoxide) may catalyze reactions among the ligands

spectrum of ÍAla ½Na˽, formed byFigure 13. ½NfR½ He/O

2FAB at 8.0 keV. This spectrum is strikingly similar to the CIDIspectrum of 4.0 keV Ala depicted in Fig. 4(b). Note that Ala rel-eased from ÍAla ½Na˽ has 4.7 keV kinetic energy.

that ultimately produce an amino acid. This hypothesiscould be tested by studying the ionÈmolecule reactionsbetween Cu` and suitable molecules.63

STRUCTURAL INFORMATION FROMNEUTRAL LOSSES

Certain isomeric ions show only minor di†erences intheir CAD spectra, because they mainly decompose tocommon fragment ions by elimination of di†erent neu-trals.34 This is true for the [M] H]` cations fromisomeric mononucleotides, whose major CAD channelyields the protonated nucleobase by elimination of dif-ferent sugar residues, as exempliÐed for the cytidine-monophosphates in Scheme 9.64 In this and in relatedinstances, characterization of the neutral CAD frag-ments may provide useful structural insight.

Scheme 9.

Owing to the relatively high mass-to-charge ratio ofthe precursor ions (324), the peak resolution in theresulting spectra is poor (cf. Fig. 14) ; nonethe-`Nf R`less, a number of structurally diagnostic features can bespotted. Qualitatively, the spectra of [2@-CMP]`Nf R`H` and [3@-CMP]H` are similar to each other anddi†er markedly from the spectrum of [5@-CMP]H`. Thedi†erences can be explained by assuming that theH-atom transferred to form the protonated nucleobaseoriginates from the acidic phosphate group. Then, theneutrals liberated from the three isomeric precursor ionswould have the bicyclic structures 1È3 shown in Scheme9. As has been mentioned, collisional ionization depos-its high average internal energies ;40,43 it is therefore notsurprising that the molecular ions of these multi-substituted neutral losses (m/z 212) are not observed. Allthree spectra are dominated by P-containing`Nf R`fragments, viz. (m/z 99, protonated phosphoricH4PO4`acid) and (m/z 81). The largestH2PO2` `Nf R`product of 1 from [2@-CMP]H` and 2 from [3@-CMP]H` appears at m/z 181 and corresponds to the loss of31 u, consistent with the presence of a hydroxymethylgroup (31 u) in these two molecules. Consecutive disso-ciations from m/z 181 can account for other high-massproducts in the reionization spectra of 1 and 2, as

CHARACTERIZATION OF NEUTRAL FRAGMENTS IN MS/MS 1083

of ÍM½H˽ from isometric cytidine-Figure 14. ½NfR½ He/O

2monophosphates, formed by FAB. (a) 2¾-CMP, (b) 3¾-CMP and(c) 5¾-CMP. The major neutral losses from these precursor ions are(a) 1, (b) 2 and (c) 3 (cf. Scheme 8).

shown in Scheme 10. Note that there is a signiÐcantdi†erence between 1 and 2 in the abundance ratio [m/z122] : [m/z 123], which is ?1 for 1 but \1 for 2(appraised by overlaying the spectra). A stable cyclicm/z 122 (Scheme 10) can arise via simple bond cleavagesonly from 1, justifying the uniqueness of this product forthe vicinal phosphate 1. Compared with 1 and 2, isomer 3from [5@-CMP]H` bears no hydroxymethyl substituentand yields no m/z 181 or any of the fragments proposedto result from m/z 181 (Scheme 10). The absence ofhigher mass ions in the reionization spectrum of 3 sug-gests that this neutral su†ers more extensive fragmenta-tion upon collisional ionization (ClDl), possibly owingto increased ring strain.

Scheme 10.

Finally, the ion of m/z 111 appears with higher rela-tive abundance in the spectrum of [5@-CMP]H` than inthe other two spectra Fig. 14(c) vs. Fig. 14(a) and (b).This product corresponds to the molecular ion`Nf R`of the nucleobase (cytosine), which is also eliminated to

a small extent from the collisionally excited [M] H]`precursor ions (\10% of the major channel which isformation of the protonated nucleobase by loss of 1È3,cf. Scheme 9). The reason for the higher abundance ofm/z 111 from [5@-CMP]H` is not known.

