Investigation of the dynamics of matrix-assisted laser desorption/ionization ion formation using an electrostatic analyzer/time-of-flight mass spectrometer

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 34, 684–690 (1999)

Investigation of the Dynamics of Matrix-assistedLaser Desorption/Ionization Ion FormationUsing an Electrostatic Analyzer/Time-of-flightMass Spectrometer

Gary R. Kinsel,1* Mary E. Gimon-Kinsel, 2 Kent J. Gillig 3 and David H. Russell31 Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, Texas 76019-0065, USA2 Sensar Corp., Provo, Utah 84601, USA3 Department of Chemistry, Laboratory for Biological Mass Spectrometry, Texas A&M University, College Stution, Texas77845, USA

A hybrid electrostatic analyzer/time-of-flight mass spectrometer was used to examine the matrix-assistedlaser desorption/ionization (MALDI) ion kinetic energy distributions for Na Y, protonated 7-hydroxy-4-methylcoumarin matrix ions and protonated bradykinin under a variety of source region accelerating electricfield conditions. Broad kinetic energy distributions are observed for the three targeted ions with the ions havingboth positive and negative kinetic energies relative to the full acceleration potential. The positive kinetic energyions are shown to have flight times in good agreement with flight times calculated from first principles whilethe flight times of the negative kinetic energy ions diverge positively from the calculated values. Analysis ofthe flight time shifts for the negative kinetic energy ions allows an entrainment velocity of∼550 m s−1 for thematerial to be calculated. A composite picture of the dynamics of MALDI ion formation is presented whichcombines promptly formed protonated matrix and analyte ions with ions formed/accelerated at delayed times inan expanding, constant velocity plume of laser desorbed material. Copyright 1999 John Wiley & Sons, Ltd.

KEYWORDS: matrix-assisted laser desorption/ionization; kinetic energy; electrostatic analyzer; mechanism; ion formation

INTRODUCTION

One parameter which has been considered important inunderstanding the mechanism of matrix assisted laser des-orption/ionization (MALDI) ion formation is a measure ofthe desorbed ion kinetic energy (IKE) distributions undervarious experimental conditions. MALDI IKE distribu-tions have been measured under zero accelerating electricfield conditions by a number of researchers.1–4 Differ-ences in flight time between promptly accelerated ionsand ions allowed to drift across a short field-free regionbefore acceleration were measured and converted fromvelocity distributions to IKE distributions. These studiessuggested that the desorbed matrix and analyte speciesattained mass independent velocities of between 500 and1200 m s�1 leading to increases in IKE with increases inmass. These studies have been augmented by evaluationof the flight time shifts in delayed extraction MALDI5

which further support a mass-independent velocity asso-ciated with desorbed matrix and analyte ions.

* Correspondence to: G. R. Kinsel, Department of Chemistry andBiochemistry, University of Texas at Arlington, Box 19065, Arlington,Texas 76019-0065, USA.E-mail: [email protected]

Contract/grant sponsor: United States Department of Energy, Divi-sion of Chemical Sciences, Office of Basic Energy Science.

Contract/grant sponsor: National Science Foundation.

MALDI IKE distributions have also been measuredunder normal accelerating electric field conditions, i.e.several tens of thousands of V cm�1. Profile analysis ofdetected MALDI ion signals has been used to suggestlarge IKE distributions associated with the laser-desorbedanalyte ions.6 Furthermore, MALDI IKE distributionshave been determined using retarding grid approaches7

and the results of these studies implicate broad IKE dis-tributions with most probable energies at a deficit rela-tive to the full accelerating potential. In addition, it hasbeen observed that this IKE deficit increases with increas-ing molecular weight of the analyte molecule. Studies ofMALDI IKE distributions using an electrostatic analyzer(ESA) time-of-flight (TOF) mass spectrometer are consis-tent with the results of the retarding field experiments.8

