2 Physical Methods and Techniques Part (ii) Mass Spectrometry By C. P. BALL and P.J. DERRICK Department of Chemistry University of Warwick Coventfy CV4 7AL UK 1 Introduction Mass spectrometry (MS)is currently enjoying a period of rapid expansion due in large part to the developments in methods of creating gaseous ions from complex molecules and molecular assemblies. Applications of mass spectrometry in structural biochemis- try are attracting a great deal of attention in particular those made possible by the advances with electrospray and Fourier transform ion cyclotron resonance (FT-ICR). The possibilities of studying non-covalent interactions between proteins and substrates are widely discussed and there have been examples of such interactions apparently surviving intact the transition from solution to vacuum (vide infra).Matrix-assisted laser desorption/ionization (MALDI)Is of great current interest and there is again an exciting possibility that through MALDI mass spectrometry will come to make significant contributions in the field of polymer science. On the more physical side of the subject the advances are in the sophistication of experiments to study the behaviour of gaseous ions. The complexity of the systems which can be studied increases steadily and the level of detail provided by spectroscopic and ion-molecule experiments steadiiy rises. Meanwhile gas-phase ion chemistry has truly come of age and knowledge from mass spectrometry on organic mechanisms and metal-organic interactions does now have relevance far beyond the bounds of mass spectrometry.2 Physical Organic Chemistry Mass spectrometry remains a wideiy-used method for the elucidation of organic reaction mechanisms particularly if short-lived intermediates are involved. Increasing- ly isotopically labelled compounds are studied nut simply to ‘tag’ particular atoms in an ion but so as to make use of kinetic isotope effects to elucidate reaction mechanism.’ Distonic ions and ion-molecule complexes have become more firmly established as important concepts in gas-phase ion chemistry over the last two years. ZeIler et a1.2 have found that long-lived radical cations of simple organophosphate esters isornerize spontaneously to more stable distonic structures in the gas phase. On the basis of cullision-induced dissociation experiments Bowen and Wright3 proposed that the P.C.Burgers C.A. Kingsmilf G.A. McGibbon and J. K. Terlouw Org. Mass. Spectrom. 1992,27 398. L. Zeller J. Fartell Jr. H.I. Kenttamaa and P. Vainiotalo J. Am. Chem. SOC.,1992 114 1205. R.D. Bowen and A. D. Wright J. Chem. Soc. Chem. Commun. 1992 96. 21 C. P. Ball and P. J. Derrick crucial step in alkyl radical loss from ionized pentenyl and hexenyl methyl esters involves a 1,Zhydrogen shift at the radical site of a distonic ion followed by y-cleavage of the resultant ionized enoI ether. The loss of water from ionized ethyl furmate produced4 the rarely seen ion -CH,CH,CO+. The distonic ion *CH,OSi* was shown to be stable by Srinivas et ~l.,~ the ions being generated by electron-impact ionization of Si(OCH,),.Holmes and coworkers" have shown that intramolecular hydrogen bonds in /?-diols and b-hydroxy ethers are not preserved in their molecular ions. The fragment ions of metastable formyl loss from ionized ethylene glycol and 2-methoxyethanol have been shown to be the conjugate acids of methanol and dimethyl ether re~pectively.~ Mechanisms involving ion-neutral complexes were ruled out and it was concluded that distonic ions were the intermediates. A new isomer of dimethyl propyl phosphate was synthesized by transfer of ionized ethylene to trimethylphos- phate in the gas phase,* and it was proposed that this ion has separate radical and charge sites. The distonic radical cation CSH5N'-C'CH2 has been generated by reacting pyridine with the radical cations of cyclopropane ethylene oxide and ketene.This species transfers (CH,)" to more nucleophilic substituted pyridine~.~ The 8-distonic ion formed by the reaction between pyridine radical cation and ethene behaves differently. It can Iose C,H by coIlisionaI activation or can ionize again giving a doubly-charged ion. This 8-distonic ion requires collisional stabilization to be visible. The ortho effects in collision-induced dissociation of closed-shell aromatic ions has been examined using both quadrupole ion traps and triple quadrupole mass spectrometers.' The meta and para isomers were found to have qualitatively similar dissociation pathways whereas the ortho isomers dissociated differently.Several isomers of C,H,O+ and C,H,S * ions were distinguished using ion-molecule reactions with isoprene and methylanisoks to show that the ions have different reactivities depending on the precursor molecule from which they are generated. l2 Epimers of methylcyclohexanol were distinguished on the basis of their relative gas-phase acidities which were obtained using a kinetic method based on the relative rates of competitive fragmentation of cluster ions.' McCarley and Brodbelt have studied the behaviour of 174-benzodiazepinsin ion-molecule reactions using a quadrupole ion trap. The reactions with dimethyl ether ions give selectively formed adducts which can be used to diagnose the benzodiazepin ~tructure.'~ Huang and Dunbar" studied the breakdown pattern of n-heptane ions by photodissociation and discovered that the m/z 71 and 43 ionabundanceswere strongly dependent upon the internal energy of the parent ion.Photodissociation and photoionization techniques have been employed very successfully to elucidate not only C.E. Hudson and D.J. McAdoo Org. Mass. Spectrom. 1992,27 1384. R. Srinivas D. K. Bohme 1. Hrusak D. Schroeder and H. Schwarz J. Am. Chem. Soc. 1992,114,1939. J. R.Cao,M.Georgqf. L. Holmes M. Sirios,J. K.Terlouw and P.C. Rurgers,J. Am. Chem.Soc. 1992,114 2017. ' H.E. Audier A. Milliet D. Leblanc and T.H. Morton J. Am. Chem. Soc. 1992 114 2020. L.K. M. Kiminkinen K. G. Stirk arkdH.I. Kenttamaa J. Am. Chem. Soc. 192,114 2027. S.J.Yu and M.L. Gross @rg.Mass. Spectrom. 1993,4 117.S.J.Yu C.L. Holliman D. L. Rempel and M.L. Gross J. Am. Chem. SOC. 1993 115 9676. T. Donovan and J. Brodbelt Org. Mass. Spectrom. 1992 27,9. l2 M.N. Eberlin T.K. Majumdar and R.G. Cooks,J. Am. Chem. SOC. 1992,114,2884. l3 T. K. Majumdar F. Clairet J.C. Tabet and R.G. Cooks J. Am. Chem. Soc. 1992 114,2897. l4 T. D. McCarley and J. Brodbelt Ad. Chem. 1993,65,2380. S.F. Huang and R.C. Dunbar Int. J. Mass Spectrom. Ion. Processes 1992 112 101. Physical Methods and Techniques -Part (ii) Mass Spectrornefry 23 fragmentations of ions but also of neutrals such as alkenes and dienes.'6-17 Energy randomization in the benzene dimer ion has been studied by Boernson and Se1zle.l' Employing isotopically mixed benzene dimers it was demonstrated that charge and energy can be totally shared.At higher energies fragmentation can however occur without communication between the two dimer halves. The kinetic energy releases of the photodissociation processes C,H,SH + hv (193nm) +C,H + SH and C,H,SCH + hv (193nm) +C,H + SCH and C,H,S + CH were measured by a time-of-flight mass spectrometric method; values for the dissociation energies of the C,H,-SH C,H,-SCH, and C,H,S-CH bonds at 0 K were obtained.'' The boryliurn ions (CH,OBOCH,)+ and (CH,BCH,)+ studied by dual-cell FT-ICR showed an unusual bimolecular reaction involving the facile abstraction of a water molecule from an organic ether." Ruscic and Berkowitz have studied the products of the reactions of fluorineatoms with MeSH by photoionization mass spectrometry.They found adiabatic thresholds at m/z 47 of 7.536 f0.003 eV and 9.262 f 0.005eV identified as the adiabatic ionization energies of CH,SH and MeS respectiveIym2 Gable Phan and Griffin have generated the iodiranium ion by gas-phase reaction of ethylene with positive ions generated from The unimolecular chemistry of methyl formate cation radical has been studied by Heinrich et aLZ3 The mobility of t-butyl ion within t-butyI substituted diphenylalkanes has been investigated by Kuck and Matthia~.*~ Metastable protonated 4-Bu'C,H4CH,Ph lost isobutane following the abstractionof a hydride by the t-butyl ion from the benzylic C-H bonds in a n compkx. Protonated 4-Bu'C,H,(CH2),Ph did not involve free t-butyl ions but did involve an ion-neutral complex.McGibbon et aL2' have determined stabilization energies of a-and fl-silyI substituents of vinyl cations by measuring the appearance energm for metastable peaks for loss of I' from Me,SiCI=CH and (E)-Me,SiCH=CHI. Enantioselectivity in fast atom bombardment (FAB) MS was established by using a complex of a selected aldohexose derivative with an enantiomeric pair of naphthyl ether ammonium ions.' The selectivity was detected and evaluated from the relative intensities of the 1 :I adduct ions (formed with an internal standard of 12-crown-4). Similar results were seen using a crown ether host with an ammonium ion guest. It has been claimed that the absolute configurations of a sample molecule can be predicted by examination ofthe FAB mass spectra of the sample measured with reagents of R and S c~nfiguration.'~ The proton affinities of 21 amines have been calculated using data from collision experiments using a hybrid tandem mass spectrometer.These results can be used to estimate the proton affinities of amino acids aside from those containing a l6 S.E.Van Bramer and M.V.Johnston,Org. Mass. Spectrom. 1992,27,949. l7 S. E. Van Bramer P. L,Ross and M. V. Johnston J. Am. SOC.Mass Spectrom. 1993 4,65. K.0.Boernsen and H. L. Selzle Chem. Phys. Lett. 1992,190,497. I9 S. Nourbakhsh H. M. Yin,C. L. Liao and C.Y. Ng Chem. Pltys. Letr. 1992 190,449. T. D. Ranatunga and H.I. Kenttamaa 1.Am. Chem. Soc. 1992 114,8600. B. Ruscic and J. Berkowitz J. Cltem. Phys. 1992.97 1818. 22 K.P.Gable T.N.Phan and D. Griffin Org. Mass.Spectrom. 1993 28 397. 23 N. Heinrich T. Drewello P.C. Burgers J. C. Morrow 3. Schmidt W. Kulik J.K. Terlouw and H. Schwarz J. Am. Chem. Soc. 1992,114 3776. *' D. Kuck and C. Matthias J. Am. Chem. Soc. 1992 114 1901. 25 G.A. McGibbon M. A. Brook and J. K. Terlouw J. Chem. SOC.,Chem. Commun. 1992 360. z6 M. Sawada M. Shizuma Y. Takai H. Yamada T. Kaneda and T,Hanafusa J. Am. Chem. Soc. 1992,114 w5. " H. Yang and Y. Chen Org. Mass. Spectrom. 1992 27 736. 24 C. P. Ball and P. J. Derrick functional group capable of stabilizing a protonated species by hydrogen bonding.28 The question of whether the McLafferty rearrangement of ketones is concerted or stepwise has been addressed by Bowie Derrick and coworkers,29 using intramolecular I3Cand 2Heffects They conclude that the isotope effects observed are consistent with a stepwise mechanism with more than one rate-determining step.Ionized 4-rnethoxyheptane exhibits just two significant fragmentations which consist of hydrogen transfer steps with interesting site ~electivity.~' Ketene has been discovered to dirnerize in the gas phase when ionized by low-energy charge exchange.31 Morton has addressed the reorientation criterion with regards to positive ion-neutral Ion-neutral complexes have been convincingly invoked in quite a number of mechanistic st~dies.~~~'~ Morton postulates two categories of gaseous ion-neutral complexes potential energy barriers prevent the separation and collapse oftype 1 complexes but only an entropic barrier prevents the collapse of a type 2 complex.Isotopic labelling has shown that the dissociation of the metastabk ion AcCH2CO+ to produce the Ac' ion is preceded by an atom exchange involving an ion-neutral ~ornplex.'~ An ion-neutral complex intermediate has been postulated for the loss of water from fatty acid carboxyhtes by Jensen Haas and Gross.36Fatty acid carboxylates formed by FAB have been found to undergo both metastable ion and collisionally activated dissociation (CAD) to eliminate water. Upon CAD the water elimination competes with charge-remote fragmentation and deuterium labelling suggested that the elimination proceeds uia an ion-neutral complex. Mass-analysed ion kinetic energy spectroscopy (MIKES) has been used to examine the unimolecular reactivity of the protonated forms of EtCN and EtNC in the gas phase.Both species were found to isomerize into weak n complexes between ethene arid CH=NH +,which may then dissociate or interconvert. The 1,2 elimination processes proposed may require the intermediacy of loosely bound ion-neutral [M -HI -ions prepared from 17#?-oestradiol- 17 fatty acid esters by negative-ion chemical ionization have exhibited interesting low collisional energy processes.38 Both phenoxide and enolate forms of the [M -HI-were seen. Evidence was produced for molecular isomerizations into iondipole complexes. The mechanism of [IH,C,N]loss from metastable molecular ions of benzene cyanides containing F CI CF, CH, OH or CH,O substituents has been investigated by deuterium and I3C labelling.39 The eliminations from the F Cl or CF,-substituted species was found to involve a randomization of the hydrogen atoms and a minor rearrangement of the CH,CN function causing expulsion of a benzylic carbon 2&30% of the time.When the substituents are CH, OH or CH30 there appears to 28 X. Li and A.G. Harrison Org. Mass. Spectrom. 1993 28,366. 29 M. B.Stringer D. J. Underwood J.H. Bowie C. E. AJIison K. F. Donchi and P.J. Derrick Org. Mass. Spectrom. 1992 27 270. 