Proton Transfer Protein Peptide

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J O U R N A L OF MASS SPECTROMETRY, VOL. 30, 339-347 (1995)

Proton Transfer Reactions of Multiply ChargedPeptide and Protein Cations and AnionsR a c h e l R. Ogorzalek Loot an d R i c h a r d D. S m i t h *Chemical Meth ods and Separat ions Group , Chemical Sciences Department , Pacific Northwest Laboratory, Richland,Washington 99352, USA

Two types of gas-phase proton tra nsfer reactions were exam ined with electrospray ionization-generated peptide andprotein ions; (i) bases reacting with multiply protonated molecules and (ii) acids reacting w ith multiply deprotonat-ed molecules. Fo r reactio ns of type (i) with bases spanning a ran ge of proton affinities, the proton transfe r reactionra te was observed to increase with increasing proton affinity of the charge transfer reagent. P roto n tran sfer was notobserved for the low proton affinity rea gents (ethyl ace tate, acetonitrile a nd water). These studie s also qualitativelymeasured for the first time the temperature dependences for reactions with multiply charged peptides and proteins.Negative temperature dependences were observed for the weaker bases and positive dependences for the strongerbases. A negative temperature dependence was also observed in the reaction of propionic acid with ( M - nH1"-ions. Two hypotheses are proposed to explain th e data. In th e first, negative temper ature dependences are attribu tedto slightly exothermic reactions, while the positive dependences may reflect contributions from a competing clus-tering pathway, a pathway which could be more dominant with the heavier reagents. Alternatively, the positivetemperature dependences may reflect the barrier in the reaction coordinate arising from the repulsion of like-charged ions, while negative temperature dependences could reflect a cluster-mediated reaction in which chargedelocalization lowers the barrier to proton transfer. In the latter cases, clustering is invoked with the lower protonaffinity reagen ts because of th e higher concentrations employed.

INTRODUCTIONThe development of electrospray ionization masss p e ~ t r o m e t r y ' - ~(ESI-MS) not only offers new capabil-ities in molecular mass and structure determinations,but also opens up new classes of gas-phase ions forreaction studies. Ion-ion and ion-molecule reactionshave been carried out previously with ESI-generatedion^.^-^' Stud ies of positive ion-negative ion reactionsat ou r laborat ory led to the development of a Y-shapedcapillary inlet-reactor which merges streams ofoppositely charged ions at near atmospheric pressuresand transfers them to the mass ~pectr om eter.~ . 'Ion-ionexperiments were pursued for which both reactant ionswere generated by ESI and in experiments where onereactant was generated by ESI and the other was pro-duced in an electrical discharge in air. Proton transfer4and charge inversion5 reactions were studied with thisapproach.Experiments probing ion-molecule chemistry havebeen more common, including (i) proton t rans-fer,6-1 2.14-1 8 (ii) hydrogen-deuterium (H/D)exchange,' 3*19 (iii) c o l l i s i o n a l d i s s o c i a t i ~ n , ~ ~ - ~ ~(iv) col-lisional scattering,' 1,20*21 and (v ) clustering30 reactionstu dies. M cL uc key an d c o - ~ o r k e r s ~ - ~were the first todemonstrate the dependence of reactivity on chargestate for proton transfer of large multiply charged ions.Their quadrupole ion-trap experiments yielded ratemeasurements for reactions with am in es6 Ot her groups

* Paper prepared for Publication in Organic Mass Spectrometry.t Present address: Protein and Carbohydrate Structure Faci l i ty,2552 MSRB 11, University of Michigan, Ann Arbor, MI 48109-0674,US A

have pursued similar studies with quadrupole massIko nom ou and Kebarle" electro-sprayed ions into a reduced-pressure reaction chambercontaining tributylamine, while Feng and Konishi"'flooded an enclosed atmospheric pressure spray regionwith am mo nia. Lin and Sun nerI8 examined positive-ioncharge distributions after exposure to gas-phase acids.Smith and c ~ - w o r k e r s ~ ~ - ~ ~used the 'Y-tube' inlet/reactor to expose ions to varying flows of reagent atnear atmospheric pressure. Based on such studies,Winger et uLl3 argued that under more extreme inter-face conditions, ESI-generated cations can transferprotons to H 2 0 , and Ogorzalek Loo and co-w o r k e r ~ ~ ~ , ' ~ , ~ ~have demonstrated that a disulfide-intact protein is more reactive to proton transfer thanan equally charged disulfide-reduced protein. Relatedion-molecule reaction studies3' in an r.f.-only collisioncell yielded several molecules of ammonia clustered tothe precursor ion and also demonstrated proton trans-fer. Iab Recently, C assady et ul . l sc measured rates forprot on transfer from ubiquitin.Gas-phase H-D exchange has been applied to multi-ply charged ions by McLaffer ty and co-w~rkers '~an dby Winger et a l l 2 using a Fourier t ransform massspectrometer and a quadrupole mass spectrometer,respectively. Th e Cornell study" observed less H-Dexchange for disulfide-linked proteins than for theirreduced counterparts; both studies indicated that of therapidly exchanged hydrogens in equally charged ions,the disulfide-linked proteins exchanged H for D a thigher reaction rates than the disulfide-reduced pro-teins. These observations and the Fourier transformmass spectrometric observations of differing exchangerates and total exchanges for ions generated from differ-

CC C 1076-5174/95/020339-090 1995 by John Wiley & Sons, Ltd. Received 7 July 1994Accepted 12 September I994


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340 R . R. OGORZALEK LOO AND R. D. S M I T Hent solution conformers argue that elements of solution-phase higher order structure are preserved in the gasphase." Recently, collisional scattering studies havesupplied further evidence for gas-phase conformationaldifferences in proteins.".20.2'

Numerous collisionally activated dissociation (CAD)studies of multiply charged ions have now been report-ed,22p29ranging from demonstrations of facile cleavageat pro line^,^^ to differences in the cleavage betweendisulfide-intact and disulfide-reduced proteins,2hs27toCAD-MS comparisns of sequence homologies amongproteins from different species.28 Collisional disso-ciation of intact proteins as large as RMM 66 00028and 15000OZ9has been reported.In this paper, we explore proton transfer reactions ofmultiply charged ions, determining the role of reagentproton affinity and reaction temperature. Reactionswith negatively charged proteins and gas-phase acidsare also considered. The role of proton affinity isworthy of examination in our inlet/reactor studiesbecause of the reported reactivity of H 2 0 with multiplycharged proteins.' O ur experimental assembly doesnot insure that we probe thermodynamics; the resultsmore likely reflect reaction kinetics. However, theexperiments are still of interest owing to the wide differ-ences observed in proton transfer rates for multiplycharged ions,' in contrast to those usually observed forsingly charged ions.

EXPERIMENTAL

Application of the inlet/reactor approach to protontransfer reactions of multiply charged ions has beendescribed ' only a brief description of theapparatus is provided here. These experiments differfrom previous studies by quadrupole ion- traph p9 andF ou rie r tran sfo rm m ass s p e ~ t r o m e t r y ' ~ ~ ~ ' ~(FTM S) in areaction occurs in the atmospheric pressure/vacuuminterface, employing higher reagent gas flows. Reactionsin the high-pressure environment can rely on both bi-and term olecular collisions.The electrospray ionization atmosphere/vacuuminterface employed Y-shaped inlet/flow reactors con-structed from three lengths of 0.16 cm od., 0.10 or 0.05cm i.d. stainless-steel tubing. Inlet/reactors were fabri-cated by silver soldering the tubing to a stainless-steelpost drilled with a 0.10 cm diameter Y-shaped~ h a n n e l . ~ - ~ . ' ~ , ' ~One arm of the 'Y-tube' transportedions while the other arm delivered the neutral protontransfer reagent. Mixing of the two gas streamsoccurred in the Y-shaped channel and the third armdelivered the mixture to the mass spectrometer. Aninteraction time of a few ms is estimated for the proteinions with the reagent. Use of the 'Y-tube' apparatus inion-molecule reactions isolates the solution a t the tip ofthe ESI needle from the reagent, preventing elevation ofsolution pH from exposure to amines.The capillary was electrically heated with currentsfrom 11.0 to 14.5 A to drive the ESI process (e.g. desol-vate the droplet^).^"^^ Capillary temperatures weredetermined by a chrome1 alumel thermocouple attachedto the outer wall of the atmospheric pressure side of the

inlet. Actual gas temperatures are expected to be lowerthan the inlet wall temperatures indicated owing to heattransfer limitations. Additional desolvation wasachieved by collisional dissociation in the capillaryoutlet-skimmer (0s)region at potential differences ofAmmonia (NH,), methylamine (MA), dimethylamine(DMA) and trimethylamine (TMA), with proton affin-ities 871.5, 918.8, 953.1 an d 973.6 kJ mol-', respec-t i ~ e l y , ~ ~were delivered through a pressure regulator (setfor 1 atm delivery) and a needle valve. Water, acetonit-rile and ethyl acetate, with proton affinities of 696.6,34789.133 and 849.4 kJ mo l- 1 , 3 3 respectively, were intro -duced as mixtures with room air by employing a heatedbubbler assembly upstream of shut-off and needlevalves. Diethylamine (DEA), triethylamine (TEA). andtributylamine (TBA), liquids at room temperature, werealso introduced in this manner. Their proton affinitieshave been determined to be 977.0, 1002.5 an d 1017.5 kJ

mol - ', r e ~ p e c t i v e l y . ~ ~Addition of hexane-l,Bdiamine,a solid at room temperature, (proton affinity estimatedto be 22.6 kJ mo l-' higher tha n that of TEA)34 andpropionic acid, a liquid at room temperature, alsoemployed the heated bubbler assembly. The gas lineswere maintained at a higher temperature than thebubbler assembly in order to prevent solidification inthe gas lines. The mass flow for particular needle valvesettings was determined by measuring the evacuationrate of air-filled calibrated gas sampling bags and cor-recting for the molecular mass of the reagent. For air-reagent mixtures, the flow rate was corrected by theaverage molecular mass of the mixture; reagent vaporpressures were estimated using the Clausius-Clapeyronequation and Trouton's rule.One of the first tests performed with our inlet/reactorwas a confirmation that protein charge-state distribu-tions did not change on addition of air flowing throughthe assembly at various needle valve settings The resultsof this test demonstrated that any shifts in proteincharge-state distributions observed with amine reagentsarose from reaction with the amine and were not arti-facts generated by the change in flow rate through theinlet/reactor.All biochemical samples and diethylamine wereobtained from Sigma Chemical (St Louis, MO, USA),with the exception of bovine proinsulin, which wasobtained from Novo BioLabs (Danbury, CT, USA) andcr-galactosidase, obtained from Calbiochem (La Jolla,CA, USA). Ethyl acetate, acetonitrile, methanol,dimethylamine, trimethylamine, triethylamine, tribu-tylamine, hexan e- 1,6-diamine and propionic acid wereacquired from Aldrich Chemical (Milwaukee, WI,USA). Ammonia and methylamine were purchased fromMatheson (East Rutherford, NJ, USA). Doubly distilled,deionized water was used for preparing protein solu-tions and as a protein transfer reagent.

0-460 V.

RESULTS AND DISCUSSIONReactionsof a range of bases with cationsProton transfer reactions of amines with multiplycharged peptides and proteins shift charge-state dis-


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342 R. R. OGORZALEK LOO AN D R. D. SMITH

Table 1. Cytochrome c, ubiquitin and myoglobin charge distributions afterproton transferProtein Reagenta Flow (mol sec-') Charging Maxim um charge

Cytochrome c - 0 7+ to 20+ 16+NH3 1.8 x 7+ to 14+ 11+NH3 6.4 x l o - " 7+ to 12+ 9+, 10+NH3 9.1 x 7+ to l o + 9+NH3 3.7 x 10-5 7+ to l o + a+, 9+NH, 2.4 x 7+ to 8+ 7+M A 4.8 x 7+ to 10+ 9+M A 6.9 x 7+ to 9+ 7+M A 2 . 8 ~ 1 0 - ~ 7+ 7+T M A 9 . 7 ~10-7 7+ 7+T M A 3.4 x 10-6 7+ 7+TEA 4.5 K 10-7 7+ to 9+ 7+TEA 6.5 x 7+ to 8+ 7+TEA 2.6 x 7+ to 8+ 7+Ubiquitin - 0 5+ to 12+ 1o +NH3 6.4 x 5+ to 10+ 7+NH3 9.1 X 54- t0 7 + 7+NH3 3.7 x 10-5 5+ to 7+ 7+NH3 2 4 x 5+ to 6+ 6+M A 4.8 x 5+ to 7+ 6+M A 6.9 x 5+ to 6+ 5+T M A 9.7 x 5+ to 6+ 5+TEA 4 . 5 ~ 1 0 - ~ 5+ 5+