Based on the data discussed, the most usefulstructural information is provided by the principalneutral losses 1È3 ; the neutral product from [5@-CMP]H`, i.e. 3, is readily distinguished from its isomers 1 and2. On the other hand, 1 and 2 behave more similarlyupon reionization but can still be di†erentiated usingthe m/z 122 product which is diagnostic for 1 (seeabove). Overall, the knowledge gained by com-NfR,bined with the small di†erences observed in the CADspectra,64 can help assign the correct nucleotide struc-ture with increased conÐdence.

OUTLOOK

Neutral fragment reionization makes it possible to “seeÏwhat neutral species are eliminated from activated pre-cursor ions. Characterization of these fragmentssupplies information that is essential in establishingcorrect decomposition mechanisms, as has been demon-strated here for a variety of systems. Understanding dis-sociation mechanisms both aids spectral interpretationand structural assignments and gives clues on how thesame ions may be formed by association of smaller moi-eties. Neutral fragment identiÐcation also providesstructural information on the dissociating precursorion ; the latter aspect is particularly important whenstructural characterization based on ionic fragments,which is the traditional MS/MS approach, is ambigu-ous or difficult.

These capabilities are currently accompanied by a fewweaknesses. SpeciÐcally, (i) collisional reionization has alow yield (see Experimental) and produces many frag-ments from each neutral loss. For certain neutralspecies, it mainly leads to low-mass products and weak,if any, molecular ions, as is the case with amino acids(Fig. 4) and the glycophosphates 1È3 (Fig. 14). (ii) Theproduct ions from di†erent neutrals may overlap,thereby complicating spectral interpretation. (iii) Lightneutral losses are more difficult to detect than equiva-lent amounts of heavier neutrals. Generally, the largerneutral fragments dominate spectra, because theirNf Rhigher kinetic energies equip them with superior reioni-zation cross-sections and transmission and collectionefficiencies. (iv) The spectral resolution is limited if thereionization products are separated by an electrostaticanalyzer (ESA), as has so far been the case in the mostCIDI and studies.Nf RThe above deÐciencies can adequately be overcomeby a softer reionization method and instrumental modi-Ðcations. A technique that forms one ion per neutral(preferably the molecular ion) would permit the identiÐ-cation of the neutral fragments directly from their mass-to-charge ratios. Chemical reionization is anappropriate alternative.65 It requires that the neutralsmove slowly and would, therefore, be ideally suitablefor tandem quadrupole mass spectrometers ; also, such

1084 M. J. POLCE ET AL .

instruments would not discriminate against light neu-trals.2,27,36h38 With sector instruments, chemical reioni-zation could be achieved by decelarating the precursorions before dissociation, so that they yield slow neutralfragments.65 Resonance-enhanced multiphoton ioniza-tion represents another soft reionization method andmight be useful for the selective and sensitive detectionof a speciÐc neutral fragment ; with pulsed lasers, thismethod would require phase-sensitive or array detec-tion.66 The other major obstacle in studies, namelyNf Rthe poor resolution, can be alleviated by linked scan-ning if a further mass-analyzing device, for example aquadrupole, is attached to the simple electrostaticanalyzer currently used for product separation.23Nf RSatisfactory answers to these problems would bothextend the applicability of neutral fragment reionizationto larger precursor ions and also substantially simplifythe interpretation of data. The promise ofNf R Nf Rrelies on the fact that there is a vast array of precursorions (besides those presented here and in earlier reviews)that await direct characterization of their neutral frag-ments, because they either decompose via mechanisms

not yet clariÐed or yield an insufficient number of ionicfragments for complete structure or sequence diagnosis.Current studies in our laboratory are aimed at estab-lishing whether protonated peptides can lose their N-termini as oxazolones ;67 this important question hasarisen after the N-terminal sequence ions were recentlyshown to be protonated oxazolones.68 Other systemsworth investigating are sodiated oligopeptides (theyundergo puzzling eliminations at the C-terminal aminoacids),69,70 lithiated saccharides (their neutral fragmentsremain unknown),71,72 peptide complexes with tran-sition metal ions (they often produce incompletesequence ion series)73,74 and various types of clusterions (to elucidate on their evaporation mechanisms).75

Acknowledgement

This work was supported by the National Institutes of Health, theOhio Board of Regents and the University of Akron. We thankJinnan Cai, Dr Marcela M. Cordero, Blas A. Cerda and Kevin J.McHale for their experimental assistance and the reviewers for theirhelpful suggestions.

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