The contrast between the group of results which sug-gest increasing IKEs with increasing mass under zeroaccelerating electric field conditions and the group ofresults which suggest decreasing IKEs with increasingmass under high accelerating electric field conditions hasbeen remarked upon.9 Two possible explanations for theobserved behavior have been discussed. In the first inter-pretation all ions are thought to be formed promptly andare either entrained in the expanding plume of desorbedmaterial under zero accelerating electric field conditionsor are impeded from acceleration by collisions with theexpanding plume under high accelerating electric fieldconditions. Reduced metastable fragmentation of MALDIions in delayed extraction experiments has been cited as

CCC 1076–5174/99/060684–07 $17.50 Received 12 December 1998Copyright 1999 John Wiley & Sons, Ltd. Accepted 10 March 1999

DYNAMICS OF MALDI ION FORMATION BY ESA/TOF-MS 685

evidence for the latter effect.5 In the second interpretationa substantial fraction of the analyte ions are thought to beformed throughout the plume of desorbed material in thegas phase above the sample probe. Under high accelerat-ing electric field conditions, this process results in ionsformed at lower potential energy surfaces in the massspectrometer source region. Coincidence of the regionof densest analyte ion formation in the gas phase abovethe sample surface with a depletion of ionized matrixmolecules has been cited as evidence of this gas-phaseprocess.10

In the present studies we examined MALDI IKE distri-butions for NaC, protonated 7-hydroxy-4-methylcoumarinmatrix ions and protonated bradykinin under a variety ofsource region accelerating electric field conditions using ahybrid ESA/TOF mass spectrometer. Ions with both posi-tive and negative kinetic energies relative to the full accel-eration potential are observed for all source region electricfields investigated with the IKE distributions narrowingas the source region electric field is reduced. Analysis ofion TOF as a function of transmitted IKE provides evi-dence of delayed acceleration and/or gas-phase formationof both protonated matrix and analyte ions. These ionsare shown to arise from either ionized or neutral mate-rial moving at a constant velocity of¾550 m s�1. Ourconclusions are consistent with previous studies from ourlaboratory.9 That is, we present a composite picture ofthe dynamics of MALDI ion formation which combinespromptly formed protonated matrix and analyte ions, withions formed/accelerated at delayed times in an expanding,constant velocity plume of laser desorbed material.

EXPERIMENTAL

The design and calibration of the ESA/TOF instrumentused in these studies have been described in detailpreviously.11 In brief, the instrument consists of a 380 mmradius, 90° turning ESA taken from an MS-9 massspectrometer (Kratos Analytical, Ramsey, NJ, USA) towhich source region modifications have been made and a380 mm drift tube at the exit of the ESA has been added.The ion source region consists of a repeller and two inde-pendently biasable stainless-steel acceleration electrodeswith openings covered by 90% transmission nickel mesh.In normal operation the second acceleration electrode ismaintained at ground and the first acceleration electrodemay be varied from ground to user selected voltages,allowing one or two stage ion acceleration.

Laser-desorbed ions travel 200 mm before passingthrough a mechanical slit at the entrance of theESA. Turning of the ions is accomplished by the applica-tion of matched positive and negative voltages to a pairof 90° arc stainless-steel electrodes separated by 12.7 mm.A second mechanical slit positioned at the exit of theESA further defines the IKE bandpass of the ESA/TOFinstrument. Transmitted ions traverse the added 380 mmfield-free drift tube and are then post-accelerated 2.25 kVbefore being detected by a dual microchannel plate. Forthe present studies the and ˇ slits were adjusted toš0.19 andš0.17 mm respectively. Using the proceduresdescribed previously, the kinetic energy bandpass of theESA was determined to be 5.9š 0.2 eV. In addition, our

previous studies have shown that absolute IKEs can bemeasured toš5 eV.11

MALDI samples are prepared by codeposition of 2µlof a 15 mg ml�1 solution of the matrix 7-hydroxy-4-methylcoumarin in methanol and 2µl of a 0.5 mg ml�1

solution of the peptide bradykinin in methanol on astainless-steel probe. After drying, the MALDI sample isintroduced to the ESA/TOF source region in the conven-tional manner such that the stainless-steel sample supportprojects through a hole in the repeller electrode to a posi-tion flush with the surface of the repeller electrode. Ionsare generated by irradiation of the sample with the pulsed337 nm output of a nitrogen laser operating at 3 Hz. Aphotodiode positioned to detect a reflection of the ioniz-ing laser pulse triggers acquisition of the TOF spectra bya LeCroy 9450 digital storage oscilloscope.

RESULTS AND DISCUSSION

Electric field dependence of ion kinetic energydistributions

Initial studies were focused on examining the IKE dis-tributions of the matrix and analyte ions produced byMALDI as a function of source region accelerating elec-tric field. In the first study the voltage applied to therepeller electrode was held constant atC7725 eV andboth the first and second acceleration electrodes wereheld at ground, leading to a source region electric fieldof C9480 eV cm�1. The voltages applied to the two elec-trodes of the ESA were set atš515.2 eV, appropriateto transmit an ion with a nominal kinetic energy ofC7728 eV (i.e.C3 eV relative to the full accelerationpotential). Hence the IKEs transmitted using these settingsrange from¾0 to C6 eV relative to the full accelera-tion potential. The sample-coated probe was then exposedto the pulsed 337 nm laser radiation at¾ 2ð thresholdfor ion formation and MALDI/TOF mass spectra wereacquired and signal averaged for 100 laser shots.

The voltages applied to the ESA electrodes were thenstepped byC0.4 eV (i.e. an increase ofC6 eV in thenominal IKE transmitted by the ESA, IKE range fromC6 to C12 eV relative to the full acceleration poten-tial), the sample probe was rotated by¾10° and a newMALDI/TOF mass spectrum was acquired. Subsequently,the ESA voltages were incrementally increased by 0.4 eVand MALDI/TOF mass spectra were recorded until reach-ing ESA voltages ofš520.0 eV (i.e.C75 eV relative tothe full acceleration potential). If, for any given setting ofthe ESA, no ion signal for the matrix or analyte could beobserved, the ESA electrode voltages were briefly returnedtoš515.2 eV to ensure that ions of the matrix and analytewere being formed. After obtaining the positive IKE scanthe ESA voltages were returned toš515.2 eV and thenscanned in incremental�0.4 eV steps while recordingMALDI/TOF mass spectra until voltages ofš510.0 eV(i.e. �75 eV relative to the full acceleration potential)were reached.

After recording the series of MALDI/TOF mass spectra,the ion signals for the target ions NaC (m/z 23), the proto-nated matrix (m/z 177–178) and the protonated bradykininparent species (m/z 1060–1062) were examined. For each

Copyright 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 684–690 (1999)

686 G. R. KINSEL ET AL.

Figure 1. IKE distributions for (a) m/z 23, (b) m/z 177 178 and(c) m/z 1060 1062 produced by MALDI and recorded usingan ESA/TOF instrument with a source region electric field of9480 eV cm�1. Horizontal error bars represent the calibrated IKEbandpass of the instrument. Vertical error bars (not shown) areof the order of 20 25% relative standard error.

target ion, the ion signal was baseline corrected and fittedby iterative regression using a Gaussian curve. The inte-grated ion signal was taken as the area under the best-fitcurve. This approach was employed both to remove biaserrors associated with experimentalist choice of baselineand to minimize errors associated with the lower signal-to-noise ion signals. The integrated ion signals were thenplotted versus the kinetic energy of the ions relative tothe full acceleration potential. Figure 1 shows the IKEdistributions of the target ions under the experimental con-ditions described.

The experiment was repeated at two lower sourceregion electric fields by employing two-stage accelerationconditions. Target IKE distributions were recorded atsource region electric fields of 6480 eV cm�1 (2445 eVapplied to the first acceleration grid) and 3380 eV cm�1

(4970 eV applied to the first acceleration grid). Figures 2and 3 shows the result of these experiments at the tworespective lower accelerating electric field conditions. Forall data shown in Figs 1–3, the horizontal error barsreflect the 6 eV kinetic energy bandpass of the ESA TOFinstrument. For clarity, vertical error bars of¾20–25%relative to the integrated ion signal (as determined byredundant measurement of the ion signal intensities at anumber of ESA voltage settings) have been omitted.

The large errors, particularly in the integrated ion signalintensities, make a detailed analysis of the IKE distri-butions difficult. It is unclear, for example, whether thefine structure observed in the IKE distributions is signif-icant or merely a reflection of the inherent shot-to-shotirreproducibility associated with MALDI compounded bythe often low ion signal intensities. (For these experi-ments errors of the order ofš25–30% relative to theaverage signal intensity are not uncommon.) However,



an overview of the electric field-dependentIKE distri-butions does allow several important general observa-tions to be made.First, the IKE distribution of the NaCions appearsto reflect two separatecomponents.A large

Copyright 1999JohnWiley & Sons,Ltd. J. MassSpectrom. 34, 684–690 (1999)


positive energy component with a most probable IKEof C3 to C12 eV is consistently observed at all sourceregion electric fields. The positive IKE distribution forNaC is similar to our previous work, although the mostprobable IKE has decreased from a value ofC19 eV.11

This may reflect an effect of the change in wavelengthused for laser desorption from 266 nm (previous work) to337 nm. Also of interest is the appearance of NaC ionshaving IKE deficits relative to the full acceleration poten-tial. These ions were not observed in our previous laserdesorption/ionization studies. Furthermore, the negativeIKE distributions of the NaC ions appear to sharpen andshift towards the full acceleration potential as the sourceregion electric field is reduced. This behavior could resultfrom two mechanisms: either a population of the NaCions is the product of gas-phase reactions in MALDI (e.g.charge separation or charge-transfer reactions) occurringat lower potential energy surfaces in the ESA/TOF sourceregion, or the NaC ions are promptly formed and experi-ence collisional kinetic energy losses which increase withincreases in the source region electric field.

With regard to the matrix and analyte IKE distribu-tions, several trends are worth noting. First, the IKEdistributions for the matrix and analyte extend to bothnegative and positive energies relative to the full accel-eration potential. However, at all source region electricfields the IKE distributions of the matrix ions are shiftedtowards the formation of positive kinetic energy ionswhile the analyte IKE distributions favor the formationof negative kinetic energy ions. Second, both matrix andanalyte IKE distributions narrow as the source regionelectric field is reduced. (Experiments performed in ourlaboratory demonstrate that this behavior continues asthe source region electric field is further reduced.) Asdiscussed for the NaC ions, the contraction of the nega-tive IKE distributions with reduced source region electricfields could implicate either gas-phase ion formation orcollisional kinetic energy loss. Less clear is an interpreta-tion of the contraction of the positive IKE distributions.We note, however, that we have previously reported anapparent source region electric field-dependent influenceon promptly formed bovine insulin cluster ions.9 In thepresent studies the contraction of the positive IKE dis-tributions may reflect either less efficient formation orless efficient collection of high kinetic energy ions at lowsource region electric fields.

Ion signal resolution and Flight time analysis

Closer examination of the ion signal profiles reveal signifi-cant differences in the signal resolution as a functionof the IKE relative to the full acceleration potential.Figure 4 shows a selection of them/z 177–178 matrixion signals obtained when using the highest accelerationelectric field. Isotopic resolution of them/z 177 and178 is observed for ions having positive kinetic energiesrelative to the full acceleration potential. Conversely,substantial degradation of the ion signal resolution isobserved for ions having negative kinetic energies relativeto the full acceleration potential. The transmitted IKEdistribution at each setting of the ESA is unchangedat 6 eV in these experiments. Thus, the degradation ofthe ion signal resolution for ions with negative kineticenergies relative to the full acceleration potential must

Figure 4. Selected ion signal profiles of the matrix ions(m/z 177 178) recorded at IKEs of (a) C33, (b) C9, (c) �9and (d) �33 eV relative to the full acceleration potential ofC7725 eV. The source region electric field was maintained atC9480 eV cm�1 during acquisition of these TOF spectra.

reflecta loss of coherencein the spatialand/ortemporalprofiles of ion acceleration/formationas comparedwiththe positivekinetic energy ions.

Further insight into this behavior is obtainedthroughan analysisof the flight times of the matrix and analyteions. From Fig. 4 and the data in Tables1 and 2, it isseenthat the flight times of the positive kinetic energymatrixandanalyteionsshift by anaverageof 7 and15 ns,respectively,as the energy of the ions is increasedbythe 6 eV increments.Conversely,the flight times of thenegativekinetic energy matrix and analyteions shift byanaverageof 22 and27 ns,respectively,astheenergy ofthe ions is decreasedby the 6 eV increments.

Tables1 and 2 also report the resultsof a first princi-ples calculationof the expectedflight times of the ionsusingoptimizedinstrumentalparameters.(Ion flight timeswerecalculatedfrom first principlesby assumingthat theions were formed at the repeller electrodesurfaceandacceleratedto total energiesof the repellerelectrodevolt-age plus the measuredIKE. All voltageswere assumedto be accuratelymeasured.Measuredion flight timeswere first correctedfor the instrumentaloffset betweenfiring of the laser and triggering of data acquisition.Subsequently,simplex regressionalanalysiswas usedtooptimize the distances(sourceregion, secondaccelera-tion region and drift tube) to obtain agreementbetweenthe calculatedand measuredflight times of the positivekinetic energy ions.All calculateddistanceswerein goodagreement(<500 µm difference)with measuredvalues.)Thesecalculationswere performedwith the assumptionthat ions arepromptly formedat the surfaceof the sam-pleprobeandacceleratedto a total energy equalto thefullaccelerationpotentialplus the relative IKE. Good agree-mentbetweenthe observedandcalculatedflight times isobservedfor the positive IKE distributions of both the



Table 1. Experimental and calculated flight times for them/z 177 178 protonated matrix ions as a functionof the relative IKE transmitted by the ESA/TOFinstrumenta

Relative Experimental Calculated Exp.� calc.IKE (eV)b flight time (µs)c flight time (µs)d flight time (µs)

C51 15.779 15.772 0.007C45 15.788 15.778 0.010C39 15.793 15.785 0.008C33 15.802 15.792 0.010C27 15.804 15.799 0.005C21 15.817 15.807 0.010C15 15.827 15.814 0.013C9 15.827 15.822 0.005C3 NA 15.830 NA�3 15.844 15.839 0.005�9 NA 15.845 NA�15 15.900 15.851 0.049�21 15.923 15.857 0.066�27 15.948 15.863 0.085�33 15.963 15.869 0.094�39 15.984 15.876 0.108�45 15.993 15.882 0.111�51 16.020 15.888 0.132

a For these experiments the source region electric field wasmaintained at 9480 eV cm�1 and the kinetic energy bandpass ofthe ESA/TOF instrument was held at 6 eV.b IKE relative to the full accelerating potential of C7725 eV.c Experimental flight time taken from the peak centroid of thecurve fit to the ion signal profile.d Calculated flight time obtained from first principles calculationusing optimized instrumental parameters. See text for details.

Table 2. Experimental and calculated flight times for them/z 1060 1062 protonated analyte ions as a func-tion of the relative IKE transmitted by the ESA TOFinstrumenta

Relative Experimental Calculated Exp.� calc.IKE (eV)b flight time (µs)c flight time (µs)d flight time (µs)

C51 38.612 38.600 0.012C45 38.624 38.617 0.007C39 38.638 38.634 0.004C33 38.650 38.651 �0.001C27 38.664 38.668 �0.004C21 38.689 38.686 0.003C15 38.714 38.704 0.010C9 38.719 38.723 �0.004C3 38.729 38.744 �0.015�3 38.769 38.766 0.003�9 38.790 38.781 0.009�15 38.825 38.796 0.029�21 38.854 38.810 0.044�27 38.883 38.825 0.058�33 38.917 38.840 0.077�39 38.938 38.855 0.083�45 38.960 38.870 0.090�51 38.983 38.884 0.099

a For these experiments the source region electric field wasmaintained at 9480 eV cm�1 and the kinetic energy bandpass ofthe ESA/TOF instrument was held at 6 eV.b IKE relative to the full accelerating potential of C7725 eV.c Experimental flight time taken from the peak centroid of thecurve fit to the ion signal profile.d Calculated flight time obtained from first principles calculationusing optimized instrumental parameters. See text for details.

matrix and analyte (average absolute errors of 8.5 and6.7 ns, respectively). For the matrix and analyte nega-tive IKE distributions, however, the experimental flighttimes diverge positively from the calculated flight timesas the relative IKE deficit increases. This behavior canonly result if additional time is required for the forma-tion and/or acceleration of the ions with negative relativekinetic energies. It should be stressed that the lengthenedflight times cannot be accounted for solely by the kineticenergy deficit of the ions.

A straightforward interpretation of the lengthened flighttime is possible, however, if the final protonated matrixand analyte ions are assumed to undergo delayed ionformation/acceleration in the gas phase at lower sourceregion potential energy surfaces. Specifically, the increasein the total flight time would represent the time requiredfor the precursor to the observed ions to move fromthe surface of the sample probe to the point of ion for-mation/acceleration. An approximation of this point ofion formation/acceleration can be determined from theabsolute magnitude of the kinetic energy deficit and thestrength of the source region electric field. Based on thisinterpretation, a plot of the difference between the exper-imental and calculated flight times versus the distancefrom the sample probe surface to the point of ion forma-tion/acceleration would give a first-order approximationof the velocity of the precursor material. (The calculationmay be considered a first-order approximation since theinitial velocity of the material requires that the neutral pre-cursor to the ions have an initial positive kinetic energy.Hence the point of ion formation must be at a lower poten-tial energy than that indicated by the IKE deficit. Iterativecalculations accounting for the initial precursor kineticenergy have been assessed and indicate that the correc-tion to the initial velocity is less than 1% of the reportedapproximate values for these low-m/z ions.)

Figure 5 shows a plot of the type described for them/z 177–178 andm/z 1060–1062 ions having negativeIKEs and produced in a source region electric field of9480 eV cm�1. Reasonably linear plots are obtained withslopes indicating precursor velocities for the matrix andanalyte ions of 400 and 480 m s�1, respectively. Table 3summarizes the evaluated precursor velocities for thematrix and analyte ions produced by MALDI under thethree different source region electric field conditions, indi-cating an overall average precursor velocity of 550 m s�1.

Figure 5. Plots of the difference in experimental and calculatedflight times for ions with negative IKEs versus the distance fromthe probe surface at which the potential energy corresponds tothe IKE for (�) m/z 177 and (ž) m/z 1060 at a source regionelectric field of 9480 eV cm�1.



Table 3. Calculated velocities of precursors to ions with neg-ative IKEs.

Protonated matrix ions Protonated analyte ionsSource region (m/z 177) (m/z 1060 1062)

electric field Velocity R2 Velocity R2

(eV cm�1/ (m s�1) (m s�1)

9480 400 0.94 480 0.986480 550 0.95 890 0.963380 500 0.96 460 0.90

Average 480 610

The nature of the precursor to the final gas-phaseformed/accelerated protonated matrix and analyte ionsis not known. Several mechanisms can be postulatedwhich would lead to the behavior observed. These mech-anisms include (1) constant-velocity plume entrainmentof promptly formed protonated matrix and analyte ionsfollowed by delayed acceleration of these ions at somepoint distant from the probe surface; (2) protonated matrixand analyte ion formation at some point distant from theprobe surface via gas-phase ion–molecule reactions; and(3) protonated matrix and analyte formation at some pointdistant from the probe surface via delayed dissociation ofneutral or ionized matrix and analyte clusters.

Although the experimental results do not allow an unam-biguous determination of the active mechanism a numberof additional comments can be made. Mechanism 1 isappealing in that all protonated matrix and analyte ionsobserved as final products are produced during the ini-tial laser excitation event. However, the experimentallyobserved IKE distributions and the implicit mass depen-dence of the total IKE deficit are difficult to reconcile withthis mechanism. In mechanism (1), the probability of form-ing an ion with a given kinetic energy deficit would beexpected to decrease monotonically as the distance fromthe probe surface increases due to the decreasing densityof the expanding entrainment plume. This behavior is notobserved, however, for either the protonated analyte or theNaC ions as both ions have non-zero most probable negativeIKE values. Furthermore, in order for the entrainment pro-cess to result in an overall kinetic energy deficit, the kineticenergy acquired as a result of entrainment must be less thanthe kinetic energy loss resulting from delayed acceleration.Specifically, this requirement would imply that entrainmentis maintained to distances further from the probe surfaceas the mass of the analyte increases. For example, in a10 000 eV cm�1 electric field, anm/z 1000 ion would needto be constrained by the 550 m s�1 entrainment plume todistances>1.6µm off the probe surface for it to exhibitan overall kinetic energy deficit. Anm/z 50 000 ion, on theother hand, would need to be constrained by the 550 m s�1

entrainment plume to distances>78 µm off the probe sur-face for it to exhibit a kinetic energy deficit.

Mechanism (2) would require that the matrix ions (orother putative proton donor molecules) remain in thevicinity of the constant velocity neutral matrix and ana-lyte molecules until a point distant from the probe surfacewhere proton transfer occurs. Without invoking constant-velocity entrainment of the proton donor (as in mech-anism (1)), however, this mechanism is difficult to rec-oncile with the experimental data. Most of the promptlyformed matrix ions, represented by the positive kineticenergy component of the matrix IKE distribution, have

velocities>550 m s�1 (corresponds to a matrix IKE ofjust 0.28 eV). Hence, the promptly formed matrix ionswould be expected to move rapidly beyond the neutralmatrix and analyte molecules in the expanding plume.

Mechanism (3), involving delayed emission of proto-nated matrix and analyte ions from larger clusters, is inmany respects analogous to a mechanism proposed forelectrospray ion formation.12 The emission of clustersduring laser desorption has been evaluated theoretically13

and the viability of intracluster proton transfer in pho-toexcited matrix–analyte clusters has been demonstratedexperimentally.14–17 For this mechanism to be consistentwith the experimental results would require either that theprecursor matrix–analyte cluster be neutral and travelingat a constant velocity, or that ionized matrix –analyte clus-ters be large enough to exhibit only modest accelerationincreases in velocity before emission of the final prod-uct ions. This latter observation may provide a consistentinterpretation of the slight curvature of the plots shownin Fig. 5. Additional studies of the dynamics of MALDIion formation, combined with studies designed to probethe specific proposed mechanisms, could provide furthervaluable insight into the MALDI process.

CONCLUSIONS

The experimental results presented in this paper providenew insights into the dynamics of ion formation in contin-uous extraction MALDI. Analysis of the field-dependentIKE distributions and ion flight times leads to a pictureof ion formation combining two groups of ions with sub-stantially different ion formation dynamics. For both thematrix and the analyte used in these studies one group ofions is formed promptly with IKE distributions equal toor greater than the full acceleration potential. The kineticenergies of these ions can reachC75 eV under strongaccelerating electric field conditions.

A second group of the matrix and analyte ions arethe product of delayed acceleration and/or ionization pro-cesses occurring above the probe surface in an expandingplume of constant-velocity material. Analysis of the flighttimes of these ions suggests a most probable velocityfor the entrained material of¾550 m s�1. A mechanismof formation for these ions requires either that promptlyformed ions are entrained, and physically impeded fromacceleration, in an expanding plume of constant-velocitymaterial, or that the ions are the product of gas-phase pro-ton transfer reactions at lower potential energy surfaces inthe ion source region. The IKE distributions suggest that,while both protonated matrix and analyte ions are formedby the gas-phase mechanism, the process appears to dom-inate slightly in the formation of the analyte ions. Cumu-latively, the picture of MALDI ion formation dynamicspresented is consistent with a mechanism previously sug-gested to explain changes in the ion signal profiles ofbovine insulin cluster ions.

Acknowledgements

This work was supported by grants from the United States Departmentof Energy, Division of Chemical Sciences, Office of Basic EnergyScience and from the National Science Foundation.

Copyright 1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 684 690 (1999)


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Documents

Investigation of the dynamics of matrix-assisted laser desorption/ionization ion formation using an electrostatic analyzer/time-of-flight mass spectrometer