30 R.D. Bowen and P. J. Derrick 1.Chem. Soc. Perkin Trans. 2 1992 1041. 31 C.H. Dass Rapid Commun. Mass Spectrom. 1993 7 95. 32 T.H. Morton Org. Mass. Spectrom. 1992 27 353. 33 V. Nguyen X. Cheng and T.H. Morton J. Am. Chem. SOC.,1992 114 7127. 34 M. Gu and F. Turecek J. Am. Chem.SOC.,1992 114 7146. 35 J. Tortajorda D. Berthomieu J. P. Mariztlr and H. E. Audier 1.Am. Chem. Soc. 1992 114 10874. 36 N. J. Jensen (3.W. Haas and M. L. Gross Org. Mass. Spectiom. 1992 27 423. 37 G. Bouchoux,M.T. Nguyen and P. Longvialle J. Am. Chem. Soc. 1992 114 1OOOO. 38 L. Debrauwer A. Paris D. Rao F. Fournier and J.C. Tabet Urg. Mass. Spectrum. 1992 27 709. 39 T. A. Molenaar-Langeveld R. H. Fokkens S. Ingemann and N. M. M. Nibbering Org. Moss. Spectrorn. 1992 21 390. Physical Methods and Techniques-Part (ii) Mass Spectrometry 25 be a rearrangement of the meta and para isomers to the ortho isomers. Tkaczyk and Harrison have deveIoped a method for the identification of esters by colIisiona1 charge inversion of their enolate ions.4o The enolate ions of m/z 115 derived from the four butyl acetates have been distinguished by their coilisional charge inversion and neutraliz- ation-reionization mass spectra.The reactions of the ion HC,=CHCH=U+CH, generated by radical loss from allylic alkenyl methyl ethers have been probed using 2H and 13C labelling experirnent~.~’ 3 Fulkrenes and Clusters Mass spectrometry has always had a connection with the field of fullerenes and these compounds have continued to generate a huge amount of intere~t.~’ Gaseous C, was studied by photoionization mass spectrometry between the ionization threshold and 40.8eV.43 Fragmentation to form C;8 was only observed at the highest energy and Ci; also became more prominent with increasing energy. An adiabatic threshold of 7.57 20.01 eV was observed for C&,.An Fe adduct with C, was obtained by carrying out contact-arc vaporization of graphite in a partial atmosphere of [Fe(CO)6].44 The properties of the molecule differ from those of a moIecule prepared with Fe outside the cage by solution methods.Wu,Christian and Anderson examined the effects of colliding a beam of Ne’ with C, vapo~r.~’ (26 has been found to add to a large variety of neutral and charged n~cleophiles,4~ and to exhibit fade reactivity with CH in both positive and negative ionization. The reaction of fullerenes with fluorine gives a variety of products which may well have the fluorine externally attached to the ~keleton.~’ C, and C, in 3-nitrobenzyl alcohol and 2-nitrophenyl octyl ether have been investigated by both positive and negative-ion FAB.48 Many protonated and hydroxylated ions were observed.Collision-induced dissociation of the fuIlerene- adduct ions indicated that the oxygen and hydroxyl were connected to the outer surface of the fullerene. A four-sector tandem mass spectrometer was used to prove that as a result of collisions small target gases such as helium and deuterium are incorporated into intact fullerene radical cati~ns?~ The internal energy of these complexes was varied by changing the centre-of-mass coIlision energy which is converted into the internal energy of the complex when the target gas is captured. The formation of endohedral (C6,He) + complexes in mass spectrometric experiments has been modelled with molecuIar dynamic sir nu la ti on^.^^^ These simulations predicted optimal complex formation at a C& incident energy of 8000eV.The results were consistent 40 M. Tkaczyk and A.G. Harrison Org. Mass. Spectrom. 1992 27 585. *’ R. D. Bowen A. W. Colburn and P.J. Derrick Org. Mass. Spectrom. 1992 27 625. *’ S. W. McElvany and M. M. Ross,J. Am. Soc. Mass Spectrom. 1992 3 268. *’ R.K. Yoo,B. Ruscic and J. Berkowitz J. Chem. Phys. 1992 % 911. 44 T. Pradeep,G. U. Kulkarrti K. R. Kannan T. N. Row and C. N. R. Rao J. Am. Chem.Soc. 1992,114,2272. *’ Z. Wu J. F. Christian and S. L. Anderson J. Chem. Phys. 1992 % 3344. 46 F. Wudl A. Hirscb K. C. Khemani,T. Suzuki P. M. Allemand A. Koch H. Eckert G. Srdanov and H. M. Webb in ‘Fullerenes’ ACS Symposium Series no. 481 ACS Washington DC 1992 p.161. *’ K. Kniaz J.E. Fischer H. Selig G. B. Vaughan W. J. Romanow D. M. Cox S. K. Chowdhury J. P. McCauley R.M. Strongin and A. 3. Smith 111 J. Am. Chem. SOC. 1993 115,6040. 48 J. M. Miller and L. 2. Chen Rapid Commun.Mass Spectrom. 1993,6 184. *9 K. A. CaIdweIl D.E. Giblin and M. L. Gross J. Am. Chem. Soc. 1992 114 3743. S. W. McElvaney M. M. Ross,and J.H. CalIahan Ace. Chem. Res. 1992 % 3344. ” R.C. Mowrey M. M. Ross and J.H. Callahan J. Phys. Chem. 1992,% 4755. 26 C. P. Ball and P. J. Derrick with a collision involving momentum transfer between He and an isolated portion of Czo.Lykke and Wurtz have reported neutral products from photodissociated C, for the first time.s2 The electron impact cross section of Ce0was determined by Baba and coworkers5’ using Knudsen cell mass spectrometric techniques to be (5.35 4 5.5) x 10-16m2at 38 eV.Doubly-charged negative ions of fullerenes have been produced by chemical ionization using isobutane as the reagent gas. The efficiency of the attachment increases with the size of the fullerene. Under Iow-energy collision conditions an electron is ejected54 from the doubly-charged C:; ion to form C;:. Knudsen cell mass spectrometry of pure C6* was carried out at 600-800K to determine5’ the vapour pressure and sublimation energies of C,o. Evidence of a ‘radical sponge’ effect of C, has been presented by McEwen McKay and Larsen when additions of H CH, and C,H to C6* were observed using methane as the source under electron attachment conditions.s6 The results are explained by a mechanism in which radicals produced in the gas phase react with c6 at a surface.Multiphoton ionization of fullerenes has shown prompt electrons resulting from direct two-photon ionization as well as a low-energy delayed electron signal following a thermal energy distribution with average energy of E-= 0.3 & 0.05 eV. The results lead to an estimate5’ of the photoionization cruss-section for C, in the lowest triplet state of (5.1 2.0) x 10-17~-2. Delayed ionization of fullerene over a timescale of 15 ps was reported in good agreement with Klots’ model for thermionic emission.58 FAB of fullerenes using helium argon or xenon beams gives characteristic patterns with helium showing abundant triply-charged peaks.” Soluble yttriurn-containing metallofullerenes YC, and Y2c82 were obtained from yttrium-carbon soot and detected using laser desorption time-of-flight (TOF)mass spectrometry which showed a series of peaks Y,C2 (2n = 84,905 2n 5 The metallofullerenesYC, and YC,o were not observed.The Yc82 compound was shown by X-ray crystallography to have a Y-Y interacton with the Y-Y neighbour distance at 4.05 0.05 A,61which might indicate that Y is not endohedral. Gas-phase Y-fullerene adduct ions (Y,C,)* (x = 1,2) with even numbered n of 60-100 were formed in a FT-ICR mass spectrometer by direct laser ionization of graphite and Y20 These compounds were found to be extremely stable towards fragmentation and oxidation and hence it was proposed that they could be endohedral.Endohedral scandium fullerenes have been produced by arc burning scandium oxide/carbon graphite rods.6 The fullerenes were confirmed as discandium fullerenes by MALDI ” K.R.Lykke and P. Wurtz J. Phys. Chem. 1992,% 3191. ” M.Sai. Baba T.S. L. Narasimhan R. Balasubramanian and C.K. Mathews Int. J. Mass Spectrom. Ion. Processes 1992 114 R1. ” A. Mandelbaum and A. Etinger Org. Mass.Spectrom. 1993 28 487. ” C. K. Mathews M. S. Baba,T. S. L. Narasimhan R.Balasubramanian,N. Sivaraman,T. G. Srinivasan,and P. R.V. Rao J. Chem. Phys. 1992,% 3565. s6 C. N. McEwen R.G. McKay and B. S. Larsen J. Am. Chem. Suc. 1992 114,4412. 57 D. Ding R.N. Compton R. E. Haufler and C. E. Klots J. Phys. Chem. 1993,97,2500. ’’ E. E. B. Campbell G. Ulmer and I.V. Hertel Z. Phys.D:At. Mol. Clrrsrers 1992 24 81. 59 M. Takayama and H. Shinohara Inr. J. Mass Spectrom. Ion Processes 1993 123,R7. 6o H. Shinohara H. Sato Y. Saito M. Ohkohchi and Y. Ando J. Phys. Chem. 1992 % 3571. 61 L. Sonderholm P. Wurz,K. R. Lykke D. H. Parker and F. W. Lytle J. Phys. Chem. 1992 % 7153. S.W. McElvany J Pbys. Chem. 1992 % 4935. 63 H. Shinohara H. Yamaguchi N. Hayashi H. Sato M. Obkohchi Y. Ando and Y. Saito J. Phys. Chem. 1993,97,4259. Physical Methods and Techniques -Part (ii) Mass Spectrometry 27 and have the formulae SC~@C~~ sc,@c8, and sc,@C,,. Propane(1 + bpropane [(C,H~)(C,H,),,] cluster ions induced by isornerization of the propyl ion have been observed by Foltin et a1.64to decay by the evaporation of up to four propane monomers as well as the normal single-monomer evaporation.This additional fragmentation is described by a new decay mechanism. Dissociative ionization of clusters has been studied using photoionization by vacuum UV laser dispersed light from synchrotron sources or discharge light SOUTCXP 4 Organometallics Mass spectrometry is being used increasingly to study reactions of metal ions with organic molecules. A review has been written by Teesch and A review has appeared comparing the primary processes undergone by transition metal complexes in photochemical excitation in solution and FAB in a matrix.67 Schroeder et d.have studied the gas-phase oxidation of methane by FeO . There + was evidence68 of four distinguishable isomers of [Fe,C,H,,O] . Fisher and Atmen-+ trout"g investigated the activation of alkanes by Cr +,employing ion-beam mass spectrometry to study the reactions of ground-state Cr' (6S) with propane butane methylpropane dimethylpropane and selectively deuteriated propane and methyl- propane.Ground state Cr+ ions were found to undergo no bimolecular reactions at therrna1 energies but did react at elevated kinetic energies. The structure and mechanisms of formation of (C,H,Fe)+ produced by electron impact on q-C,H,Fe(CO),CH were examined by Uggerud and coworkers7' using collisional activation. The ions consist of a 1 1 mixture of benzene-iron and fuhene-iron structures and two different fragmentation pathways were observed. Fe' was shown to mediate in [4 + 21 additions of buta-1,3-diene with ethyne and propyne.The proposed mechanisms involve formation of [Fe(benzene)f'for the ethyne reaction and [Fe(toIuene)] for the reaction with propyne. [FeCp] was generated at low pressure and its sequential reactions with ethylene oxide and related heterocycles monitored. Molecular mass determination of ferrocenyl-aryl-plumbanes has been carried out by californium-252 plasma desorption combined with TOF analysis.72 Positive and negative plasma desorption mass spectra of methylbenzenethiol cadmium salts which are three-dimensional non-molecular materials were found to contain fragment ions directly related to the compound lattice struct~re.'~ A reverse-geometry tandem mass spectrometer was used to analyse the unimolecular and collision-induced dissociation of doubly-charged porphyrins containing nickel and vanadyl ions.The major reactions were classified and illustrated with appropriate 64 M. Foltin V. Grill T. Rauth Z. Herman and T. D. Maerk Phys. Reo. Lert. 1992,68 2019-22. 65 J.A. Booze and T. Baer J. CAem. Phys. 1992,% 5541. "L. M.Teesch and J. Adams Org. Muss.Spectrom. 1992 27 931. '' T.J. Kemp Coord. Ckem. Rev. 1993 125 333. D. Schroeder,A. Fieidler J. Hrusak and H. Schwarz J. Am. Chem. SOC. 1992 114 1215. 69 E. R. Fisher and P.B. Armentrout J. Am. Chem. Soc. 1992 114 2039. 'O D. Ekeberg G. Hvistendahl Y. Stenstroem and E. Uggerud Actu Chem. Scund. 1992 46,92. 'I R. Bahktiar J.J. Drader and D. B. Jacobson J. Am. Ckem. SOL 1992 114,8304. l2 G. Ailmaier 0.Roepstroff and K. E. Schwarzhans Rapid Commun.Mass Spectrom. 1992 6 58. l3 A.G. Craig D. Kinny R. Garbutt 1.G.Dance and P. J. Derrick Org. Mass.Spectrom. 1992 27 1019. 28 C. P. Ball and P. J. Derrick spectra and information from tandem quadruple experiments was presented.74 Electrospray(ES) and four-sector tandem mass spectrometry has been used to study complexes of lanthanides and trifl~oracetylcamphorates.~Electrospray mass spectra of mercury phosphine complexes has been used to examine the cation exchange that readiIy takes place with these species.76 5 Polymers Developments in electrospray and more particularly MALDI are revolutionizing the mass spectrometry of polymers. Mass discrimination has been reported however in the laser desorption-FT-ICR mass spectrometry cation-attachment spectra of poly-mer~.~~ It is unclear whether mass discrimination occurs with laser desorption and FT-ICR of polymers in general.The interactions by collision-induced decomposition and ion-molecule reactions of poly(ethy1ene glycol) and ethoxylated alcohols have been studied by FT-1CR. PoIy(ethy1ene glycol) when protonated can transfer protons to both polymer and alcohol and measurements of the proton affinity and entropy of protonation of the ethoxylated alcohols give evidence for a strong intramolecular hydrogen bond.78 Plasma desorption mass spectrometry has become less popular in recent years but work is still being carried out using this technique on polymers. Coincidence-counting techniques were used with TOF mass spectrometry to study the impact of MeV/amu ions on surfaces.79 Correlations were observed between proton emission and certain hydrocarbon-ion emissions from polymer samples. 252Cf fragments were used to probe the surface of polystyrene/PVME blends by coincidence-counting TOF mass spec- trometry.8o Nohmi and Fenn have shown that ions formed from electrosprayed poly(ethy1ene oxide) with molecular weights from 200 to 5ooOOOO can be mass ana1yseds8' The number of charges per unit length decreased with increasing molecular weight for molecular weight below 2OOOO. Above this figure the number of charges per unit length increased. Above 5 OOOOOO the number exceeds that allowed by the electrostatic model. Two different mechanisms were proposed for formation of ions from charged droplets.MALDI-TOF has been used to examine the absolute masses and mass distribution of poly(styrene sulfonic acid) and poly(acry1ic acid).*' MALDI was used successfully to characterize polyethylenes PMMA polystyrene and poly(ethy1ene glycols) by the use of appropriate matrices and the addition of metal salts. Molecular masses up to 70000Da are observed but no fragmentati~n.'~ It has also been discovered that A1+ Cr+ Fe' and Cu' all attach efficiently to polystyrene and poIy(ethy1ene glycol) in 74 G.J. Van Berkel S. A. Mctuckey and G. L.Glish J. Am. SOC.Mass Spectrom. 1992,3,235. 75 J. M.Curtis P.J. Derrick A. Schnell E. Constantin R.T. Gallagher and J. R. Chapman Org. Mass. Spectrom. 1992 27 1176. '' R. Colton and D. Dakternieks Inorg.Chim. Acra 1993,208,173. 77 J.D. Hogan and D.A. Laude Jr. Anal. Chem. 1992,64 763. '* H.Y. Lin,A. Rockwood M. S. B. Munson and D.P. Ridge Anal. Chem. 1993,65 2119. 79 M.A. Park E.A. Schweikert and E. F. Da Silveira J. Chem. Phys. 1%2,% 3206. '' D. B. Cox M. A. Park G.R. Kaercher and E.A. Schweikert Anal. Chem. 1992,64,843. T. Nohmi and J.B. Fenn J. Am. Chem. SOC. 1992,114 3241. *' P. 0.Dank D. E. Karr F. Mayer A. Holle and C.H. Watson Org. Mass. Spectrom. 1992,27,843. 83 U.€3ahr A. Deppe M.Karas and F. Hillenkamp Anal. Chem. 1992,64,2866. Physical Methods and Techniques -Part (ii) Mass Spectrometry 29 laser desorption TOF mass Spectrometry. The metals can be added either by applying the salt to the matrix or by desorption from a metal support.84 6 Bioorganic Compounds Using ES mass spectrometry it has been possible to demonstrate the attachment of coenzymeA to a flavodoxin by a modified strain of Escherichia coli.8sIon-spray mass spectrometry has been used to characterize lipopeptides with high sensitivity and simple sample preparati~n.~~ Molecular mass determination up to 6000 Da with an accuracy of I Da was routinely possible.Using 266 nm laser desorption foIlowed by 255 nm multiphoton ionization a variety of structurally significant sequence fragment ions have been obtained for pep tide^.'^ The fragment ions were mostly obtained during the 266 nm Iaser desorption. A variety of immoniurn ions were also formed at higher ionizing power densities which gave an overview of the amino acid residues present.Fragment ions observed in mass spectra produced during resonant 266 nm laser desorption may originate as neutral fragments formed from the intact molecule during laser desorption and subsequently post- ionized.'' MALDI-TOF and ES ionization were used to investigate the interactions of copper@)ions with a 26-residue sequence of amino acids found on the surface of the human plasma metal-transport protein histidine-rich gIycoprotein.89 The peptide (GHHPH),G was reacted with copper and manganese and was observed to bind one copper ion per repeat unit. Manganese showed no reaction. New reagents are frequently proposed for the Edrnann degradation and one such has been heavily studied by mass spectrometric methods." 3-[4'-(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanatewas detected as amino acid derivatives with high sensitivity by ion-evaporation mass spectrometry.Sensitivity was subferntomolar and amino acids of all types of side chain were detected with equal facility. The polar nature of the reagent required covalent immobilization of polypeptides prior to automatic sequence analysis. The veterinary drugs dimetridazole and ipronidazole and their metabolites were detected in turkey muscle by HPLC followed by thermospray tandem mass spec- trornetryg' at the ppb Ievel. Tandem mass spectrometry confirmed the identity of the drugs and their metabolites. TaxolQ cephalornannine and baccatin HI@ have been detected by tandem mass spectrometry in crude plant extracts at less than 500pg q~antities.~~ No interferences were observed.Diciofenac@ an antiinflammatory drug has been investigated by means of electron impact chemical ionization and FAB tandem mass spectrometry along with hydroxylated metabolite^.^^ GC-MS with C. F. Llenes and R. M. OMalley Rapid Commun. Mass Spectrom. 1992 6 564. " R. N. F. Thorneley C. Abell G. A. Ashby M. H. Drurnmond R.R.Eady S. Huff C.J. Macdonald and A. Schneier Biochemistry 1992 31 1216. 86 J.W. Metzger W. Beck and G. Jung Angew. Chem. 1992 104 235. '' G. R. Kind 3. Lindner and J. Grotemeyer 1.Chem. Phys. 1992 96 3157. 88 G. R. Kinsel 1. Lindner and J. Grotemeyer J. Chem. Phys. 1992 96,3162. 89 W.T. Hutchens R. W. Nelson,M.H. Allen C. M. Li and T.T. Yip Biol. Mass Spectrom. 1992,21 151. 90 R.Aebersold E.1.Bures M.Namchuk M. H. Goghari B. Shushan and T. C. Covey Protein Sci. 1992,1 494. 91 J. E. Matusik M.G. Leadbetter C.J. Barnes and J. A. Sphon J. Agric. Food Chem. 1992 40 439. 92 S.H. Hoke 11 J.M. Wood R.G. Cooks X.H. Li and C.J. Chang Anal. Chern. 1992,64 2313. 93 F.M. Rubino and L. Zecca Biol. Mass Spectrom. 1992 21 109. 30 C. P. Ball and P. J. Derrick selected ion monitoring has been employed to detect cortisol and cortisone in human plasma. C2H2]cortisol and [2H,]cortisone were used as internal standards and the determination was based on the [M-3I] fragment ions of the rnethoxine-trimethyl- silyl derivative^.^^ Macrolide antibiotics have been examined by capillary zone electrophoresis combined with ES. Increasing the ES skimmer voltage gave structurally significant fragment ions.This method gave a better limit of detection than any other currently in use.95 Tandem mass spectrometry has been used to demonstrate that halopridoe@ a neuroleptic drug is metabolized into at least eight different compounds.96 The purification of saxitoxin the parent compound in the family of toxins responsible for paralytic shellfish poisoning (PSP) has been monitored using flowinjection analysis and capillary electrophoresis-mass spectr~rnetry.~ Tandem mass spectrometry was used to obtain structural information. Quilliarn ef aLg8have analysed okadaic acid and dinophysistoxin-1 ,the principle toxins implicated in cases of diarrhetic shellfish poisoning by a liquid chromatography/mass spectrometric method which provided a mass detection limit of 0.4 ng for each toxin.The trace level analysisg9 of sulfonylurea herbicides has been reported using thermospray. Enan- tiomers of chiral octachloranes in tissue samples from aquatic vertebrates in Antarctica"* were separated using chiral high resolution GC and detected by electron capture negative ionization mass spectrometry. TaxoP and related diterpenoids have been analysed by microbore BPLC coupled with electr~spray.'~~ 100 pg of all taxanes were readily detected using secondary ion monitoring. Vitamin D metabolites have been characterized by cycloaddition with 4-phenyLl,2,4-triazoline-3,5-dione, followed by continuous-flow FAB-MS.'02 The spectra are dominated by peaks corresponding to a protonated molecule and by side-chain fragments.The siderophores of the fire blight pathogen Erwinia arnylouoria a group of proferrioxamines have been detected and characterized by LC-ES. In addition six hydroxamate compounds were characterized and another five detec- ted.'03 Tomatine a glycoalkaloid has been identified at the femtomole level by four-sector tandem mass spectrometry and scanning-array detection. 'O4 CapilIary eIectrophoresis combined with electrospray was used to make the intriguing discovery that the venom of the black mamba contains at least 70peptides and has a molecular mass range of 5OOO-9000 Da.'" Peptides and Proteins-Mass spectrometryis now fairly well established as a refiable 94 H. Shibasaki I. Arai T. Furuta and Y. Kasuya J. Chromotogr.I992,576,47. 9s C.E.Parker J. R. Perkins K. B. Tomer Y. Shida K.O'Hara and M. Kono,J. Am. SOC. Mass Spectrom. 1992 3 263. 96 J. Fang J. W. Gorrod M. Kajbaf 3. H. Lamb and S. Naylor,J. Mass Spectrorn.Ion Processes 1992,122 121. 97 S.Pieasance S. W. Ayer M. V. Laycock and P. Thibault Rapid Commun. Mass Spectrom. 1992,5 14. 98 M.A.Quilliarn S. Pleasance and J.C. Marr Rapid Commun. Mass Spectrom. 1992 6 121. 99 M.L. Shalaby F. Q. Bramble Jr. and P. W. Lee,J. Agric. Food Chem. 1992,40 513. 100 H.R,Buser M.D. Mueller and C. Rappe Enuiron. Sci. TechnoL 1992,26 1533. 101 F. Bitsch W. Ma F. Macdonald M. Nieder and C. H. L. Shackteton J. Chromntogr. Biomedical Appl. 1993,615 273. 102 B. Yeung P. Vouros,and G. Satanarayana Reddy J. Chromatogr. 1993,645,115.103 G.J. Feistner D.C. Stahl and A. H. Gabrik Org. Mass. Spectrom. €993,28 163. f 04 S. Evans R. Buchanan A. Hoffman F. A. Mellon K.R. Price S. Hall F. C. WaIls A. L. Burlingame S. Chen and P.J. Derrick,Org. Mass. Spectrom. 1993 28 289. 105 J. R. Perkins C. E. Parker and K.B. Torner Electrophoresis (Weinheim) 1993,14. Physical Methods and Techniques -Part (ii) Mass Spectrometry and sensitive method of sequencing peptides. The rapid deveIopment of ES and MALDI of proteins and advances in techniques for interfacing mass spectrometers with separation methods such as capillary zone electrophoresis(CZE)has led to more and more mass spectrometry being undertaken in the protein field. Antigens recognized by T-cells are expressed as peptides bound to major histocom- patibility complex (MHC) molecuIes.Microcapillary HPLC-ES tandem mass spec- trometry has been used to sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1.'06 Of 200 species quantitated eight were sequenced and four found in cellular proteins. All were nine residues long and shared a distinct structural motif. The enzyme 6-pyruvoyl tetrahydropterh synthase which occurs at very low levels in the metabolic disorder atypical phenylketonuria was purified and characterized from salmon liver digested and sequenced by tandem mass spec- trometry.'*' Biemann and coworkers'a8 showed from the m/z ratio of ions of the tryptic peptide of myoglobin encompassing the disputed amino acid 122 (previously commonly held to be asparagine) derived from horse heart and horse skeletal rnyoglobins the apomyogIobin of the latter and the tryptic and chymotryptic peptide of sperm whale myoglobin that amino acid 122of the myoglobin tryptic peptide is aspartic acid rather than asparagine.This finding was confirmed by the collision-induced dissociation spectra of the EM + HI+ ions of the tryptic peptides. Thus the correct relative molecular mass of horse myoglobin is 16 951.49 (not 16950.5 as previously reported) and that of the sperm whale protein is 17 199.91 (not 17 199 as previously reported). Since myoglobin is used as a common mass calibrant this finding is of importance. Destruxins (cyclodepsipeptides) were sequenced using FAB by Lange st a1.,'*' with deuteriation used to overcome problems caused by isobaric fragment ions.Lumb et a[.' * have studied D52S hen lysozome oligosaccharide complexes by ES. They found that in the spectrum of a mutant form with a catalytically important residue Asp-52 replaced by serine the most intense peaks arise from uncomplexed protein indicating that dissociation takes place in the mass spectrometer. Peaks from minor species corresponding to complexes between the protein and oligosaccharide were also observed.The leucine zipper found in several DNA-binding proteins has been shown by HPLC-ES and tandem mass spectrometry to dimerize both in solution arid in the gas phase. These dimers are stable in the gas phase for minutes."' The infectious scrapie prion PrP'" has been sequenced from endoprotease digests by mass spectrometry and Edman degradation data.I Species-selective effects have been observed in the production of peptide ions by ES. For Arg-Arg-Arg and Lys-Lys in methanol-water (80:20 by volume) the relative intensities of (Ch,OH,)+ ions were found to decrease faster than those of other ions with distance from the capillary tip to Io6 D. F. Hunt R.A. Henderson J. Shabanowitz K. Sakaguchi H. Michel N.Sevilir A. L. Cox E. Appella and V. H. Engelhard Science (Washington D.C.) 1992 255 1261. lo' C. R. Hauer W. Leirnbacher P.Hunziker F. Neukeiser N. Blau and C. W. Heimann Biuchem. Biophys. Res. Cammun. 1992 182 953. lo' J. Zaia R.S. Annan and K. Biemann Rapid Commun. Mass Spectrom. 1992,5 32. Io9 C. Lange C. Mulheim A. Vey and M. Pais Biol.Mass Spectrom. 1992 21 33. 'lo K.J. Lumb R.T. Aplin S.E. Radford D. B. Archer D. J. Jeenes N. Lambert D.A. MacKenzie C.M. Dobson,and G. Lowe FEBS Lerters 1992,2% 153. I' I G.S. Gorman and J.I. Amster Org.Mass. Spectrom. 1993 28 437. N. Stahl M.A. Baldwin D.B. Teplow L.Hood B. W. Gibson A. L. Burlingame and S. B. Prusiner Biochemistry 1993 32 1991. 32 C. P. Ball and P.J. Derrick interface plate. These ions are believed to have maximum intensities in the central region of the spray cone whilst ammonium and singly charged oligopeptide ions are found in the peripheral region. The results are interpreted in terms of the volatility of ions in the charged liquid A detailed study of the field desorption of valinomycin found that the ratio of the molecular ion to the protonated molecule was independent of the emitter heating current and the nature of particular additives to the solution.In contrast the presence of the [M + Na] + ions was very strongly dependant on the heater current. There was also found to be no relationship between the abundance of cationized species and the selectivity of valinomycin for monovalent species in solution.' l4 Tandem mass spectrometry of valinornycin has been studied with a number of ionization methods; these show a marked difference between [M + Na]' spectra following different methods of ionization."' SmalI peptides react with alkaline earth metals under FAB conditions to form metal-bis(peptide) complexes [2 peptide + metal -3 H]-. The metal ion binds to the deprotonated C-terminal carboxylate group of both peptides and to a deprotonated amide nitrogen of the constituent peptides.This gives rise to fragments containing metal ions tightly bound to the C-terminal end of the peptide with few N-terminal fragment ions.' l6 A similar study has been described for tripeptides. I' Fragmentation mechanisms for the formation of several commonly occurring product ions in high-energy collision-induced decomposition spectra of [M + Ca-H]+ ions have been evaluated by using deuterium-labelled peptides.' I8 The [M -171peak observed in the muitiphoton ionization mass spectra of dipeptides has been explained as the elimination of ammonia from the molecular ion. The [M-181 peak is generated from the ionization of the thermally decomposed dipeptide.' l9 Tripeptide stereoisomers have been distinguished successfully by tandem mass spectrometry on a hybrid instrument by using CAD.The tripeptides were of the form X-L-Pro-L-Phe where X is L-AIa D-AI~ L-As~ or asp. The differences in fragmentation were correlated with force-field calculations on hydrogen-bonded tautomers."* Abundant ions due to the cleavage of Asp-Pro and Asp-Xxx ions following MALDI have been observed by TOF. Using four-sector tandem mass spectrometry abundant metastable decomposition of Asp-Pro bonds takes place in larger peptides and proteins. The implication is that the Asp-Pro bond is especially The gas-phase basicities of 22 peptides have been estimated by using MALDI to generate prutonated peptides which were then reacted with reference compounds to bracket the gas-phase basicity.Neutral peptides were desorbed using substra-te-assisted laser desorption and reacted with protonated references to confirm proton affinity values. The results are consistent with an intramoIecuIar hydrogen bond between the N-terminus nitrogen and the arnide carbonyl oxygen of a dipeptide which 'I3 K. Hiraoka K. Murata and I. Kudaka Rapid Commun. Muss Spectrom. 1993,7 363. li4 M.M. Sheil and P.J.Derrick Org. Mass. Spectrom. 1992 27 1OOO. 'I5 M. M. Sheil G.W. Kilby J. M. Curtis C. D. Bradley and P.J. Derrick,Org. Mass. Spectrom. 1993,28 574. 'I6 P. Hu and M.L. Gross,J. Am. Chem. SOC. 1992,114,9161. 'I7 P. Hu and M.L. Gross J. Am. Chem. SOC. 1992,114 9153. 'I6 H. Zhoa and J. Adams Int.J. Mass Spectrom. Ion Processes 1992 125 195. 'I9 D. S. Nagra J. Y. Zhang A. P. L. Wang and L. Li Int. J. Mass Spectrom. ion Processes 1992,116 127. 12' B. L. Schwartz R. D. McQain B. W. Erickson and M. M. Bursey Rapid Commun. Mass Spectrom. 1993 7 339. W. Yu 5.E. Vath M. C. Huberty and S.A. Martin Anal. Chem. 1993 65 3015. Physical Methods and Techniques -Part (ii) Mass Spectrometry 33 increases in strength with the strength of the basicity of the C-terminal amino acid.lZ2 Bean and Carr used tandem mass spectrometry and high-energy CAD to sequence through the cystine bridge of intermolecularly disulfide-bonded peptides. 23 Using this method they found that a characteristically-defined triplet of intense fragment ions is observed. FAB tandem mass spectrometry of protonated and metal-cationized peptides was foundlZ4to give evidence of intramolecular rearrangements in the gas phase."0 was used to label the C-terminal carboxyl group and suggested a mechanism where the C-terminal amino acid group is lost with retention of the original carboxyl residue. Using two steps of tandem mass spectrometry (MS/MS/MS) analysis the intermediate formed in the process was found to be the same for both the ["O,] and the [180]-labelIed rearrangement products. The probable intermediate was concfuded to be almost certainly cyclic. The fragmentation of the peptide luteinizing of hormone releasing-hormone measured in plasma desorption mass spectrometry (PDMS) and keV-atom bombardment has been compared with the field desorption (FD) mass Kenny has proposed a mechanism for the formation of y ions in the collision-induced dissociation spectra of protonated peptides produced by FAB,l z6 in which a hydrogen atom attached to nitrogen migrates during the amide bond cleavage.Evidence was obtained by Ackermann et a/. 27 for a lysine-specific fragmentation in FAB of peptides. Fragment ions from peptides containing lysine (or other amino acids with a free amino group on a side chain) were found 16Da below the b ions normally resulting from C-terminal fragmentation. Using liquid secondary ion mass spec- trometry (LSIMS) the peptide somatostatin-14 (SS-14) was found to undergo unsymmetrical disulfide bridge cleavage. The mechanism for c" ions from protonated peptides produced during fast atom bombardment and collisonal activation has been eiucidated.Neither the carboxylic proton nor a hydrogen atom from a nitrogen is involved in the process of hydrogen migration that gives rise to the c" ion. The most feasible position from which the hydrogen originates is that at the first C-atom in the adjacent amino acid side chain.'. 28 FAB-MS/MS of a disulfide-containing enkephalin [Pen]-enkephalin gives a large number of fragment ions in contrast to other disulfide peptides. Most of the fragments are due to backbone cleavages with unsymrnetric cleavage of the disulfide bond giving charge retention at the N-terminus; the fragment ions rarely include a sulfur atom. The N-terminal ethyltriphenylphosphonium derivative however shows very little frag- mentation due to its suggested charge localization at one end of the peptide.1.29 The disulfide bridges in recombinant human macrophage-stimulating factor a 49 kDa homodimeric protein have been assigned through FAB after cleavage using BrCN.Papayannopoulos and Biemann have suggested a reason for the non-detection of 122 G.S. Gorman and J.I. Amster J. Am. Chem. Suc. 1993 115 5729. M.F. Bean and S.A. Carr Anal. Biochem. 1992 201 216. K. D. Ballard and S.J. Gaskell J. Am. Chem. Soc. 1992 114 64. IZ5 A. G. Grey H. Bennich and P. J. Derrick,Ausr. J. Chem. 1992,45,403. P.T.M. Kenny Rapid Commun. Mass Spectrom. 1992,6,95. B. L. Ackermann R. J. Barbuch J. E. Coutant J. L. Krstenansky and T. J. Owen Rapid Commun. Muss Spectrom.1992,6 257. W.D. Van Dongen C.G. De Koster W. Heerma and J. Haverkamp Rapid Commun. Mass Spectrom. 1993,7,241. '29 Y. S. Chang D. A. Gage and J.T. Watson Biol. Mass Spectrom. 1993 22 176. 130 M.O. Giocker B. Arbogast J. Schreurs and M. L. Deinzer Biochemistry 1993 32,482. 34 C.P. Ball and P. J. Derrick components in peptide mixtures in FAB-MS. This phenomenon has always been attributed to the suppression of ionization of some of the mixture components by other components; a change in the sample probe temperature has been suggested as a method of overcoming this effect. I Derivation has been used to increase negative ion sensitivity and positive ion sequence information for LSIMS of peptides. The analyte is acylated at unprotected N-termini tyrosine and lysine by application of pentaf- luorobenzoyl The obervation that certain preparations for FAB yield an intense puIse of peptide ions at the start of ionization hasprompted the development of a source incorporating a probe that permits mechanical movement of the sample stage and mimics a pulsed atom-beam.Following bombardment initiation sample ions are preferentially desorbed with respect to matrix ions. Using an array detector the system allows significantly enhanced sensitivity up to an improvement in the detection limit of three to four orders of magnitude.133 A computer program has been developed specificaIly for interpretation of four-sector tandem mass spectra of peptides.'34 The sequencing algorithm uses a pattern based on the polymeric nature of peptides to classify spectra1 peaks.An algorithm has been described which uses both Edman and mass spectrometric data for the simultaneous detection of the amino acid sequences of several peptides in a mixture. The algorithm utilizes Edman data to detect all hypothetical sequences with a catculated mass equal to the observed mass of one of the peptides present in the mixture. These sequences are then assigned figures of merit according to how well each of them accounts for the fragment ions in the tandem mass spectrum of the pe~tide.'~~ Carbohydrates.-The analysis of carbohydrates by mass spectrometry is receiving considerable attention partly because of the introduction of the ES and MALDI techniques. Harvey has reviewed the roIe of MS in glycobiology,136 with particular emphasis on oligosacc harides and glycoconj ugates.Chai er c-11.'~' identified22 neutral 0-linked oligosaccharides in bovine submaxilli- ary gland mucin glycoprotein using LSIMS. The sequences and linkages of sub-nanomolar amounts of thyroid gland oligosaccharides have been confirmed using FA3 and MS/MS. The [M + HI+ and [M + Na]' ions were compared revealing several ions in the [M + NaJ+ spectra not present in the appropriate protonated form in the EM + HI+ spectra. The utility of metal attachment for MS/MS analysis of oligosac- charides has been demonstrated by Leary and coworkers who used dilithiated disaccharides labelled with l80and 'H to determine the Iinkage positions of three isomeric disaccharides.' 38 Centrifugal size-exclusion chromatography and membrane filtration have been used to remove saIts from carbohydrates prior to analysis resulting in an increase in the sensitivity and in the number of structurally significant peaks.139 Ammonia desorption chemical ionization MS of peracylated gentiobiose and 13' I.fapayannopoulos and K. Biemann int. J. Mass Spectrorn. Ion Processes 1992,122,43. 13' J. P. Kiplinger L. Contillo,W. L. Hendrick,and A. Grodski Rapid Commun. Mass Spectrom. 1992,6,747. 133 A. N. Tyler L.K. Romo M. H. Frey B. D. Musselman J. Tamura and R.B. Cody J. Am. SOC.Muss Spectrom. 1992 3 637. 134 W. M.Hines A. M. Falick A. L. Burlingame and B. W. Gibson J. Am. SOC.Mass Specfrorn. 1992,3,324. 135 R.S.Johnson and K. A. Walsh Protein Sci. 1992 1 1083.D. J. Harvey Glycoconjugate J. 1992 9 1. 13' W.Chai E. F. HounselI G.C. Cashmoqe J. R.Rosankiewicz,C.J. Bauer J. Feeney T. Feizi and A. M. Lawson Eur. 1.Biochem. 199? 203 257. A. Staempfli 2.Zhou,and J. A. Leary J. Org. Chern. 1992 57 3590. 139 T. F. Chen H. Yu,and D. F. Barofsky Anal. Chem. 1992,64 2014. Physical Methods and Techniques-Part (ii) Mass Spectrometry isotopically labelled forms showed that the (M + NH -42f+ ion is not formed directly from [M + NH,J* by loss of ketene but by way of a nucleophilic acyl substit~tion.'~~ FT-ICR has been used to examine the origin of fragment ions created during desorption-ionization of disaccharides avoiding the interference of matrix Three major product ions were observed corresponding to loss of water loss of monosaccharide and a combination of both.Mass spectrometry of high-rnannose oligosaccharides after trifluoroacetolysis and periodate oxidation was examined by FAB and electron irnpact.l4* The conditions used were designed to degrade terminal N-acetylglucosarnine residues and binding positions between monosaccharide resi- dues were deduced. GC-MS has been used to study the carboxymethyl groups in 0-(carboxymethy1)ated(1 -,3)-a-~-glucans and (1 43)-a-~-glucans. 143 The results showed a distribution characteristic of the glucatn type especially at position 2 on the D-glucosyl residues. Three thyroid gland oligosaccharides have been characterized by FAB-MS/MS. The saccharides were derivatized with p-aminobenzoic acid ethyl ester and detected in subnanomolar quantities.The [M + Na] + ions in some cases allowed assignments of the linkage positions between the sugar residues.'44 MS/MS has been used to analyse the B -+ C linkage position in neutral permethylated trisaccharides of the formulae galactose(B-1-+ 4)glucose(/3-1+ X)glucose where X = 3 4 and 6. The relation of daughter ion to parent ratios was used to predict the linkage position in glu- cose@-1-+ X)gl~cose.'~~ This method has been used for the [M + Na]' ions of permethylated oligosaccharides. The linkage positions in one compound may be detected by the presence or absence in a single spectrum of specific fragment ions arising from the cIeavage of two ring bonds. The behaviour of the [M + Na] +-ions of permethylated oligosaccharides at low energy leads to preferential cleavage of the glycosidic linkages which become more efficient if argon replaces helium as the collision gas.'46 Distinctions between the linkage types 1 -2 1 + 3 and 1 + 4 of xylobioses can be achieved on the basis of the unimolecular decomposition spectra of the oxonium ions of the per-0-acylated methyl glycosides.This allows the detection of linkages between xylose residues. 14' Ohgosaccharides have been analysed using on-line liquid chromatography/ther- mospray mass ~pectrometry.'~~ Molecular mass information on oligomers of up to a degreeof polymerization of 10was obtained using a mobile phase containing 0.1 mmol sodium acetate which was compatible with thermospray ionization and gave sodiated and disodiated ions without fragmentation.Van der Greef and coworkers'49 have shown how thermospray and ES are useful for the detection of intact oligosaccharides using selected ion monitoring. The on-line coupling of high-performance anion- ''' J. M. Peltier R. W. Smith D. B. McLean and W. A. Szarek Org. Mass. Spectrorn. 1992 27 31. 14* J. A. CarroIl and C.B. Lebrilla Org. Mass. Spectrom. 1992 27 639. '02 P. Lipniunas A. S. Angel K. Erlansson F. Lindh and B. Nilsson Anal. Biochem. 1992 ZOO 58. S. Ukai I. Yoshida A. Honda K. Nagai and T. Kiho Carbohydr. Res. 1992 224,201. D.A. Gage E. Rathke C. E. Costello and M. Z. Jones Glycoconjugate J. 1992,9 126. E. Yoon and R. A. Laine Biol. Mass Spectrom. 1992,21,479. J. Lemoine B.Fournet D. Despeyroux K.R. Jennings R. Rosenberg and E. de Hoffrnann J. Am. SOC. Mass Spectrom. 1993 4 197. L. Blok-Tip A. van der Kerk-van Hoof' W. Heerma,J. Haverkamp V. Kovacik and J. Hirsch Biol. Muss Spectrom. 1993,22 474. W. M. A. Niessen R. A. M. Van der Boeven J. Van der Greef H. A. Schols and A. G. J. Voragen Rapid Commun. Mass Spectrom. 1992 6 197. W. M. Niessen R. A.M. Van der Hoeven and J. Van der Greef' Org. Mass. Spectrom. 1992,27 341. 14' 36 C.P.Ball and P. J. Derrick exchange chromatography and thermospray MS has been described for the analysis of neutral and acidic oligosaccharides obtained as degradation products of cell wail dige~tion.''~ Lipids and Steroids.-Negative-ion mode FAB/MS/MS has been applied to 24 naturally occurring and synthetic diacylglycerylphosphocholines.'51 The method of selecting EM -151-as the parent ion and using the relative abundances of the carboxylate daughter ions to distinguish the esterification positions was found to be unreliable in the cases where the fatty acid group at S,1 was much larger than that at 5,2.By use of the [M -861-ion as the parent the relative intensities of the detected fragments resulting from the Ioss of the free fatty acids at SNland S,2 were found not to depend on the nature of the fatty acid groups present. Van Dorsselaer and coworkers have used negative FA3 on phosphodiesters linked to oxysterols and nucleosides.' 52 The effect of structure on the electron-capture mass spectrometric response of steroids has been in~estigated.'~~ Combinations of double bonds carbonyl groups epoxides and halogens with a steroid nucleus were correlated with their elec- tron-capture negative ionization responses.An epoxy keto steroid has a relative response 40 times greater than the corresponding enone. A method to reduce degradative losses of steryl fatty acyl esters in high-temperature GC-MS employs selective deuteriation of the double bonds in the fatty acid moiety using a homogeneous catalyst.ls4 GC behaviour is improved and structural inforrna- tion is preserved by labelling each doubIe bond present in the original moiety. 16-Androsterones and their isomeric saturated analogues have been analysed by GC-MS using selected ion monitoring.' 55 Desorption chemical ionization triple quadrupole mass spectrometry has been used to study the coliision-induced dissociation of permethylated glycosphingolipids.The dissociation of positive ions produced mainIy sugar sequence ions. A rearrangement reaction was suspected €or unsaturated ceramides as no fatty acid daughter ions were seen for compounds with unsaturated bonds at the C-4-C-5 position.' 56 Lipids from Entobacter agglornerans a Gram-negative bacterium found in cotton have been identified using plasma desorption (PI))-MS. 57 FAB-MS/MS has been established as a useful technique for the identification of intact glycexophosphatidylethanolamine phospholipids giving information about the polar head group and about fatty acid constituents. Scanning fur a loss of 141mass units was the strategy used to confirm the presence of a phosphoethanolarnine polar head group but this scan has now been found to discriminate against the common subgroup of phopholipids with a 1-0-alkyl-1'-enyl linkage (the plasmalogens) and against a 1-0-alkyl ether specie^."^ Is* W.M. A. Niessen R. A. M. Van det Hoeven J. Van der Greef H. A. Schois,G. Lucas-Lokhorst A+G.J. Voragen and C. Bruggink Rapid Commun.Mass Spectrom. 1992,6,474. Is* Z. H. Huang D. A. Gage and C.C. Sweeley J. Am. SOC. Mass Spectrom. 1992,3 71. X. Pannecouke A. Van Dorsselaet and B. Luu Org. Muss.Spectrom. 1992,27 140. 153 H.K. Mayer W. Reusch and J. T. Watson Org. Mass. Spectrom. 1992 27 560. Is' R.P. Evershed M. C.Prescott and J. L. Goad .I.Chromatogr. 1992 590 305. T.K. Kwan D. B. Gower D. J.H.Trafford and H. L. J. Makin Bid. Mass Spectrom. 1992 21 160. J. S. Duh and G.R. Her Bid. Mass Spectrom. 1992 21 391. 15' R. B. Cole L.N. Domeksmith C. M. David R. A. Laine and A. J. DeLucca Rapid Commun. Mass Spectrom. 1992 6 616. 15' K.A. Kayganich and R. C. Murphy Anal. Chem. 1992,64 2965. Physical Methods and Techniques -Part (ii) Mass Spectrometry 37 DNA and Nucleotida-McLuckey et a).' 59 have examined multiply charged ions derived from ES ionization of the sodium salts ofsmall oligonucleotides (n = 4 to 8) by MS/MS in a quadrupole ion trap. The ions were observed to dissociate with high efficiency. In every case the most facile reaction was the loss of the adenine anion followed by the cleavage of the C-3-0 bond of the phosphodiester linkage of the sugar from which the adenine had been lost.The collision-induced dissociation of protonated adenine and of rnonomethylated adenines has been studied by MS/MS spectrometry and isotopic labelling.' 6o A DNA quadruplex has been detected by negative-ion ES at a low nozzle-skimmer bias (-150V) but at higher bias values it is not seen.The complex is only seen in lOmM sodium phosphate (pH 7.5) with 0.1 mM EDTA. Without the metal only single-stranded DNA is seemi6' MALDI ofoligodeoxythyrnidylic acids has been investigated by Huth-Fehreet a].;16* using ferulic acid sinapinic acid or 2,5-dihydroxybenzoic acid as matrices for pure oIigonucleotide P~(T),~ and a mixture of pd(T), through pd(T)18. 2,5-dihydroxyben- zoic acid gave the least fragmentation and adduct formation and highest mass resolution.3-hydroxypicohic acid has been evaluated as a matrix far MALDI of oligonucleotides and found to show good characteristics with regard to signal-to-noise ratio mass range available and ability to handle mixed-range oligomers. Spectra taken at 266 nm were found to give the best results in negative-ion mode.163 RNA oligomers and transcripts up to 104nucleotides iong have been analysed successfuIly by MALDI. RNA is found to be more stable to MALDJ than oligothymidyIic acids as the N-glycosidic bond seems to be broken during the desorption and bases are MALDI has been used to determine the molecular masses ofsynthetic methylphosphon- ateoligodeoxyribonucleotidesofup to 18 nucleotidesin length.16' TOFmass spectra of DNA fragments as massive as 18 OOO Da have been produced using laser ablation.'66 Low picomole levels of nucleoside-arcinogen adducts were detected using FA3 and MS/MS.167Chemical derivatization by trimethylsilyl neutral Ioss scans and daughter ion scans were all used to probe the structural specificity of the adducts.FAB and MS/MS have been used to examine the interactions between the anticancer drug cisplatin and DNA.168FAB gives useful structural information and high energy collisianal-induced dissociation shows the location of the platinum binding sites. For aglycon-halogenated nucleosides an inverse linear reIationship exists between the extent of dehalogenation during FAB and the electron affinity of a given nucleoside. 69 159 S.A.McLuckey G.J. Van Berkel and G.L. Glish J. Am. SOC. Mass Spectrom. 1992 3 60. 160 C.C. Nelson and J.A. McCloskey J. Am. Chem. Soc. 1992,114,3661. 161 D. R. Goodlett D. G. Camp IT C.C.Hardin M.Corregan and R. D. Smith Biol. Mass Spectrom. 1993 22 181. 16' T. Huth-Fehre J.N. Gosine K. J. Wu and C.H. Becker Rapid Commun. Mass Spectrom. 1992,5,209. 163 K. J. Wu A. Steding and C. H. Becker Rapid Commun. Mass Spectrom. 1993,7,142. E.Nordhoff R. Cramer M. Karas F. Hillenkamp F. Kirpekar K. Kristainsen and R. RoerpstorfF Nucleic Acids Res. 1993,21 3347. 16' T.Ke0ugh.T. R. Baker R. L. M. Dobson,M.P. Lacey T. A. Riley J. A. Hasselfield and E. P. Hesselberth Rapid Commun. Mass Spectrorn. 1993,7 195. *" P. Williams D. Schieltz R. W. Nelson C.W+ Chou C.W. Luo and R.Thomas in 'Proceedings of Advances in DNA Sequencing Technology' 1993 p. 86. 167 M. S. Bryant J.O. Lay Jr. and M. P. Chiarelli J. Am. SOC. Mass Spectrom. 1992,3 360. C,E. Costello K. M.Comess A. S. Plaziak D. P. Bancroft and S.J. Lippard int. J. Mass Spectrorn. Ion Processes 1992,122,255. S. M. Musser and J. A. Kelley Org. Mass. Spectrorn. 1993,28,672. 38 C. P. Ball and P. J. Derrick Narcotics and Drugsof Abuse.-Mass spectrometry is recognized as being a formidable tool for the analysis of controlled substances particularly in conjunction with GC and a wealth of information on techniques for the analysis of narcotic compounds in particular has been published. A review of opiate MS has been given by Cone and Darwin.I 70 Using GC-MS in electron impact (EI)mode the metabolites of danazoI an anabolic steroid banned by the International Olympic Committee were detected in a urine sample.Ethisterone was identified as a nonconjugated metabolite and the two stereoisomersof 2-hydroxymethylethisteronewere observed one in a conjugated form and the other primarily unconjugated.’ 71 Derivatives of seven commonly used sympathomimetic amines and two designer amines were isolated from urine separated from amphetamine and methamphetamine by GC and detected by MS using seIected ion monitoring. False positive were only seen for methamphetamine and only when >5 pg of ephedrine was present in the ~arnples.”~ Several methods for introduction derivitization and ionization of lysergic acid diethylamide (LSD) iso-LSD and N-dimethyl-LSD in body fluids have also been evaluated for MS/MS.Fragmentation pathways derived from low-energy collision-induced dissociation of LSD and derivatives have been proposed. Positive ion chemical ionization of these compounds provide a high degree of specificity for their identification in blood and urine at low pgml-levels whilst negative ion chemical ionization and GC-MS/MS has been found to be of use in determining rnetab01ites.l~~ 7 Electrospray Electrospray atmospheric pressure ionization and ion-spray are all variations on a common theme. Comparison between ES and MALDI is inevitable but not straightforward. Green’74 has criticized a paper by Allen et al. that sought to compare ES and MALDI for analysis of protein mixtures.In Green’s opinion the paper did not compare the techniques impartially but detracted from the utility of the ES technique. ES was used to show the difference in the conformational states of a zinc-finger DNA-binding protein domain. Evidence for the induction and stabilization of a different conformational state with bound copper as opposed to zinc was revealed by a characteristic shift in the electrospray charge envelope.’ 75 A similar zinc-finger protein that binds to oestrogen was demonstrated to have a high affinity for copper which inhibits its function by the use of E5.l7‘ It was also found that non-covalentIy bound polypeptide and protein dimer ions are relatively common as low level contributions to ES mass spectra. The detection of these compounds requires gentle electrospray conditions yielding low levels of internal excitation but these conditions can lead to artifacts in MS/MS due to incomplete solvation of ions.’77 The haem-apomyoglobin complex which is non-covalently bound and has a mass of 17568Da has been detected by ES at femtomolar quantities.The complex does not fragment under 170 E.J. Cone and W.D. Darwin J. Chromutogr. I992,=,43. D. De Boer E.G. Jing and R.A.A. Maes J. Anat. Toxicol. 1992,16,14. ”* E. M. Thuman M.J. Pedersen R.L. Stout and T. Martin J. Anal. Toxicol. 1992,16,19. 173 CC. Nelson and R.L. Fottz Anal. Chem. 1992,64 1578. 17’ 3.N. Green Biochem. J. i992 284 603. If’ W. T. Hutchens and M. H. Alien Rapid Commun. Mass Spectrom. 1992,6 496. lf6 W.T.Hutchens M.H. Allen C. M. Li and T.T. Yip FEBS Lett. 1992,309,170. 177 R.D. Smith K.J. Light-Wahl B. E. Winger and J.A. Loo Org. Mass. Spectrom. 1992,27,811. Physical Methods and Techniques -Part (ii) Mass Spectrometry 39 kilovolt energy conditions.' 78 Allen and Vestal developed an ES system using heat as a means of desohation and no counterflow of heated gas."' The ability to obtain fragmentation data by collisional-induced dissociation was demonstrated clearly. ES mass spectra of human haemoglobin chains and transferin were acquired on a magnetic sector mass spectrometer.''' The observed isotopic distributions for the haemoglobin ion were in good agreement with the theoretical distribution within a resohtion of 2000. Accurate mass measurements have been obtained for masses in the 500-16000 range using a double-focusing magnetic-sector instrument and ES ionization.Magnetic-field scans and accelerating-potential scans were used and gave mass accuracies in the low ppm range in both positive and negative-ion modes. ES ionization has been used in a study of the reactions of multiply protonated melittin molecule ions. Flowing the ions entrained in gas through a heated metal capillary inlet served as a reaction vessel for measurements of ion reaction rates.IB2 Differences in activation energies found for different charge states were attributed to Coulombic repulsion effects. ES has been used to detect a ternary complex between the dimeric enzyme HIV-1 protease and a substrate-based inhibitor.183 Under denaturing conditions only the monomeric HIV-1 protease could be observed by mass spectrometry (see aIso ref.€84). Using the tightly binding competitive inhibitor JG365 the ternary complex was detected and this was dissociated by raising the declustering potential at the ion sampling orifice. Electrospray has been used to detect and differentiate betweenthe free human H-ras protein and its non-covalently bound GDP complexform. 85 Analysis of recombinant proteins has been performed using high-performance capillary elec- trophoresis interfaced with an electrospray ionization mass spectrometer.186 The detection Iimit was 100 fmol the standard deviation of peak migration times was less than 1YO,and mass assignment was within 1 unit in 22 0o0. The results showed that individually neither CZE nor ES ionization could detect all impurities but coupling enhanced the abilities of both.Oligosaccharides digested from asparagine side chains of glycoproteins have been analysed by ES and MS/MS. a Oligosaccharides containing sialic acids were detected best by negative-ion mode whilst others were best in the positive-ion mode. In positive-ion mode the addition of sodium or ammonium acetate greatly enhanced the ion abundances. MS/MS was used to detect primary structural features of the oligosaccharides. Boyle and Whitehouse coupled an ES source to a reflectron time-of-flight mass spectrometer achieving a resolving power of over loo0 by orientation of the ion source 178 J.A. Loo,A. B. Giordani and H. Muenster Rapid Commun.Mass Spectrom. 1992 7 186. 179 M. H. Allen and M. L. Vestal J. Am Soc. Mass Spectrom. 1992,3 18. Y.Wada J. Tarnura B+D.Musselman D. 8. Kassel. T. Sakurai and T. Matsuo Rapid Commun. Mass Spectrom. 1992,6 9. "' R.B. Cody J. Tamura and B,D. Musselman And. Chem. 1992,64 1561. lS2 M.Busman A. L. Rockwood,and R. D. Smith J. Phys. Chem. 1992,% 2397. lS3 M. Baca and S,B. H. Kent J. Am. Chem. Soc. 1992 114 3992 lS4 U.A. Mirza S. L.Cohen and B.T. Chait Anal. Chem. 1993 65 1. la' A. K. Ganguly B.N.Pramanik A. Tsarbopoulos T. R. Covey E. Huang and S.A. Fuhrman J. Am. Chem. Soc. 1992 114,6559. la6 K. Tsuji L Baczynskj and G.E. Bronson Anal. Chem. 1992,64 1864. Is' K.L. Duffin J.K. Welpy E. Huang and 3.D. Henion Anal. Chem. 1992,64 1440. 40 C.P.Ball and P. J. Derrick perpendicular to the field-free drift region.'*' The instrument was also sufficiently sensitive to allow the routine detection of low picomole quantities of multiply chafged cy tochrornes. 8 Matrix-assisted Laser Desorptlonflonization Matrix-assisted laser desorptionjionization (MALDI) remains one of the fastest- moving branches of mass spectrometry. MALDI in combination with time-of-flight (TOF) has been used for an extremely wide variety of applications normally involving high molecular mass compounds but some work involving smaller molecules has also been performed. The coupling of MALDI and FT-ICR has produced remarkable results; using laser fluences close to the MALDI threshold resolutions of 30000 have been presented for bovine insulin rising to 100OOO for lower mass proteins.Detection of a doubly-charged carbonic anhydrase trimer (m= 87OOO) has also been achieved. Optimum deceleration times for a singly-charged MALDI-generated protein ion are a linear function of Jm when a 9.5 V deceleration potential is used.leg Nelson and Hutchens used MALDI-TOF to examine the affinity of a 25-residue synthetic peptide for copper(I1) ions.'g0 The chemistry was performed on the probe tip and results showed the binding of up to one copper ion per histidine residue. MALDI was also used to identify metal-binding proteins in pruteolytic digests.'" His-tidine-rich glycoprotein a plasma and milk metal-transport protein was purified and digested with trypsin; the fragments were analysed by MALDI-TOF.Copper@) ions were added to the digest mixture and at least one metal-binding peptide was seen. This is the first direct observation by MS of differential peptide-metal digests in protein digest maps. A TUF mass spectrometer with an electrostatic mirror has been used to measure the kinetic energy distributions of molecular ions ejected into an externaI field by MALDI. An energy deficit was observed by this experiment (ix.the ions had less kinetic energy than imparted by the field) of around 24 eV in the typical case of insulin. This deficit increases with molecular ion signal intensity and with molecular weight of the protein. It is aIso larger for negative ions than fur positive ions.'92 Pan and Cotter investigated the initial kinetic energy distributions of arginine-vasopressin molecule-ions gener- ated by UV-MALDI.lg3 The measured molecule ion kinetic energy distribution from vasopressin was found to be much broader than that from gramicidin S and was characterized by a high-energy tail possibly resulting from entrainment of analyte ions in the matrix ions as they leave the surface.Ehring Karas and Hillenkamp have studied a large number of small polar organic molecules which have a strong resonant absorption at the laser ~avelength.'~' In most cases radical cations and ion species formed by single and multiple hydrogen cleavages and additions appeared as strong signals. A model was proposed which explains these features as a product of initiaI photoionization. J.G. Boyle and C. M. Whitehouse Anal.Chem. 1992,64 2084. J. A. Castor0 and C.L. Wilkins Anai. Chem. 1993,65,3015. R.W. Nelson and W.T. Hutchens Rapid Cornrnun.,Moss Spectrom. 1992,6,4. 191 R. W. Nelson W.T. Hutchens and T.T. Yip FEBS Lett. 1992,2% 99. 192 J. Zhou W. Ens K.G. Standing and A. Verentchikov Rapid Commun. Mass Spectrom. 1992 6,671. Y. Pan and R. J. Cotter Org. Mass. Spectrom. 1992 27,3. 194 H. Ehring M. Karas and F. Hilienkarnp Urg. Mass. Spectrom. 1992 27 472. Physical Methods and Techniques-Part (ii) Mass Spectrometry 41 Chan er al. demonstrated that cluster ions extending to very high masses (>500000Da) can be formed and detected from proteins using the liquid 3-NBA matrix for UV-MALDI.'95 Excellent shot-to-shot reproducibility was reported for this liquid matrix.An organic dye added to the matrix was found to increase the efficiency of visible laser systems. The addition of Rhodamine 6G to 3-nitrobenzyl alcohoI allowed the production of protein ions of mRup to 250000 using a 532 nm It has been found that the UV (248nm) desorption of neutral peptide moIecules is greatly enhanced by the application of a thin layer of the sample on top of a UV-absorbing organic substrate (sinapinic acid).' Using this technique gramicidin S is desorbed as an intact neutral with approximately 1eV less internal energy than if desorbed from a metal surface. a-Cyano-4-hydroxycinnaminicacid when used as a matrix,'99 has been found to enhance the tendency of proteins to protonate multiply. Ionic and neutral products of matrix-assisted laser desorption have been detected simultaneously in a TOF mass spectrometer the neutrals being photoionized by coherent UV radiation.The ratio of neutra1:ions has been calculated at around 10OOO :1 with desorption irradiances close to the threshold for ion formation. This ratio decreases with increasing irradiance from the laser beam. This dependence is consistent with a colIisiona1 mechanism for analyte-ion formation during the desorption initiated by photoionization. Matrices giving large signal intensities of directty formed analyte ions gave large intensities of neutral ions as Metastable decay of laser-desorbed ions taking place in the first field-free drift region of a reflectron TOF mass spectrometer has been successfully monitored to obtain structural information from the larger jxptides.201 Fragments from such metastable decay are mass analysed by adjusting the potentials of the ion reflectron according to the kinetic energies of the ions.It has been discovered that post-source decay in MALDI is only 10% with nitrogen as the residual gas compared to An excimer-pumped dye Iaser generating ultra-short pulses (560fs full width at half maximum) in the UV (248nm)and visible (496 nrn) has been used to desorb protein ions from different matrices.203 The existence of a threshold energy for the MALDI of insulin ions by the ultra-short pulses was established and found to be similar to that observed at much longer pulse lengths. The amount of laser energy deposited into the sample not the laser power thus appeared to be important in MALDI.ResuIts obtained frum MALDI work involving bovine insulin in 3-nitrobenzyl alcohol or sinapinic acid matrices suggest that the threshold fluences for the formation of positive and negative ions are the same for a given matrix.204 Laser generated plumes such as those observed in MALDI have been imaged by fluorescence measurements giving concentration maps at time-intervals of 100 19' D.T. W. Chan A. W. Colburn. and P. J. Derrick Urg. Moss. Spectrom. 1992,27 53. 196 D.S. Carnett M.A. Duncan and J. I. Amster Org. Mass. Spectrorn. 1992,27 831. 19' D.S. Carnett M. A. Duncan and J.I. Amster Anal. Chem. 1993,65,2508. 198 J.P. Speir and J.I. Amster Anal. Chem. 1992 64 1041. 199 R. C. Beavis T. Chaudhary and B.T.Chait Org. Mass. Spectrorn. 1992,27 156. 2oo C. D. Mowrey and M.V. Johnston Rapid Cummun. Mass Spectrum. 1993,7 569. B. Spengier D. Kirsch R. Kaufmann and E. Jaeger Rapid Commun. Mass Spectrom. 1992,6,105. '02 B. Spengler D. Kirsch and R. Kaufmann. J. Phys. Chem. 1992,% 9678. *03 P. Demirev,A. Westman,C.T. Reirnann P. Hakansson D. Barofsky B. U. R. Sundqvist Y. D. Cheng W. Seibt and K. Siegbahn Rapid Commun. Mass Spectrom. 1992 6 187. '04 P.Y. Yau D.T. W. Chan P.G.Collis A. W. Colburn,and P.J. Derrick Chern. Phys. Lett. 1993,202,93. '05 T. W.Heise and E. S. Yeung Anal. Chem. 1992 64 2175. 42 C. P. Ball and P. J. Derrick Biemann and coworkers have studied MALDI using a double-focusing magnet- ic-sector mass spectrometer. Pulsed ion signals were generated by irradiation of a mixture of sample and matrix (2,5-dihydroxybenzoic acid) with either an XeF laser (353 nm)or a nitrogen laser (337 nm) and were detected by a focal-plane detector.A resolution (full width at half maximum) of 4500 was achieved for substance P (m/z = 1347.7) 2500 fur CsI cluster ions at m/z = 10005.7 and 1250 for the isotope cluster of horse cytochrome c (m/z = 12360); 11fmol of substance P was detected.'06 A simple method for the off-line coupling of CZE and laser desorption has been described with a detection limit of 1OOfmoI for bovine IactaIb~rnin.~~~*~~~ The challenge of interfacing liquid chromatography with MALDI has been exarnined.'O9 Liquid sample has been introduced directly into the ion source of the mass spectrometer through a capillary.210 Evaporative cooling was used to produce ice at the end of the capiflary which could be extruded using resistive heating.Immobiliz- ation procedures were used to improve the MALDI of hydrophobic cytochrome P-450 proteins containing high concentrations of buffer. A mass accuracy of 0.075% was achieved using bovine serum albumen as an internal standard.21 Smalf fragments of DNA up to a 34-mer have been analysed with various Both positive and negative ions were seen with little fragmentation. A similar series of experiments have been performed on oligodeoxyribonucleotides as various matrices were examined for their efficiency in analysis.213 Mixtures of low picomole amounts of oligodeoxyribonucleotideswith sizes from 6 to 30 units have been e~amined.~ I4 Again molecule-ions were observed again with little or no fragmenta- tion.MALDI has been used to analyse crude synthetic pep tide^.^'^ A highly hydrophobic six-helix bundle template-assembled synthetic protein a 1Prner peptide with an amphiphilic a-helix and a 20-mer thrombin inhibitor were all successfully anaIysed. MALDI was used to verify the insertion of glycine into a C-terminal a-helix thus solving a crystallographic problem in the inability to pinpoint the nature or location of an anomaly in a protein sequence.* t6 MALDI was used to identify rapidly sensitively and sequence-specifically phosphopeptides in unfractionated digests of pho~phoproteins.'~ Beavis produced a review of MALDI covering topics such as sample preparation the nature of the spectra nomenclature and matrices.21 9 Fourier-transform Ion Cyclotron Resonance Electrospray has been combined with Fourier-transform ion cyclotron resonance '06 R.S.Annan H.J. Kochhng J.A. HilI and K. Biemann Rapid Commun.Mass Speccrom. 1992,6 298. 207 T. Keough R.Takigiku M.P. Lacey and M. Purdon Anal. Chem. 1992,64 1594. J. A. Castoro R.W. Chiu C. A. Monnig and C. L. Wilkins J. Am. Chem. SOC. 1992 114 7571. '09 L. Li A. P. L. Wang and L.D. Coulson And. Chem. 1993 65 493. 'lo E.R. Wilhams,G. C. Jones L.L. Fang,T. Nagata R.N. Zare Proc. SPIE-lnt. SOC.Opt. Eng. 1992,172. S. Lewis K. K. Korsrneyer and M. A. Correia Rapid Commun. Mass Spectrom. 1992 7 16. 'I2 K. Tang S. L. Allman and C.H. Chen Rapid Commun. Mass Spectrom.1992,6 365. E. Nordhoff A. Ingedoh R. Cramer A. Overberg 8. Stahl M. Karas F. HiIlenkamp and P. F. Crain Rapid Commun. Mass Spectrorn. 1992,5 771. G.R. Parr M. C. Fitzgerald and L. M. Smith Rapid Commun. Mass Spectrorn. 1992 6 369. 215 V. Steiner K.O. Boernsen M. Schaer E. Gassmann S. Hoffstetter-Kuhn H. Rink and M.Mutter Peptide Res. 1992,5 25. 'I6 L. J. Keefe E. E. Lattman C. Wolkow A. Woods M. Chewier and R. J. Cotter J. Appl. Crystallogr. 1992 25 205. 217 T.T. Yip and W.T. Hutchens FEBS Lett. 1992,308 149. 'la R. C Beavis Org. Mass Spectrom. 1992 27 653. PhysicaI Methods and Techniques -Part fii) Mass Spectrometry 43 (FT-ICR) with the ion source positioned in the bore of the superconducting magnet adjacent to the trapped ion 1x11.~’~ An electrospray current of 375 pA could be detected from gramicidin S at the cell which is two orders of magnitude higher than an external ion source.Five stages of differential pumping within a series of concentric tubes of increasing radius were used to reduce pressure to lom7torr in the trapped cell. Wilkins and coworkers explored the utility of ‘screened’ electrostatic ion trapping techniques. The observed rate of change of ion cyclotron resonance (ICR) frequency with trapping potential is independent of mass and charge fin the range of 1185-17 OOO Da) and using a magnetic field of -7 T the electric potential within a screen cubic trap is reduced decreasing frequency shifts as a function of trapping potential by a factor of 25. The average resolution for a bovine insulin spectrum was 10OO0.220 Hofstadler and Laude have interfaced an electrospray ionization system with FT-ICR.In their system the electrospray occurs at atmospheric pressure in a 1.5Tfield and the FT-ICR detection takes place 25cm away at 3.0Tin one or two cells separated by a conductance limit and maintained at a pressure differential of 5 x lo-’ and 2 x torr. The trapped ion cell wits filled up to the ion space charge limit within a few hundred milliseconds. The rate of ion accumulation is independent of background pressure; hence the primary trapping mechanism is not solely colIision dependent.22 Trapped ion cells which substitute open rectangular trapping electrodes for trapping plates perpendicular to the magnetic field have been evaluated.z2z Radical trapping electric fields are somewhat larger in the centre of the open cells but axial excitation fields are a factor of two smaller compared with the closed cell.Axial ejection is significantly reduced in the open celI compared to the closed cell. The question of poor high-mass (high m/z) performance in FT-ICR has been addressed.z23 Theoretical calcuIations and experimental data showed the existence of resonances involving the z-motion and the radid motion of the ion which result in energy transfer between modes expansion of the ion cloud and a signal loss. Polypeptide ions formed by MALDI have been found not to be in thermaI equilibrium with their surroundings but to exhibit a non-BoItzrnann initial ion velocity distribution about a most probable ion velocity which is independent of polypeptide mass.Radical and axial m/z upper limits and optimum trapping potentials were derived and discussed.224 Analytical expressions fur the probability ofradial or axial loss of thermal ions from a hyperbolic ion trap were developed and gave rise to the observation that the probability of radial ion loss increases with m/z ratio and ion thermal energy whilst axial ion loss is inversely proportional to trapping potential and directly proportional to thermal energy. This model is based on the assumptions that non-interacting ions are formed initially along the trap y axis with a Maxwell-Boltzrnann velocity distribution and trapped by a quadrupolar electrostatic potentiaLZz5 The absoiute number of trapped ions that produce a time-domain signal in FT-ICR has been estimated by comparing the experimentally observed signal voltage to that calculated ”’S.A. Hofstadler and D. A. Laude Jr. Anal. Chem. 1992,64 569. ”* J.A. Castoro C.Koster and C. L. Wilkins Anal. Chem. 1993,65 784. ”’ S.A. Hofstadler and D,A. Laude Jr. J. Am. SOC.Mass Spectrom. 1992 3 615. 12’ S.C. Beu and D.A. Laude Jr. int. J. Mass Spectrom. Ion Processes 1992 112 2. 223 C.L. Holliman D. L. Rempel and M. L. Gross J. Am. SOL Mass Spectrom. 1992,3,460. 224 T.D. Wood,L. khweikhard and A.G. Marshall Anal. Chem. 1992,64 1461. 225 M. A. May P. B. Grosshans and A. G. Marshall Int. J. Mass Spectrom. Ion Processes 1992 120 193. 44 C.P. Ball and P. J. Derrick for a single ion orbiting at the ICR orbital radius of the ion packet.This gives an estimate of -177singly charged ions in a cubic trap at 3.0T.226 Detection limits have been reduced by subjecting a single population of multiply charged protein ions formed by ES to multiple FT-ICR without promoting ion loss from the trapped ion cell. This process is non-destructive and allows at least 250 consecutive remeasurements of a single population of protein ions. A 25 :1 signal :noise ratio has been observed for 30fmol of bovine albumin di~ner.~’~ A two-dimensional charged-disk model was used to explain Coulomb-induced frequency shifting in FT-ICR MS.228 The model consists of a uniformly charged disk whose excited cyclotron motion is perturbed by another uniformly charged disk with excited cyclotron motion.This model gave a better agreement with the experimental data than the charged-point model. Control of ion kinetic energy in ICR spectrometry with respect to very low-energy collision-induced dissociation has been examined by Boering et aL2” Ions were continually accelerated and decelerated over a period of hundreds of milliseconds by repeatedly shifting the phase of the ratio frequency driving signal by 180” using an external electronic circuit. At low kinetic energies a succession of collisions which transferred small amounts of energy favoured the lowest energy fragmentation pathway and allowed ions with large activation energies to fragment at energies very close to threshold. Solutions have been reported for the frequency shift due to the induced image charges for both cylindrical and tetragonal ion traps as a function of ion axial and radial position.For a cubic trap the shift for an ICR orbital radius of one half the trap radius is -10p Hz for one ion. Thus a frequency shift in the ppm range can be expected for typical ICR experiments involving 1000-100 O00 ions at low m/z ratio (60-600).This has important implications for calibration.230 A new 20-dimensiona1 FT-ICR experiment stored-waveform ion modulation or SWIM 2-D-FT-ICR-MS/MS has been developed. Each row of the two-dimensional data array is obtained by the use of a single stored excitation waveform whose frequency-domain magnitude spectrum is a sinusoid the frequency of which changes from one row to the next.The conventional FT-ICR spectrum appears along the diagonal of the two-dimensional mass spectrum with off-diagonai peaks correspond- ing to ion-neutral reactions whose ionic components may be identified by horizontal and vertical projections to the diagonal spectrum. Fragmentation due for example to collision-induced dissociation results in a peak on only one side of the diagonal; bidirectional processes give peaks on both sides. All ion-molecule reactions can therefore be seen without any prior knowledge of the Self-assembly alkanethiol monolayers on gold films have been studied by laser desorption at 193 nm radiation.232 Adding sugars as comatrix elements permitted higher resolution than without sugar in MALDI-FT-ICR. Mass resolution of 6-10 OOO was obtained for melittin bovine insulin and bovine insulin 3chain which rose if ribose or fructose was added to the matrix.233 Capillary electrophoresis has been ”‘P.A. Limbach P. B. Grosshans and A.G. MarshalI And. Chem. 1993 65 135. 227 2.Guan S.A. Hofstadler and D.A. Laude Jr Anal. Chem. 1993 65 1588. ’’’ S. P. Chen and M.B. Comisarow Rapid Cornmun. Mass Spectrom. 1992,6 1. 229 K.A. Boering,J. Rotfe and J. I. Eirauman Rapid Cornmun. Mass Spectrom. 1992,5 303. 230 X. Xhang P.B. Grosshans and A.G. Marshall Int. J. Mass Spectrom. lon Processes 1993 125 33. 231 C. W. Ross 111 S. Guan P. B.Grosshans T.L. Ricca and A. G. Marshall J. Am. Chern.Soc. 1993,115 7854. 232 Y. Li J. Huang R.T. Mclver Jr and J.C. Hemmtnger J. Am. Chem. SOC. 1992 114 2428. 233 C. Koster,J.A. Castoro and C.L. Wilkins J. Am. Chem. SOC.,1992,114 7572. Physical Methods and Techniques -Part (ii) Mass Spectrometry 45 successfully interfaced with FT-ICR using an electrospray interface and a high speed cryopurnping arrangement. Initial results show that spectra with high sensitivity and excellent resolution (30ooCr50000)can be obtained every six FT-ICR has been used as a post-ionization technique for separation of electros-prayed protein mixtures.235 The relative order of trapping of the ions is determined by the application of potential to the skimmer cone and the ion velocity. The optimum trapping potential has been found to depend primarily on the m/z ratio within the electrospray envelope but also on protein-specific features including velocity slip in the expansion.10 Tandem Mass Spectrometry The combination of electrospray and FT-ICR provides possibilities for tandem mass spectrometry (MS/MS) and hence elucidation of molecular structure through collision-induced dissociation in the actual ion source (so called ‘nozzle dissociation’) in the case of very large ions (104Da).236 The difficulties inherent in the analysis of large multiply-charged proline-containing proteins have been addressed by Smith and coworkers.z37Low resolution tandem mass spectrometric data can be ambiguous due to the tendency of proline-containing proteins to form multiply-charged product ions. Collisional dissociation of proteins of up to 22 OOO Da ionized by ES was considered. Fragmentation due to cIeavage of the peptide bond near proline residues was usually dominant and this process was found to be the only dissociation pathway observed for many large proteins.Multiply-charged ions of an IgG class monoclonal antibody anti(human a,-acid glycoprotein) which has a weight of 149600 Da were produced by pneumatically assisted ES and then subjected to coilisionally activated dissociation with argon at collision energies of 5-12 keV in a triple-quadrupole mass spectrometer. The structurally significant light-chain fragment (23 720 f30 Da) was collisionally released. There is evidence that this took place near an energetic threshold and the energy required may well have come from the many collisions encountered by the anti body ions. Peptide and caesium iodide cluster ions decomposing to certain fragment ions following collision-induced decomposition have been shown to exhibit energy losses independent of transmission whilst parent ions of certain other fragments show strong dependence of energy loss upon transmission.These results have been interpreted in terms of energy redistributions taking place in single collisions by direct momentum transfer.239 MS/MS results for high mass ions (m/z 1000-2500) comparable to those obtained by four-sector instruments were obtained using a magnetic sector/quadrupole instru- men t . 40 Kenny and Orlando have reported MS/MS analysis of peptides at the femtomole 234 !LA. Hofstadler J.H. Wahl J.E. Btuce and R.D. Smith J. Am. Chem. Suc. 1993 115 6983. 235 S. A. Hofstadler S.C. Beu and D. A. Laude Jr Anal. Chem. 1993,65 312. ‘’‘ J,A. Loo J. P. Quinn S.I.Ryu K. D. Kent M.W. Senko and F. W. McLafferty Proc. Natl. Acad. Sci. U.S.A. 1992 89 286. 237 J. A. Loo C.G. Edmonds and R.D. Smith And. Chem. 1993,665 425. 238 R. Feng and Y.Konishi Anal. Chem. 1993 65 645. 239 C.D. Bradley and P.J.Derrick Org. Mass. Spectrom. 1993 28 390. 240 C. D. Bradley J. M. Curtis P. J. Derrick and B. Wright Anal. Chem. 1992,64 2628. 46 C.P. Ball and P.J. Derrick leveI using a four-sector instrument and array dete~tion.'~' Emptoying continu- ous-flow FAB the structures of several peptides (9W2000Da) at the 9OOfrnol to 5.8 pmol level were elucidated without interference from the background. Metabolites in a crude plasma extract from spontaneously hypertensive rats were identified without chromatographic separation by FAB MS/MS.242 The calcium-entry blocker AJ-2615 was detected and the structure elucidated.Compound-specific sequence ions were identified in the collision-induced dissociation spectra of the immonium ions of two isomeric hexaglycosyl~erarnides.~~~ Using standard EI the spectra had been too complex to distinguish the isomers from one another. Structure-specific collision- induced fragmentations of ceramides has been achieved by complexation of the analytes with alkali metal ions. Structures of individual components of complex mixtures could be detected with detection limits as low as 8 prn01.~~~ A number of different devices that can be used for surface-induced dissociation (SID) in tandem quadruple instruments have been evaluated on the basis of fragmentation of (C6H6)+*,[W(CO),]'* and (Me,N)+.Spectra obtained by in-line and 90" instruments were found to be similar.24s The overall efficiency for even-electron ions was higher than for odd-electron ions of similar structure. High average internal energies and narrow distributions of energies were said to be imparted to the parent ions in SID. SID of angiotensin I renin tetradecapeptide porcine pancreastatin fragment 3349 and ACTH fragment 1-17 has been observed in a four-sector MS/MS spec-tr~rneter.'~' Abundant fragment ions were seen facilitating determination of the amino acid sequences. SID has been applied to smal1 protonated peptides generated by The relative abundances of daughter ions were found to depend critically upon the energy of the ion-surface collision.At approximately 13eV collision energy the number of decomposition processes was maximized for the peptides studied. For the S1D process the kinetic energy distribution of the parent ions was said to be converted into a wider spread of centre-of-mass collision energies leading to a larger variety of decomposition processes compared to collision-induced dissociation. A comparison of the molecular and quasi-molecular ions of valinornycin formed using EI FAB field desorption (FD) and ES four-sector MS/MS has been made by Curtiset aLZ4'Large differences were observed between the FD and FA3 tandem mass spectra of [M + Na]" ions and between FD and EI spectra of M" ions.This was ascribed to formation of ions of different structures. A number of small peptides (500-1800amu) with various different masses were fragmented by SID in a tandem mass spectrometer as a comparison with collkional activation. The results allow the formulation of a model to explain the easy cleavage of 24i P. T. M. Kenny and R. Orlando And. Chem. 1992,64,957. 242 M. Kurono A. Itogawa K. Yoshida S. Narto P.Rudewicz and M. Kanai Biof. Moss Specrrorn. 1992.21 17. 243 J. M. Curtis P.J. Derrick J. Holgersson B. B. Samuehson and M. E. Breimer J. Am. Soc. Muss Spectrorn. 1992 3 353. 244 Q. Ann and J. Adams Anal. Chem. 1993,65 7. "'V.H. Wysocki,M. J. Ding J. L. Jones J. H. Callahan and F. L. King J. Am. Soc. Moss Spectiom. 1992,3 27.2*6 A. D. Wright D. Despeyroux K. R. Jennings S. Evans and A. Riddoch Urg. Mass. Spectrom. 1992,27 525. 247 R.B.Cole S. LeMeillour and J.C. Tabet Anal. Chem. 1992,64,365. ''' J. M.Curtis,C. D. Bradley P.J. Derrick and M. M. Shiei Org. Mass. Spectrom. 1992,27 502. Physical Methods and Techniques-Part (ii) Mass Spectrometry 47 the amide bond charge-remote backbone fragmentations and the influence of intramolecular hydrogen bonding.z49 Collision-induced dissociation was again com- pared with surface-induced dissociation (at 10eV) for peptides of mass 1300-1850. Only a small amount of the kinetic energy of the precursor ions is converted into internal energy for SID as the spectra resemble low energy collision-induced dissociation rather than the high energy spe~tra.’’~ A quadrupole ion trap storagelreflectron TOE; mass spectrometer has been developed as a detector for ES ionization.A continuous ES beam is converted into a pulsed beam for TOF measurement. Sensitivity in the low ferntomole region was demonstrated with small peptides. The spectrum can be acquired in less than one second.251*252 A reflectron TOF mass spectrometer for laser photodissociation has been described by Cornett et a1-253Mass selection was performed by pulsed deflection plates at the end of the initial drift section. Laser photodissociation of ions was effected at the turning point of the ion trajectory in the reflectron and the transit time through a second drift section defined the fragment masses (see also ref. 254). Scan laws havebeen derived for the detection of fragment ions produced by coIlisiona1 activation of a multiply-charged precursor in a floated collision cell of a four-sector tandem mass ~pectrometer.~~ ’ A review of collisional activation in MS/MS has been given by McL~ckey.~~~ A review of trace analysis of organics in mixtures has been made by Bieman~~~’ 11 Ion Trap The ion-trapping technique holds great promise as an inexpensive relatively high-performance mass spectrometer.The selectivity of methykne substitution in a low-pressure quadrupoie ion trap was elucidated by collisionally-activated dissociation.258 Dimethyl ether and ethylene the covalently bound adducts found at [M+ 451 and [M+ 411 respectively are direct + precursors to the methylene substitution product ions at [M+ 131+.It was discovered that at low helium pressures collisional-activated dissociation shows that the average internaI energy deposition is higher and sensitivity is not lost if a collisional cooling delay is added.259 Heavy target gases improved the trapping efficiency of caesium iodide clusters when added in small amounts to the helium buffer gas.260 Trapped ions were activated by applying a short DC voltage pulse to one end of a quadrupole ion-trap mass spectrometer.High internal energy deposition was obtained as evidenced by peak ratios in excess of 20 fur the m+ = 91 :m+ = 92 ions for the dissociation of the n-butylbenzene ion.26 249 A. L. McCorrnack A. Somogyi A.R. Dongre and V. H. Wysocki Anal. Chem.1993,65,2859. D. Despeyroux A. D. Wright and K. R. Jennings tnr. J. Mass Spectrom. ion Processes 1993 126 95. 25’ S. M.Michael B. M. Chien and D. M.Lubman Anal. Chem. 1993 65 2614. 252 S. M. Michael B. M. Chien and D. M. Lubman Rev. Sci. instrum. 1992,53 4277. 253 D.S. Cornett M. Peschke K.LaiHing P. Y. Cheng and K.F. Willey Rev. Sci. Inslrum. 1992,53,2177. 254 D. R. Jardine J. Morgan D. S. Alderdice and P.J. Derrick Org. Mass. Spectrom. 1992 27 1077. Is’ R. Orlando and R.K.Boyd,Urg. Mass. Spectrum. 1992 27 151. 256 S.A. Mchckey J. Am. SOC.Mass Spectrim. 1992 3,599. ’” K. Biemann Pure Appl. Chem. 1993,65 1021. ’” T. Donovan C.C. Liou and J. Brodbelt J. Am. SOC. Mass Spectrom. 1992,3 39. 259 C. Paradisi J.F.J. Todd P.Traldi and U. Vettori Org. Muss.Spectrom.1992 27 251. 260 K. L. Morand K. A. Cox and R.G. Cooks,Rapid Commun.Muss Spectrom. 1992,6 520. ”’ S.A. Lammert and R. G. Cooks Rapid Commun. Mass Spectrom. 1992 6 528. 48 C.P. Ball and P. J. Derrick The effect of the radial and axial dimensions on the Mathieu stability diagram (in terms of DC and RF voltages) has been discussed in relation to the stretched geometry employed in a commercial quadrupole ion-trap mass spectrometer.262 The effects of storing ions of different values of the stability parameters a and q were studied in a quadrupole ion trap using helium or argon A region was localized near the boundaries of the stability diagram where the ions experience an increase in their kinetic energy reflected by the occurrence of fragmentation due to collisional activation and a certain extefit of ion loss due to unstable trajectories.These effects known as boundary effects depend on the buffer gas and on q of the ions it is rep~rted.'~' Simultaneous data obtained from each of the two steps of a MS/MS experiment show that the collisional history of the ions before the isolation process can greatly influence the degree to which ions can survive isolation. The facility with which ions absorb energy from the field within the trap or whether the field is derived from the radiofrequency potential or a supplementary potential can determine the extent to which ions are retained in the field.265 The fullerenes C, and C, were found to be ionized by desorption from a liquid matrix upon bombardment by caesium ions of 7 KeV kinetic energy.266 The resulting radicaI cations when activated in the ion trap by collisions with Xe target gas undergo extensive dissociation by loss of multiple C2 units.Expressions for the ion-resonance ejection line shape have been derived incorporat- ing terms for ion-molecule collisions and variation with time of the resonance excitation frequency.267 The results show that for any non-zero scan rate there is an optimum collision damping value corresponding to a resolution maximum. Schwartz and Jardine discovered that the same techniques used for generation of high-resolution mass spectra on a quadrupole ion trap can be used for selection or storage of a very narrow mass window with nearly 100% efficiency.268 Resonant ion ejection in a quadrupole ion trap was used to obtain mass measurement accuracy of better than 0.015% on gramacidin SZ6'Traldi Catinella and Bortolini suggested that mass displacements seen in the ion-trap mass spectra of certain fluorinated benzene derivatives were due to an interaction with the permanent dipole moment of the ion within the radiofrequency field.270 It was found that at specific q values higher-order fieId effects caused a decrease in the intensity of chemical ionization reagent ions due to inefficient storage.These are found to occur continuously along iso-j? lines for fi2 = 1/3 and 2/3.27' McLuckey et al. found that random noise applied to the end caps of quadrupole ion traps was effective in inducing collisional activation of trapped ions independent of m/z ratio and number of ions.272 Efficiency was a factory of three less "'J.V. Johnson R. E. Pedder and R. A. Yost Rapid Commun. Mass Spectrom. 1992,6 760. 263 K. L. Morand S. H. Hoke 11 M. N. Eberlin G.Payne and R.G. Cooks,Urg. Mass. Spectrom. 1992,27 248. 264 C. Paradisi and P. Traldi Rapid Commun.Mass Spectrorn. 1993 7 690. ''' R. E. March M. R.Wier M. Tkaczyk F. A. Londry R,L. AIfred A. M.Franklin and J. F.J. Todd Urg. Mass.Spectrom. 1993 28 499. 256 H. F. Wu and 3. Brodbelt int. J. Mass Spectrorn. Ion Processes 1992 115 67. 2c7 D. E,Goeringer W. B. Whitten M.J. Ramsey S. A. McLuckey and G. L. Glish Anal. Chem. 1992,64 1434. J. C. Schwartz and I. Jardine Rapid Commun. Mass Spectrom. 1992 6 313. 269 J. D. Williams and R.G.Cooks Rapid Commun.Mass Spectrom. 1992 6 524. 270 P. Traldi S. Catinella and 0.Bortolini Urg. Mass. Spectrom. 1992 27 927. 271 D. M.Eades and R. A. Yost Rapid Commun. Mass Spectrom. 1992,5 573. 27f S.A. McLuckey D. E. Goeringer and G. L. Glish Anal. Chem. 1992,64 1455. Physical Methods and Techniques -Part (ii) Mass Spectrometry than for conventional methods and there is evidence for discrimination against low m/z product ions for multiply-charged biomolecules however. A quadrupole ion-trap mass spectrometer was used to examine internal energy deposition and fragmentation pathways of ionized pyrene and anthracene.”’ Through multiple collisional-activation steps large amounts of energy can be deposited and multiple-stage dissociation reactions are observed.High-energy pathways can also be accessed in single stage MS/MS experiments by introducing a small amount of xenon as a target gas with the helium buffer which also imparts a high dissociation efficiency. A capillary SFC instrument with direct on-column injection was connected to a quadrupole mass filter/ion trap by a custom buiIt interface.274 The ion source was a standard electron impact-chemical ionization and operated using conventional ion trap electronics. This enterprising system was characterized and optimized. A sample of 80 fmol of anthracene provided a molecular-ion peak with a signal-to-noise ratio of 20. The interfacing of particle beam analyses with ion traps has been performed The particle-beam interface was directly coupled with a quadrupole ion-trap mass spectrometer without the use of an external ion source and vaporization ofthc particle beam and ionization by electron impact are all accomplished in the ion trap.Lower limits of detection for pesticides were seen than for conventional particle-beam systems using quadrupole mass analysers although non-linear calibration curves seem to be the rule.27’ Quantitative analysis by ion-trapping was investigated by Strife and Simms when they used one gram plasma samples spiked with antiinflammatory drugs at levels of 1-100ng (with a standard at 50ng).276 Crude extracts of the sample were analysed using scan voltages combining radiofrequency (r.f.) and d.c. voltages and an ion-trap tandem mass spectrometer. Ion-trap chemical ionization performance has been improved by the application of a modified scan function for the rejection of undesired electron-ionization-like ions formed at the beginning of the ionization period.277 Desorption-ionization has been accomplished in a quadrupole ion trap by using a fibreoptic interface with Gramicidin C as the reagent.278 Trapping was found to be most efficient using a high gas pressure (21mtorr) a long storage delay (250 rns) prior to detection and a low radio-frequency trapping potential during the desorption pulse.Ion-molecule reactions were also observed during the experiments. A MALDI instrumentfor use with ion trap mass spectrometry has been developed. The laser beam and the sample are brought through holes in the ring electrode allowing the laser-desorbed ions to expand and fill the ion trap.Detection limits in the ferntornole range have been achieved for many small polypeptides such as bradykinin neuro- medim U-8 and leucine-enkephalin. However a loss of sensitivity was seen at m/z above 3000,279 273 B.D. Nourse K.A. Cox K.L. Morand and R.G. Cooks,1.Am. Chem. SOC. 1992,114,2010. 2 74 J. D. Pinkston T. E. Delaney K. L. Morand and R.G. Cooks,Anal. Chem. 1992,64,1571. ”’ B.L.Kleintop D. M. Fades and R. A. Yost Anal. Chem, 1993,65 1295. 276 R.J. Strife and J. R. Simms J. Am. SOC,Mass Spectrom. 1992,3,372. 277 T. Cairns K. S. Chiu E. Siegmund and M. Weber,Rapid Commun. Mass Spectrom. 1992,6,449. 278 A. McIntosh T. Donovan and J. Brodbelt Anal. Chern. 1992,64,2079. 279 D. M.Chambers D. E.Goeringer,S.A. McLuckey. and G.L. Glish Anal. Chern. 1993,65,14.