Ionization

Aug 2, 2010 - Vernadskogo 86, Moscow, 117571, Russia. ReceiVed: May 7, 2010; ReVised Manuscript ReceiVed: July 5, 2010. The initial ionization reactio...
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J. Phys. Chem. B 2010, 114, 10853–10859

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Initial Ionization Reaction in Matrix-Assisted Laser Desorption/Ionization Bo-Hong Liu,† Oleg P. Charkin,‡ Nina Klemenko,§ Chiu Wen Chen,† and Yi-Sheng Wang*,† Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang District, Taipei 115, Taiwan, R. O. C., Institute of Problems of Chemical Physics, Russian Academy of Sciences, Institutskii pr. 18, ChernogoloVka, Moscow oblast, 142432, Russia, and LomonosoV State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 117571, Russia ReceiVed: May 7, 2010; ReVised Manuscript ReceiVed: July 5, 2010

The initial ionization reaction in matrix-assisted laser desorption/ionization (MALDI) was examined on the basis of the appearance of photoelectrons. The threshold laser fluence for the ejection of photoelectrons from 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), and trihydroxyacetopheone (THAP) on stainless steel (SS) substrates was 0.05, 0.41, and 8.39 mJ/cm2, respectively. These values are considerably lower than those for MALDI ions, indicating that the electron detachment likely precedes other ionization reactions. The SS substrate played an insignificant role in the production of photoelectrons because suspended DHB produced a photoelectron signal similar to DHB on the SS surface, and decreasing the DHB thickness on the SS reduced the photoelectron intensity. For crystalline DHB and SA, the photoelectron intensity increased with the laser (337 nm) fluence in a relationship of less than second order, suggesting considerable reductions of ionization potentials in comparison with free molecules. According to ab initio calculations, the ionization potential of DHB clusters reduces as the cluster size increases from monomer to octamer. The impact of these abundant electrons on the ion production in MALDI is discussed. Electron disproportionation

Introduction Elucidating the initial ionization reaction, an unresolved puzzle, is fundamental to extending the applications of matrix-assisted laser desorption/ionization (MALDI). Most mechanistic studies have examined the production of ions to establish possible reaction models,1-4 in which the charge redistributions after the initial ionization is a common interpretation for the final product formation.4-6 However, conceptual differences exist as to the production of initial ions, such as whether they are produced by multiphoton ionization3,4,7,8 or charge disproportionations,2,8-12 or even whether they may be preformed in crystals.1,13 Unfortunately, information about initial ionization is obscured by extensive ion-molecule reactions, especially if high laser fluences resulted in a harsh ionization condition and produced complicated spectral patterns. It is thus important to study the initial reaction from the early ionization stage, including the production of photoelectrons. Photoexcitation of matrix molecules is a generally accepted concept and is described extensively in the literature, especially for 2,5-dihydroxybenzoic acid (DHB).11,14-19 Equations 1-3 describe the three fundamental pathways of initial ionization induced by laser photons.

Photoionization

M + nhν f M•+ + e-

(1)

* Corresponding author. Phone: 886-2-27871272. Fax: 886-2-27899931. E-mail: [email protected]. † Academia Sinica. ‡ Russian Academy of Sciences. § Lomonosov State Academy of Fine Chemical Technology.

Proton disproportionation

2M + nhν f M•+ + M•- (2) 2M + nhν f [M + H]+ + [M - H]-

(3)

where M denotes the matrix molecules. The practical MALDI may involve multiple processes in parallel. Knochenmuss and co-workers have determined that the ionization potential (IP) of gaseous (DHB)1-10 clusters via eq 1 levels off at roughly 7.85 eV, an energy above two laser photons with commonly used wavelengths (337 nm ) 3.68 eV, 355 nm ) 3.48 eV).20 Since a three-photon process demands very high laser fluence and is impractical in MALDI, it was proposed that the energy pooling of two excited DHB molecules and the internal energy compensates for the energy deficit for ionization.5,21 In contrast to the leveling of IP of DHB trimer and larger clusters, a continuous reduction of IP was experimentally and theoretically found for DHB-(proline)n clusters.18 Such a contradiction has not been examined, probably due to the absence of reliable simulation of large DHB clusters. Laser-induced charge disproportionations2,9,11 are also possible ionization pathways (eqs 2 and 3), but our recent study using a synchronized dualpolarity ion detection method showed no apparent correlation between positive and negative DHB ions.12 In fact, intense photoelectron signals were seen in this work, even though a laser fluence below the ion appearance threshold was used. This result suggested that the electron-detachment was a dominant reaction before the ejection of ions, and there was an immediate concern for the source of electrons and the connection between electrons and negative ions.22 The impact of electrons on MALDI ion generation has been discussed in the literature,23-29 although the origin of the electrons is not clearly understood. With typical laser fluences,

10.1021/jp104178m  2010 American Chemical Society Published on Web 08/02/2010

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free electrons were found to be involved in the production of negative ions via electron-capture reactions26 or to suppress positive ions due to charge neutralization.13,23,24 Some of the photoionization models proposed that electrons are ejected mainly from metal-matrix boundaries.24,29-31 It was also reported in this series of literature that photoelectron production was negligible when dielectric substrates were used to support the matrix, and no photoelectrons were ejected from an empty stainless steel (SS) substrate.24 However, a rational reason for the enhancement of the ion signal with dielectric substrates is missing in this postulation if the metal-matrix boundary possesses the highest ionization efficiency. Indeed, our previous observation showed an intense photoelectron signal from DHB on both SS and dielectric substrates.12 To accurately explain the ionization reaction, new experimental evidence is critically needed. This work studied the origin of photoelectrons in laser fluence regions lower than the ion appearance threshold. Experiments were conducted using a mass spectrometer optimized for electron detection. We report the appearance of photoelectrons near threshold fluence for three commonly used matrices, including DHB, sinapinic acid (SA), and trihydroxyacetophenone (THAP). Further studies on the relationship between photoelectron intensity and laser fluence, substrate interaction, and matrix thickness were conducted using DHB. The density functional theory calculation for the stable geometries and IP values of (DHB)n clusters, where n ) 1-8, are also reported for the first time. Combined with previous observations, a qualitative ionization sequence is proposed on the basis of the results in this work. Experimental Section A laboratory-made MALDI time-of-flight (TOF) mass spectrometer was utilized for the measurements. It comprised a special substrate geometry, allowing the use of regular matrix crystals on substrates or suspended matrix wafers. The ion source resembled conventional MALDI sources in that it consisted of a sample, an extraction, and a grounded electrode, but an additional electrode was installed behind the sample electrode to adjust the electric field when suspended matrix wafers were used. The typical electric potentials of the sample, the extraction, and the back electrodes were (10, (9, and (10.2 kV, respectively. The extraction voltage was adjusted to optimize the signal. All the electrodes were installed coaxially and equally spaced at 10 mm apart from each other. Except for the sample electrode, all other electrodes were washer-shaped SS discs (65 mm o.d., 5-10 mm i.d.). Ions and electrons produced in the source region were mass-separated by a 75 cm TOF region and detected by a chevron-type microchannel plate detector. The data used were averages of 50 laser shots. Figure 1a shows the schematic diagram of the ion source. The sample probe consisted of two types of removable metal plates to accommodate the samples. For regular MALDI samples on metal substrates, a 2-mm-thick rectangular SS plate was used (Figure 1b). For suspended crystals, a 0.15-mm-thick rectangular SS foil with a 5-mm-i.d. central hole was used to support the wafer-shaped crystals. This thin foil with the crystal wafer was sandwiched between the sample probe and another 2-mm-thick metal plate identical in shape to the SS foil apart from the thickness (Figure 1c). Pictures of thick DHB on SS and suspended DHB are presented in Figure 1d and e, respectively. Experiments used a 0.8 mm circular area of a nitrogen laser beam (337 nm, 3 ns pulse width, VSL 337ND-S, SpectraPhysics Inc., Mountain View, CA) selected with an iris

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Figure 1. Schematics of ion source and sample morphology. (a) