Stepwise evolution of protein native structure withelectrospray into the gas phase, 10�12 to 102 sKathrin Breukera and Fred W. McLaffertyb,1

aInstitute of Organic Chemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 52a, 6020 Innsbruck,Austria; and bDepartment of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853-1301

Edited by Jack Halpern, University of Chicago, Chicago, IL, and approved September 4, 2008 (received for review July 21, 2008)

Mass spectrometry (MS) has been revolutionized by electrospray ionization (ESI), which is sufficiently ‘‘gentle’’ to introduce nonvola-tile biomolecules such as proteins and nucleic acids (RNA or DNA) into the gas phase without breaking covalent bonds. Although insome cases noncovalent bonding can be maintained sufficiently for ESI/MS characterization of the solution structure of large proteincomplexes and native enzyme/substrate binding, the new gaseous environment can ultimately cause dramatic structural alterations.The temporal (picoseconds to minutes) evolution of native protein structure during and after transfer into the gas phase, as pro-posed here based on a variety of studies, can involve side-chain collapse, unfolding, and refolding into new, non-native structures.Control of individual experimental factors allows optimization for specific research objectives.

electrospray ionization � gaseous proteins � mass spectrometry � protein conformations � proteomics

Until the late 1980s, it was be-lieved by many researchers inthe field of mass spectrometry(MS) that the transfer of

large biomolecules into the gas phase,without breaking covalent bonds, wasjust impossible. Then biomolecular MSwas revolutionized by the introductionof the ‘‘soft’’ desorption/ionizationmethods matrix-assisted laser desorption(MALDI) (1) and electrospray ioniza-tion (ESI) (2) that produced intact bio-molecular ions. Even more surprisingwas early evidence that ESI could pre-serve noncovalent bonds, producingmasses corresponding to undissociatedprotein complexes (3–5). These exam-ples for retention during ESI of nonco-valent bonding initiated great enthusi-asm about the potential of massspectrometry for studying biologicallyrelevant systems, but at the same timeraised the question of whether or notESI is generally capable of transferringbiomolecules and their noncovalentlybound complexes into the gas phasewhile preserving their solution structure.Ever since, the effect of gaseous envi-ronment on noncovalent bonding andhigher-order biomolecular structure hasbeen hotly debated. Should the uniquestructure of a native protein survive theremoval of its aqueous environment,with the loss of hydrophobic bondingoffset by the strengthening of its electro-static interactions? In his high-impact1997 review article, Joseph Loo (6) saysabout the use of ESI for the study ofnoncovalent complexes that ‘‘there arethree camps of opinion: believers, non-believers, and undecided. I am a cau-tious believer.’’ Today, in 2008, substan-tial evidence for retention of nativestructure has been presented for ESI ofsystems such as large protein (7–9) andnucleic acid (10, 11) complexes. How-

ever, globular proteins such as cyto-chrome c and ubiquitin (12) appear toundergo a temporal evolution of struc-tures after ESI: side-chain collapse, un-folding, and refolding into multiple gas-eous structures in 10�12 to 102 s.Solution structure and experimental fac-tors can affect each of these steps, indi-cating ESI optimization possibilities forspecific control of the resulting gaseousconformers for a variety of proteinstudies.

ESI MS has been used to characterizecomplex protein machinery such asGroEL (�800 kDa) (13) or to deter-mine relative binding energies of pro-tein/ligand complexes (6, 14). Here, it isimperative that any modifications to thesolution structure occurring in the gasphase do not appreciably affect themeasurement, e.g., the binding energiesin the gas phase must be proportional tothose in solution. On the other hand,characterizing the structural transitionfrom solution to gas phase and the sta-ble gaseous conformers is crucial for afundamental understanding of howchemical environment affects biomolec-ular structure and stability and for de-veloping efficient strategies for manipu-lation of gaseous ion structure insophisticated MS experiments. The com-bined data from a wide variety of exper-iments and computational results nowallow us to draw a clearer picture ofhow desolvation affects biomolecularstructure and stability and lead us tobelieve that temporal evolution of struc-ture during and after the phase transi-tion is the key factor for understandingapparently conflicting MS data. Nowpossibly Loo’s question in 1997 can berephrased: ‘‘For how long, under whatconditions, and to what extent, can solu-tion structure be retained withoutsolvent?’’

The evidence to be discussed for astepwise structural evolution is summa-rized in Fig. 1. It covers the removal ofwater molecules from the native proteinconformation and the period of picosec-onds to many minutes in which the sta-ble gaseous structures are formed.

Final Water Loss from the Protein and ItsCharged Side ChainsIn the transition from solution to gasphase during ESI MS, biomolecules suchas proteins experience vastly differentphysicochemical environments that canpotentially affect their structure. TheESI process starts with the productionfrom solution of electrically chargeddroplets that undergo a series of succes-sive evaporation and droplet fissionevents at atmospheric pressure and am-bient temperature. Accumulating evi-dence suggests that ESI of native, globu-lar proteins proceeds via the chargeresidue model, in which solvent evapora-tion from very small (nanometer-sized)droplets containing only one protein (orprotein complex) eventually results inthe formation of ‘‘dry’’ ions (15).

Because of small droplet size, the fi-nal desolvation step has yet escaped ex-amination by experiment, but was re-cently studied with molecular dynamics(MD) simulations for native cytochromec surrounded by a monolayer of watermolecules (16). This study finds no ap-preciable changes in the overall proteinstructure during water evaporation on a

Author contributions: K.B. and F.W.M. designed research,performed research, analyzed data, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1To whom correspondence should be addressed. E-mail:fwm5@cornell.edu.

© 2008 by The National Academy of Sciences of the USA

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nanosecond time scale. Moreover, thelast water molecules to evaporate aggre-gate around charged sites that pointaway from the protein surface in thenative structure (Fig. 1 A and B),thereby stabilizing the solution structureuntil the very last stages of desolvation.In the absence of energy exchange withthe environment, solvent evaporationwas also found to result in a significantreduction of protein ion temperatureand an associated decrease in waterevaporation rate. For a cytochrome cion initially surrounded by 182 watermolecules at 110°C, after 200 ps ofevaporation it contains 102 H2Os at80°C, and after 400 ps it contains 81H2Os at 65°C (16).

In reality, however, the partially hy-drated protein ions from ESI are in con-stant energy exchange with their envi-ronment via inelastic collisions with gasmolecules and surfaces and via black-body infrared radiation. Because theenergy exchange process may be tooslow for full desolvation of the protein(or protein complex) ions before enter-ing the mass spectrometer, most ESImass spectrometers allow for additionalheating in the region interfacing atmo-spheric pressure and the first vacuumstage (for example, a heated capillaryinlet or a counterflow of heated gas).Kebarle has estimated a time scale ofhundreds of microseconds for the for-mation of nanometer-sized dropletsfrom initial ESI droplets of 3 �m diam-eter, with droplet temperatures �10°C

lower than that of the ambient gas as aresult of evaporative cooling (17), al-though the resulting ions may havesomewhat higher temperatures (18). Sol-vent evaporation from and fission oflarger droplets (20–50 �m in diameter)studied in ‘‘ping-pong’’ experiments wasincomplete even after 250 ms (19).However, only parent, not offspring,droplet behavior has so far been moni-tored in experiments.

Collapse of Charged Side Chains(Picoseconds)According to new MD calculations onnative cytochrome c ions, full desolva-tion (Fig. 1C) is followed almost instan-taneously, on a time scale of a few pico-seconds, by specific structural changeson the exterior of the native protein(20). These generally involve chargedside chains (Fig. 1D) such as protonatedlysine or arginine and deprotonated glu-tamic or aspartic acid residues. The sim-ulations show that charged side chainsform salt links between each other, orionic hydrogen bonds with backboneheteroatoms (e.g., Fig. 2; N�H3 ofLys-79 to amide oxygen of Tyr-48). Thenumber of electrostatic interactions ofthe 21 positively and 12 negativelycharged side chains in equine cyto-chrome c increased from 11 in solutionto �35 after 10 ps, with some of thecharged side chains participating inmore than one electrostatic interaction.The multiple interactions result in across-link-like network of electrostatic

interactions on the protein surface,thereby stabilizing the native fold. Thissurface interaction, along with the ioncooling resulting from solvent evapora-tion, produces transiently stabilized pro-tein ions; the backbone fold was foundto stay essentially the same as that insolution during the 4-ns duration of cal-culations (20). A common feature ofproteins denatured by addition of or-ganic solvent such as methanol is a high�-helix content of their solution struc-ture; collapse of the charged side chainsshould strengthen the H-bonding of�-helices in the gas phase by interactionof charge with the helix dipole (21–23)(see below).

Loss of Hydrophobic Interactions andSubsequent Dissociation of ElectrostaticBonds (Milliseconds)Consistent with the MD results, experi-mental evidence also indicates that thisbasic structure (Fig. 1D) can remain es-sentially unchanged for up to millisec-onds. Ion transfer into the MS vacuumyields not only cooling by solvent evapo-ration and adiabatic expansion but alsoheating by collisions and surface contact(18, 24). In our ESI system (Fig. 3), aninlet capillary heats the ion beam aswell as drops the pressure from atmo-spheric to �1 mbar. The ion residencetime in a similar capillary was estimatedby Mirza and Chait (25) as �10�3 s.Stepwise unfolding here of cytochromec is demonstrated by the effect of capil-lary temperature on fragment ion abun-

Fig. 1. Stepwise evolution after ESI of the structure of a globular protein (e.g., cytochrome c, ubiquitin). (A) Native protein covered with a monolayer of H2O,followed by nanosecond H2O loss and concomitant cooling. (B) Native protein with exterior ionic functionalities still hydrated undergoes ns H2O loss and cooling.(C) The dry protein undergoes �10-ps collapse of its exterior ionic functionalities. (D–G) The exterior-collapsed ‘‘near-native’’ protein undergoes thermalre-equilibration (D), millisecond loss of hydrophobic bonding (E), and millisecond loss of electrostatic interactions (F); the transiently unfolded ions form newnoncovalent bonds in seconds, folding to more stable gaseous ion structures (G); these stabilize to energy minima conformers in minutes.

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dance in native electron capture dissoci-ation (NECD) (26, 27).

NECD spectra of cytochrome c showfragment ions from protein backbonecleavage next to residues that are incontact with the heme in the nativestructure, whereas no fragment ions arefound for nonheme proteins under theexact same experimental conditions.This unusual phenomenon was observedwith ESI of relatively concentrated (�40�M), aqueous cytochrome c solutions atnondenaturing pH (26), from which itwas concluded that noncovalently bounddimers of native protein are required forNECD. Analysis of the fragment ioncharge states suggested that protontransfer occurs between the two mono-mers (26–28), thereupon causing elec-tron transfer via the heme group andbackbone cleavage in a mechanism simi-lar to that of electron capture dissocia-tion (ECD) (29). ‘‘Asymmetric chargepartitioning’’ was also observed withcollisionally activated dissociation(CAD) of cytochrome c homodimers(30). Apparently, one of the monomersin the dimer unfolds faster than theother, causing proton transfer from thestill compact monomer (Fig. 1D) to thepartially unfolded monomer (Fig. 1E).The lack of NECD cleavage productsfrom the terminal helices and the�-loop (Fig. 4) identified the regions ofcytochrome c that unfold first (Fig. 4) tostart the charge asymmetry process.

These regions are generally stabilizedby hydrophobic bonding between eachother and the heme and are the moststable ‘‘fold-ons’’ in the native solutionstructure (32, 33). In the gas phase,however, these become the least stable(Fig. 1E). Now the order of disappear-ance of specific NECD fragment ionswith increasing capillary temperaturedirectly indicates the order of furtherunfolding (Fig. 1F). This further unfold-ing involves separation of electrostaticprotein/heme interactions, which havebeen strengthened by removal of water

that could otherwise compete for (ionic)hydrogen bonding. By loss of hydropho-bic bonding and strengthening of elec-trostatic interactions, removal of waterhas reversed the order of regional pro-tein stability, making the newly desol-vated gaseous ions (Fig. 1 C and D)thermodynamically unstable. This rever-sal causes disintegration of the nativefold, making possible the formation ofnew intramolecular interactions that arenecessary for the formation of stablegas-phase structures.

Elegant ion mobility spectrometry(IMS) studies by Clemmer and cowork-ers (34) also show unfolding of cyto-chrome c ions in the milliseconds timedomain (Fig. 5). The IMS drift timeprovides collision cross-section valuesfor the corresponding ions. In Fig. 5Left, the conformer assigned as B ofcytochrome c 9� ions has a drift timenearly as short as that calculated for thenative structure. B represents the majorconformer present when the ions arestored for up to 30 ms, corresponding tothe limited structural evolution of Fig. 1A to D. Longer storage allows rear-rangement into multiple conformerswith longer drift times such as unfoldedconformer E, consistent with the unfold-ing of Fig. 1 E and F. The substantiallylonger times required for conformerchanges in these IMS experiments ver-sus those observed with NECD couldresult from lower IMS ion temperaturesand/or collisional activation in theNECD experiment.

New Noncovalent Bond Formation(Seconds to Minutes)In Fig. 5, however, after ion storage forfractions of a second, the proportion ofthe most open conformers decreases infavor of more compact forms (Fig. 1 Fand G). ‘‘Drying’’ the native conformerhas caused hydrophobic and electro-static bonds to dissociate until the struc-ture is sufficiently open (Fig. 1F) to al-low refolding into more stable

structures. To study the kinetics of suchgaseous ion refolding, 7� ubiquitin ionswere stored and thermally equilibratedin the cell of a Fourier-transform massspectrometer (21), presumably to forman approximation of the ‘‘stable gas-phase structures’’ of Fig. 1G. These ionswere unfolded with a 0.25-s pulse of aCO2 IR laser, and their ECD spectrameasured every 0.2 s (Fig. 6). Con-former denaturation, shown by increas-ing abundance of all ECD fragments,occurred in �0.2 s, after which first-order refolding in the regions aroundresidues 24, 51, and 54 occurred in 1 s.Although refolding in other regions oc-curred more slowly, and by separate ki-netic steps (including the regions aroundresidues 24 and 51 at 65°C), most re-turned to near initial values in 10 min.However, the regions around residues 51and 48 underwent refolding into con-formers allowing less and no ECD prod-uct formation, respectively. Althoughlaser activation did not immediately un-fold the residue 68 region, it thenfolded, unfolded, and dramatically re-folded to prevent all ECD fragment ionseparation.

This variety of new conformers wasformed by activating and cooling 7�ubiquitin ions that already had substan-tial opportunity for equilibration. Thusmany conformers must be in local en-ergy minima on the very bottom of the32 kJ/mol energy well for the overallunfolding of 7� ubiquitin ions (21). IMScross-section measurements (34–40) alsoindicate that a variety of ubiquitin ionconformers are formed by ESI not onlyfor 7� ions, but also for most chargestates from 4� to 15� (Fig. 7).

Thus with sufficient time and activa-tion, singular native protein conforma-tions passing into the gas phase can un-dergo side-chain collapse, unfolding, andfolding to a variety of 3D structures sub-stantially different from the structuretailored by evolution for a specific pur-pose in solution. The extent and timingof the events in Fig. 1 are controlled bya variety of experimental factors and thenature of the biomolecules being elec-trosprayed. Optimization of these factorsdepends on the objectives of the specificESI application, as discussed furtherbelow.

Structural Features of MultiprotonatedGaseous BiomoleculesBasic knowledge of the effect of solventremoval on the secondary and tertiarystructure of proteins, DNA, RNA, andother biomolecules should contribute tothe understanding of their biologicalfunctions. A variety of methods haveprovided important data for the charac-terization of gaseous conformations of

Fig. 2. In ESI of native cytochrome c, juxtaposition of the �-N�H3 of Lys-79 versus the amide oxygen ofTyr-48. (A) Local native structure just after complete H2O loss. (B) Ten picoseconds later, the near-instantaneous collapse forming ionic hydrogen bonds between these adjacent (in structure, but notsequence) sites. [Reproduced with permission from ref. 20 (Copyright 2008, Wiley).]

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proteins (12, 41): H/D exchange labelsexposed reactive sites (42–44), IMS (seeabove) directly indicates their collisionalcross section (34–40, 45, 46); ECD (seeabove) of backbone bonds does not de-stroy, and thus helps characterize, non-covalent bonding (21, 29, 47); and gas-phase infrared photodissociation (IRPD)spectroscopy measuring O–H/N–Hstretching vibrations directly character-izes H-bonding (22, 48). The initial side-chain collapse (Fig. 1 C and D) illus-trates the common high tendency foundfor ionic H-bonding of the protonatedresidues Lys and Arg to amide oxygenor nitrogen of the gaseous protein back-bone. This finding offers an explanationfor an apparent dichotomy of early re-sults of two experimental approaches.Increasing the charge of an ion increasesits collision cross-section (Fig. 7), with�1,000 and �2,000 Å2 values (35–39)for the 6� and 13� ions, respectively,of ubiquitin (nominally, the 13� ion isfully protonated, with seven Lys, fourArg, one His, and N-terminal NH2 asprotonation sites) (12). In contrast, theextent of gaseous H/D exchange dramat-ically decreases with increasing charge,with �70 D atoms exchanged for 7�and �16 for 13� ions (22, 43). Theseamino/imino groups of the side chainsundergo ready H/D exchange when neu-tral, but adding a proton that bindsthem to the backbone apparently pro-tects most of their H atoms (even fourof five guanidinium Hs on Arg) fromexchange. The �2,000 Å2 cross-sectionvalues found for the 13� ions corre-spond to that calculated for an �-helicalstructure (35–39); ECD of the 13�ubiquitin ions is highly specific for cleav-age at the second, third, and fourth resi-

dues toward the N terminus from a ba-sic residue, corresponding to collapse ofits side-chain N�–H to the nearestamide oxygens in an �-helix (21, 23).

More detailed evidence of this inter-action comes from 3,050–3,800 cm�1

(O–H/N–H stretching region) IRPDspectra of several electrosprayed proteinions (e.g., mellitin, ubiquitin, cyto-chrome c, and ribonuclease) that showabsorption only in the 3,300–3,400-cm�1

region (ref. 22 and unpublished work)that mainly represents amide A bands.The N�-H bonding to the �-helix ap-pears to increase its charge density suffi-ciently so that other side-chain OH/NHgroups are also strongly H-bonded, red-shifting their absorbencies by �300cm�1 to �3,050 cm�1. In fact, these H-bonding tendencies appear to be rela-tively insensitive to total charge, withonly minor qualitative differences in theIRPD spectra of 7� to 13� ubiquitinions.

From IMS (34–40, 45) and ECD (21–23) evidence, H� removal from an �-he-lical peptide or protein ion allows bend-ing at the charge removal site. From the13� ubiquitin ions that apparently havea linear �-helical structure, the 11� ionshave several bent isomers, the 9� arefolded over at one end (stabilized by theopposite dipoles of the overlapping heli-ces), and the 7�, 6� ions have bothends folded over into a three-helix bun-dle (22) whose compact ion cross-sec-tion closely resembles that calculated forthe native structure (37–40). Despite thereduction of total charge, the 7�, 6�conformer (or mixture of similar con-formers) has apparently maintained asufficiently high charge density to retainthe IRPD spectral characteristics of ex-

tensive hydrogen bonding. Removal ofwater molecules that solvate the chargesites in solution not only allows side-chain collapse to the central structure,but by removing charge and thus lower-ing electrostatic repulsion, the anhy-drous secondary structure such as a he-lix can itself collapse further to a varietyof more compact conformers (Fig. 7) inlocal energy minima (Fig. 1 F and G)that bear little resemblance to the nativestructure of ubiquitin.

Other techniques for further charac-terization of such gaseous conformationsare obviously needed. Some reviewers(49, 50) have suggested trying direct as-says of native protein functions, such asmeasuring fluorescence of a green fluo-rescent protein, binding of CO to hemo-globin, or gas-phase enzymology. Thetransition from solution to gas-phasebehavior could even be probed withprotein/complex/(H2O)n ions of differentn values.

Gaseous Protein ComplexesElectrospray at very low temperatures,into a very cold mass spectrometer,could in principle result in preservationof solution structure on at least a lim-ited time scale. However, freezing theESI droplets prevents desolvation, whichis an endoergic reaction and requiresenergy input to proceed (24). JosephLoo (6) pointed out that ‘‘there is avery fine line between sufficient desolva-tion of the gas-phase complex and disso-ciation of the complex.’’ Nevertheless,transfer of a native biomolecular struc-ture to a gaseous environment may notimmediately result in global conforma-tional changes if ion activation by colli-sions with background gas and surfacesis outweighed by ion cooling from sol-vent evaporation and adiabatic expan-sion (24). As discussed above, a nativefold can actually be stabilized, althoughtransiently, by formation of new electro-static interactions on the protein surface(Fig. 1D). For protein complexes, someof the new electrostatic interactions(Fig. 1D) could form between complexpartners and thus stabilize the complexin the gas phase. Depending on experi-mental conditions and nature of thecomplex, a time window of �10�3 s canbe available for complex detection inthe mass spectrometer. Then ESI MScan give information on complex stoichi-ometry and, after complex dissociation(MS/MS), reveal the identity of bindingpartners (6–11).

Protein/Ligand Relative Binding EnergiesHowever, ‘‘solution binding affinitiesmay or may not be accurate predictorsof their stability in vacuo’’ (50). Also inhis 1997 review, Loo (6) raised the im-

Fig. 3. Electrospray of a protein solution (from right) at atmospheric pressure with �1-kV accelerationof ESI droplets into a heated capillary. Exiting ions are accelerated (�50 V) into a lower pressure (�1 mbar)region to undergo variable low energy (�1 eV) collisions, pass through a skimmer into a lower pressureregion for possible higher energy (�1 eV) collisions, and are conducted into and trapped inside themeasurement cell (�10�9 mbar) of the Fourier-transform mass spectrometer.

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portant question: ‘‘Can a gas-phasemeasurement reveal solution-phasebinding characteristics?’’ At the time,encouraging and discouraging examplesfrom gas-phase experiments using CADand blackbody infrared radiative dissoci-ation (BIRD) had been published.Among the discouraging examples werecomplexes with weak electrostatic bind-ing in solution that become unusuallystrong in the gas phase, up to a pointwhere covalent bond breaking is favoredover complex dissociation. For othersystems, it was found that ligand hydro-phobicity, although greatly affecting so-lution binding, did not affect proteincomplex stability in the gas phase. Loo(6) concluded that ‘‘although some fea-tures of the solution structure may bepreserved by the gas-phase ions, the sta-bility of the gas-phase complex ion maynot be reflected by the solution-phasebinding constant.’’ In their 2002 reviewon the use of MS for quantitative deter-mination of noncovalent binding interac-tions, Zenobi and coworkers (14) statethat ‘‘with a few exceptions, no agree-ment exists between solution-phase andgas-phase binding energies.’’ Then in2003, the Klassen group (51) reportedevidence from BIRD experiments thatprotein–carbohydrate complexes fromnonspecific association during ESI canactually be more stable in the gas phasethan the specific complexes that weretransferred intact from solution. It isnow generally agreed that the strength-ening of electrostatic interactions andthe loss of hydrophobic effect in thegaseous environment of a mass spec-trometer result in vastly alteredstrengths of interactions compared withthose in solution (41), consistent withthe above NECD data showing an es-sentially reversed order of stability inthe gas phase. Moreover, as discussedabove, the new electrostatic interactionsformed almost immediately after desol-

vation (Fig. 1D) can result in additionallinks between protein and ligand andfurther transfigure binding energies.

Prefolding Dissociation of Large ProteinsAs described elsewhere in this issue ofPNAS (52), most MS protein analysis(‘‘proteomics’’) is done by first digesting(e.g., with trypsin) the protein mixture,subjecting this more complex peptidemixture to MS, and using MS/MS of theseparated peptide molecular ions to ob-tain extensive sequence information. Inthis ‘‘bottom-up’’ approach, these pro-tein fragment sequences are matchedagainst the possible proteins predictedby their precursor DNA; matching pep-tide sequences can give up to 95% se-quence coverage and 99% identificationreliability. The ‘‘top-down’’ approachhas far higher identification reliabilityand capabilities for locating sequenceerrors and posttranslational modifica-tions (PTMs), although it requires in-strumentation such as Fourier-transformMS of far higher resolving power andexpense than instruments used previ-ously for bottom-up proteomics (52–54).In this, individual molecular ions of aprotein in a mixture are separated byMS-I and dissociated (MS-II) to givefragment ions indicative of that protein’ssequence and PTM positions. ECD (29)is especially helpful for MS-II, yieldingfragment ions containing either the Nor C terminus that often provide com-plete sequence coverage and detailedPTM positioning. A serious problem isthat discussed above with Fig. 1 F andG; the extensive folding taking place in�1 s in the Fourier-transform MS ioncell makes protein ions highly stable.Proteins larger than �50 kDa, althoughelectrosprayed under denaturing condi-tions, yield few or no fragment ions byCAD, IRMPD, or other ion dissociationmethods. The prefolding dissociation(PFD) approach instead activates the

ions �1 s after ESI while they are stillin open, more labile conformations (Fig.1F), which provides useful fragmenta-tion information from proteins as largeas 229 kDa (55). Activation to delay thefolding and dissociate bonds is accom-plished by combinations of capillaryheating and ion acceleration in both the�1- and 10�3-mbar regions before andafter the skimmer (Fig. 3); the lattercollision energies generally effect disso-ciations of noncovalent and covalentbonds, respectively.

Combinations of these three activa-tion steps in 21 PFD mass spectra gaveproduct ions from 287 different inter-residue cleavages of formylglycinimideribonucleotide amidotransferase (PurL),1,314 residues, covering 238 and 248residues of the N and C termini, respec-tively (37% overall coverage). Althoughno cleavages were in the 828-residuecenter of the protein, the fragment ionsfound provided detailed sequence infor-mation (cleavage at 59% of interresiduebonds) in the termini. For example, thepredicted N-terminal Met was shown tobe absent, correcting the predicted mo-lecular weight value to 143,504 versusthe 143,500 � 23 experimental value.For the human complement C4 glycop-rotein (1,714 residues) of three subpro-teins (longest, 767 residues) joined byS–S bonds, the PFD spectra identifiedall 27 Cys residues as having –SH or S–Sbonds. ESI of mycocerosic acid syn-thase, 2,153 residues, gave molecularweight � 228,934 � 60 versus 228,936predicted after correcting for a missingN-terminal Met. This sequence informa-tion was shown in five PFD spectra indi-cating 62 cleavages up to 134 and 182residues from the N and C termini, re-spectively (15% coverage).

Note that the smaller 144-kDa linearprotein underwent greater (37%) dena-turation in the steps of Fig. 1 D–F un-der the vigorous CAD conditions (e.g.,capillary temperatures up to 345°C)(55).

For these solution-denatured largeions, the further collapse of the ions inFig. 1D has been suggested to form a‘‘ball of spaghetti’’ structure (55), forwhich vigorous activation either extendsits free ends or keeps them extended,consistent with the dominant PFD frag-mentation in the protein termini. Weare now attempting to affect ECD onthe exterior of the ball of spaghetti tocreate new ends; with activation, thesecould unravel and fragment to providesequence/PTM characterization of theinterior of the protein.

Electrospray AdditivesA serendipitous discovery of the PFDinvestigation was that specific additives

Fig. 4. Cytochrome c structures. (A) Native state, based on NMR data (31). (B) Initial gas-phase unfolding,based on NECD data (27). The order of regional stability in the gas phase, based on the reverse of the orderof unfolding determined in NECD experiments, is Y48/T49 � W59 � K79/M80 � F46 � N52 � F82 � T40 �L68 � I85 � L35 � K13.

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to the electrospray protein solutioncould increase the number of backbonecleavages by 50% (55). The most effec-tive additives are the ammonium salts ofsaturated dicarboxylic acids such asN�H4

�O2CCH2CH2CO2�N�H4 and de-

rivatives. Related unsaturated and aro-matic compounds are less effective,whereas similar sulfonates, phosphates,polyhydroxy compounds, and othersmall molecules are far less effective orinactive. A possible explanation is thatin solution the additive forms multiple

noncovalent bonds to one or more basicresidues, displacing water, so that thesolvent monolayer of the structure inFig. 1B structure will instead consistmainly of the far more strongly bondedadditive molecules of far lower volatility,some even of net negative charge. For adenatured protein with negligible ter-tiary structure, this should not only pre-vent immediate side-chain collapse (Fig.1 C and D), but also delay the participa-tion of these charged side chains in elec-trostatic folding to the very stable gas-

phase structures (Fig. 1 F and G) ofthese large proteins. However, the vola-tility of the additives is sufficient thatnone remain in the PFD mass spectra(see above) to confuse interpretation.

Such additives can also be useful forMS studies in which it is desirable toretain the solution conformation in thegas phase. In drug discovery, a commontechnique for lead compound identifica-tion is by ESI characterization of nonco-valent ligand–macromolecular targetinteractions. (6–11, 14) Here, it is neces-sary during electrospray to preservethese noncovalent interactions, whichcould be disrupted in the unfoldingsteps of Fig. 1 D–F. As reported byKlassen and coworkers (56), the solu-tion-stable complex of serine proteasetrypsin and its competitive binding in-

Fig. 6. Relative log intensity values of ECD products from cleavage next to indicated residues of ubiquitin7� ions versus time after unfolding by an IR laser pulse (from ref. 21). Apparent first-order rate constantsfor initial refolding that prevents ECD product separation at the indicated residues are shown in the box.Note that overall refolding can be both single-step and two-step, even folding and unfolding, notnecessarily returning to the same degree of folding as before unfolding with the IR laser, and with atemperature increase changing the folding process from one-step to two-step. [Reproduced with per-mission from ref. 21 (Copyright 2002, American Chemical Society).]

Fig. 7. Unique collision cross-section values ofubiquitin ions of various charge states, indicatingmultiple gaseous conformers, from the referencesindicated. [Reproduced with permission from ref.12 (Copyright 2006, Wiley).]

Fig. 5. Ion mobility drift times of 9� cytochrome c ions stored after ESI at 27°C and 1.3 mbar for various trapping times. Drift times are the smallest for themost compact ions and are directly related to ion collision cross sections. (Left) Drift times characterizing compact conformer B, partially unfolded conformersC and D, and unfolded conformer E. (Right) Relative conformer abundance as a function of trapping time. [Reproduced with permission from ref. 34 (Copyright2005, Elsevier).]

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hibitor benzamidine does not surviveESI/MS introduction (56). However, ESIsolution additives such as imidazole canpreserve the complex, possibly becauseof enhanced cooling from imidazoleevaporation that delays the dissociation.In a recent innovative approach to asimilar problem, Robinson and col-leagues (57, 58) show that detergent mi-celles can protect membrane proteincomplexes from dissociating during ESI.The micelles could prevent or retard allof the steps in Fig. 1 C–G that disinte-grate the native fold.

ConclusionsCan ESI transfer biomolecules and theirnoncovalently bound complexes into thegas phase while preserving their solutionstructure? For several cases, the answernow to Loo’s cogent 1997 question (6) isa ‘‘yes,’’ at least to the degree necessaryto answer important questions. But theopposite question, ‘‘can the solutionstructure be lost?’’ may also deserve a

yes. In fact, a variety of research ap-proaches now appear to provide a farbroader understanding of the changes inprotein ion conformation that can beeffected by its transfer from solution tothe gas phase, as summarized in Fig. 1.The picoseconds side-chain collapse(Fig. 1 C and D) on the protein surfacecan transiently stabilize the native foldof large, compact protein complexes.Critical conformer stabilization duringESI can also be provided by noncova-lent binding of intermolecular additives(56–58). For a native structure with sub-stantial stabilization by hydrophobicbonding in solution, ESI denaturing canform an unstable transient structure(Fig. 1 D–F) that then refolds and equil-ibrates (e.g., stronger electrostaticbonds) to a more stable gaseous con-former (Fig. 1 F and G). The native so-lution structure can bear little resem-blance to that of this refoldedcounterpart, with the study of suchstructures representing an exciting ex-

tension of the field of gaseous ionchemistry. For applications needing aprotein conformation less stable thaneither the solution form or the equili-brated gaseous conformers, the mostopen conformers appear to be formedduring entrance into the mass spectrom-eter, tens of milliseconds after ESI (Fig.1F), making possible top-down dissocia-tion methods (PFD) effective for �200-kDa proteins (55).

Of basic importance, this stepwisevisualization of the effect of solvent losson protein conformation should not onlyprovide further insight into the role ofthe aqueous environment on the stabil-ity and function of conformation, butalso serve as a temporal framework forfurther experimental studies of theseeffects using new methodologies.

ACKNOWLEDGMENTS. This work was supportedby Austrian Fonds zur Forderung der wissen-schaftlichen Forschung Grants V59 and Y372 toK.B. and the U.S. National Institute of GeneralMedical Sciences, National Institutes of Health,Grant GM 16609 to F.W.M.

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