Myoglobin - 0 10+ to2 5+ 19+NH, 6.4 x 10+ to 18+ 16+NH3 9.1 x 10+ to 17+ 14+NH3 3.7 x 10-5 9+ to i s + 12+, 13+M A 4.8 x 9+ to 14+ 12+M A 6.9 x 9+ to 12+ 10+T M A 9 . 7 ~ 10 - 7 g + t o i i + 9+T M A 3.4 x 10-6 9+ to 10+ 9+TEA 4.5 x 10-7 9+ to l o + 9+TEA 6.5 x 10-7 9+ 9+

NH3 2 . 4 ~ 1 0 - ~ 9 + t o l l + 9+, 10+

a M A = methylamine; TMA-trimethylamine;TEA = triethylamine.

probably contribute substantially to proton transfer inthese systems. Proton transfer reactions with ammoniain a triple quadrupole mass spectrometer have also beenreported recently.' 1cv18bIn the light of the reactivity of ammonia, we won-dered about the behavior of weaker bases. Reactionswith the lower proton affinity reagents ethyl acetate(839.7 kJ mol-I), acetonitrile, (788.3 kJ mol- I ) andH,O (6 96 .6 k J m 0 1 - l ) ~ ~were examined. No evidence ofcharge-shifting reactions was observed for cytochromec, myoglobin and ubiquitin with ethyl acetate flowsranging from 0 to 1.6 x lo-' mol s - ' or acetonitrileflows ranging from 0 to 2.4 x lo- ' mol s - ' . Bothreagents showed adduct formation (e.g. protein-ethylacetate complexes) for the high charge states of th ethree proteins (see Fig. 3). These adducts arise fromclustering in either the expansion or in the near-atmospheric pressure region of the capillary inlet, orfrom both regions. Regardless of where the adductsform, at modest capillary-skimmer biases they bear thesignature of an ion-molecule interaction-more addu ctsreside on high than on low charge-state protein ions.Similar behavior has also been observed with the aminereagents.

Th e reactivity of water at flow rates ranging from 0 to8.3 x mol s p l was explored with horse heart cyto-chrome c . With stable H,O flow rates, shifts in theprotein charge-state distribution were not observed, incont ras t to an ear l i e r repor t f rom our l ab~ra tory. '~Recent FT M S studies' also failed to observe proto ntransfer to water from multiply protonated ubiquitins.The previous study was conducted under more extremeconditions (reaction time), which may account for thedifferent behavior observed. In this work, shifts in thecharge distribution were only observed for occasionalscans and appeared to correlate with transmission ofwater droplets through the reagent line. In those cases,the resulting charge state distributions peaked at the[M + 10H]"+ charge state, independent of the vaporflow rate, behavior inconsistent with a proton transferreaction. These results indicate that under the presentconditions, observable proton transfer from multiplycharged proteins requires bases stronger than ethylacetate.A previous study from our laboratory employed aquadrupole mass filter with an m/z 45000 range toexamine to extent of proton transfer from proteins toTEA, TMA and hexane-1,6-diamine.I7 The lowest


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PROTON TRANSFER REACTIONS

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Figure 3. Bovine ubiquitin (RMM 8565) in 1 : 1 methanol-5%acetic acid-water electrosprayed wi th a methanol sheath with acapillary temperature of 115C and 0s = +135 V. (a) Withoutethyl acetate; (b) with 9.5 x mol s-' ethyl acetate. Thebimodal charge state distribution reflects contributions from twoconformer^.^'-^^number of charges observed was close to the number ofarginine residues present in the proteins. Limited to anm/z 2000 range for these experiments, we observed thatthe abundance of the melittin [M + 2HI2+ ion wasonly weakly affected by increasing the flow of DM A,implying that this ion is unreactive to DMA. Melittinhas two arginines, three lysines and a free amino ter-minus; hence a shift to the 2 + charge state is consistentwith the remaining charges residing on the two argin-ines. However, the melittin sequence has four potentialcharge sites in close proximity (residues 21-24; KRKR-),and so another explanation for the stability of the dica-tion might be based on electrostatic (i.e., Coulombic)considerations allowing only one charge site within resi-dues 21-24 and another on the lysine at residue 7. TheN-terminal glycine is another potential charge site, butits proton affinity is expected to be less than that ofD M A , while the values of the isolated amino acidslysine and arginine are higher than that ofD M A . ~3. 35,36

Temperature dependences of proton transfer reactionsCharge-state distributions for various peptides and pro-teins were examined with a nearly constant flow ofamine reagent as the capillary temperature was varied,as shown by examples given in Figs 4 and 5. (Because ofgas dynamics through the inlet, there is actually a smalldecrease in flow with increasing temperature, but theeffect is small compared with the temperature depen-dences observed.) Interface conditions other than thecapillary temperature were held constant. As is appar-ent from the measurements summarized in Table 2,weak bases display a negative temperature dependencein their reactions with proteins, while stronger basesdisplay a positive temperature dependence. The base atwhich proteins change the direction of their tem-perature dependence is protein dependent.

a(M+7H)7+I

d ZFigure 4. Temperature dependence of proton transfer frombovine ubiquitin to 6.4 x mol s- ' NH, with a capillary inlettemperature of (a) 55 and (b) 150C. The protein was electro-sprayed from 1 :1 methanol 5% acetic acid-water wi th a methanolsheath and with 0s= +175 V.

For these studies, a negative temperature dependenceis defined as one in which the charge state distributionshifts to higher charge with increasing temperature, i.e.proton transfer reaction rates decrease with increasingtemperature. Similarly, for a positive temperaturedependence, proton transfer reaction rates increase withincreasing temperature and the charge-state distributionshifts to lower charge. Temperature dependences werealso measured without amines for acidic solutions ofmyoglobin, cytochrome c and ubiquitin, to confirm thatthe charge distributions did not shift in the absence of aproton transfer reagent. Particular care must be taken

30 l a (M+1 0H)I c+I

4020 I 10+ jI1 IL0600 1000 1400 1800d Z

Figure 5. Temperature dependence of proton transfer fromequine myoglobin (RMM 16951.5) to 9.7 x lo- ' mol s - ' TMAwith a capillary inlet temperature of (a) 65 and (b) 160C. Theprotein was electrosprayed from 5% acetic acid-water with amethanol sheath and with 0s= +330 V.


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344 R . R . OGORZALEK LOO AND R . D. SMITH

Table 2. Temperature dependences for proton transfer from proteins to aminesReagentNH,

M A

D M AD EA

T M A

TEA

ProteinaCytochrome c

Cytochrome cMel i t t i nUbiqu i t i ndM y og l ob i nRibonuclease ACytochrome cUbiqui t inM y og l ob i nCytochrome cAlbuminA lbumin"LysozymeLysozymeeAprot in inAprotinin'Cytochrome cCytochrome c 9Ubiqu i t i ngUbiqu i t i ngM y og l ob i nCytochrome cUb i qu i t i nM y og l ob i nM y og l ob i nCytochrome cUb i qu i t i n

Lower temperatureb10+/11+9+/10+3+7+15+/16+


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345P R O T O N T R A N S F E R R E A C T I O N Swould have negative temperature dependences. The cal-culated repulsive potential based onH,Nt CH2CH2CH,NH2 + HNR, +

-+ H,NfCH2CH,CH2NH2.-.HNR3+yielded values of + 138.62 kJ mol-' for R = H and+ 144.43 kJ mol- ' for R = CH, .43 Based on these cal-culations, it does not seem likely that an argumentcould be made for an increase in barrier height forthe reaction with TEA versus NH, that would belarge enough to alter the temperature dependence of thereactions.It is interesting to speculate whether there may betwo different reaction mechanisms. Certainly the pres-ence of two mechanisms would easily explain why thereactions of diethylamine with ubiquitin show negativetemperature dependences below 180"C and positivetemperature dependences above 180"C.In one scenario,the negative temperature dependences may reflect anexergonic reaction, while the positive temperaturedependences may reflect competition from a clusteringpathway, less favorable at higher temperatures. Clus-tering may be more of a factor with the heavier basesbecause of the larger number of states in the complex.McLuckey et d9 observed increased clustering forhexane- 1,6-diamine in ion-trap experiments.An alternate scenario attributes the positive tem-perature dependences to a barrier in the reaction coor-dinate, while the negative temperature dependences areattributed to cluster-mediated proton transfer, a mecha-nism which would be most favorable at low tem-peratures, when cluster dissociation would be lessened.Should a charge-state neighbor many ammonia mol-ecules, or primary or secondary amines (i.e. fo r heavilyclustered or 'solvated' species), it might be possible toinvoke charge delocalization at the transition in whichthe amine cluster acquires a proton. Doing so couldeffectively increase the distance of the repulsive inter-action, decreasing the height of the barrier to protontransfer. Cluster formation leading to proton transferneed not limited to the capillary inlet/reactor; it willalso arise in the supersonic expansion where lower tem-peratures arising from the cooling in the expansionenhance cluster stability. Inlet temperatures still relateto the amount of clustering occuring downstream in thesupersonic expansion, as is evident from the fact thatheated nozzles are used in molecular beam studies tominimize van der Waal's c~mplexation?~The possi-bility for reactions facilitated by cluster formation in thesupersonic expansion would be unique to our inlet/reactor approach; thus, a different experimentalapproach may eliminate reactions with negative tem-perature dependences. We also point out that ourexperiment would be particularly susceptible to clustercontributions for the weaker bases because the tem-perature dependences of the same charge-state popu-lations were examined with different proton transferreagents. With weak bases, much higher reagent flowswere required to shift the distributions to low chargestates, making reagent-protein complexes more likely.Amine adducts are readily observed in these experi-ments at low temperatures, low interface voltages orhigh reagent flows. At a given inlet temperature, we gen-

erally adjust interface conditions downstream to lessentheir contributions to our spectra, but they could easilycontribute to the reaction pathway.We therefore offer two hypotheses to explain the tem-perature dependences observed, both invoking clus-tering. The first argues that competition from clusteringreduces proton transfer rates, the second argues thatclustering increases proton transfer rates by loweringthe barrier in the exit channel. The first hypothesisargues for enhanced clustering with the heavier bases(despite their lower concentrations in the experiment),whereas t h e second argues enhanced clustering with thelighter bases because of the much higher flowsemployed. Future measurements of proton transferrates with various bases may differentiate between thesemechanisms.These experiments did not investigate whether thetemperature dependences varied with charge state. Suchan investigation is potentially interesting. Clearly, theamount and location of charge will affect the energeticsof reactions, which could also explain why the base atwhich proteins charged their charge dependence wasprotein dependent. Steric effects may also modulate thecontributions of different channels.Yet another explanation supporting two differentmechanisms for proton transfer might differentiatebetween proton transfer to different amino acids,arguing, for example, different behavior for transferfrom lysine versus histidine. That explanation appearsto be invalid, particularly when one considers that cyto-chrome c, which displays base-dependent temperaturedependences, has 19 Lys but only 2 Arg and 3 His. Inthe cytochrome c charge-state population for which weprobe temperature dependences, it seems likely that wesee transfers from lysines primarily. Moreover, becauseour temperature dependence studies focus on the samecharge-state populations for reaction with differentbases, it seems unlikely that different residues wouldreact to yield the same product charge-state distribu-tions depending on which base was employed.Proton transfer from propionic acid to anionsReactions were also explored in which a gas-phase acid(propionic acid) transfer protons to [M - nH]"-protein ions generated by ESI. Previous studies'employed an ion trap to explore proton transfer tomultiply deprotonated ions. A-chain insulin (oxidizedRMM 2532), equine myoglobin (RMM 16951.5) andporcine pepsin (RMM 34584), selected on the basis ofour previous negative-ion ESI studies of polypeptidesand protein^:^ were electrosprayed from 3% ammonia-water solutions with a methanol sheath. The resultsfrom these reactions, summarized in Table 3 , demon-strate that proton transfer from gas-phase acids tomultiply deprotonated polypeptide and protein ions canbe studied with the inlet reactor. For myoglobin andpepsin, the reactive groups should be the acidic residuesGlu and Asp and the C-terminus. Oxidized A-chaininsulin has two Glu residues, a C-terminus and fourSO; groups as potential reaction sites.The temperature dependence of the reaction of propi-onic acid with A-chain insulin (oxidized) was explored


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346 R. R. OGORZALEK LOO AND R. D. SMITH

Table 3 A-chain insulin, pep in and myoglobin charge dis-tributions after proton transfer with propionic acidFlow Maximum

Protein (mol s-') Charging charge

A-chain insulin 0 3- to 6- 5-(oxidized) 1. 8 x 3- to 6- 5 -3.9 x 10-5 3- to 4- 3- = 4-Pepsin 0 18- to36- 28-3.9x 18- to 29- 25 -Myoglobin 0 9- to 1 7- 1 3 - ~ 1 4 -3 . 9 ~ 1 0 - ~ 9- t013- 11-

for a propionic acid flow of 3.9 x mol SKI,asshown by the example in Fig. 6 . At a capillary tem-perature of 220"C, the [M - 3HI3-an d [M - 4HI4-ions were observed, with the maximum intensity o n the4- ion, but by 140C, the charge state of maximumintensity had shifted to the 3- ion, and by 110C the4- ion had vanished and only the 3- ion wasobserved. The 110 "C capillary temperature may havereduced the propionic acid flow through condensation(it is lower than the boiling point of propionic acid), butsuch an action would have been observed as a positivetemperature dependence, and not the negative depen-dence indicated. Hence we conclude that the reaction ofoxidized A-chain insulin with propionic acid yields anegative temperature dependence for [M - 3HI3- and[M - 4HI4- from 110 to 220C. With data for onlyone gas-phase acid, we find it premature to explain thecause of the negative temperature dependence.However, by analogy with the amine data, and in thelight of the high prop ionic acid flows employed, contri-butions from clustering are plausible.

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CONCLUSIONThese experiments demonstrate that our inlet/reactorprovides a suitable basis for studying proton transferfrom gas-phase bases to multiply protonated polypep-tides. These studies also extend previous pro ton transferstudies to gas-phase acids reacting with multiply d epro-tonated molecules. In a comparison with bases span-ning a range of pro ton affinities, we find tha t the pr otontransfer reaction rate increases with increasing protonaffinity of the charge-transfer reagent, consistent withFTMS measurements."' Pr oto n transfer was notobserved for the low proton affinity reagents ethylacetate, acetonitrile and water, although ethyl acetateand acetonitrile preferentially formed adducts with thehigh charge states of the proteins examined.These studies also measured the first temperaturedependences for reactions with multiply charged pep-tides and proteins. Based on our examination of reac-tions with ammonia, methylamine, dimethylamine,diethylamine, trimethylam ine and triethylamine, we findthat the temperature dependences are negative for theweaker bases and positive for the stronger bases, withthe base at which the temperature dependence shiftsfrom negative to positive being protein dependent.Moreover, the reaction of diethylamine with ubiquitindisplays a negative temperature dependence below180 "C and a positive temperature dependence above180C. Based on these results, we suggest that twomechanisms for proton transfer operate in these experi-ments. Positive temperature dependences may reflectthe barrier to proton transfer arising from the nature ofthe transition state, where two like-charged ions in closeproximity repel. A cluster-mediated proton transfermechanism is proposed to explain the negative tem-perature dependence with weaker bases; the dependencearises because clusters dissociate a t higher temperatures.It is suggested that charge delocalization possible withthe cluster mechanism could lower the barrier to protontransfer by effectively increasing the distance betweenthe like charges in the transition state. The inlet/reactorapproach encourages cluster formation by the highreagent flow employed, particularly with weaker bases.In addition, clusters can form readily in the supersonicexpansion downstream of the inlet/reactor. Alternative-ly, the negative temp erature dependences may reflect anexergonic reaction, while the positive temperaturedependences may reflect competition from a clusteringpathway, less favorable at higher temperatures. In thiscase, the clustering could be more of a factor with theheavier bases because of a larger number of states in thecomplex. Negative temperature dependences were alsoobserved in reactions of multiply deprotonated poly-peptides and p roteins with propionic acid.AcknowledgementsWe thank Scott McLuckey for proposing alternative explanations forthe temperature dependences. Joseph Loo, Harold Udseth, JohnFulton, Bill Robins, and Philip Andrews also provided helpful dis-cussions. We acknowledge support of the US Department of Energythrough the Oflice of Health and Environmental Research andthrough internal Exploratory Research of the Molecular ScienceResearch Center (Contract DE-AC06-76RLO 1830). Pacific North-west Laboratory is operated by Battelle Memorial Institute.


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PROTON TRANSFER REACTIONS 347REFERENCES

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Proton Transfer Protein Peptide

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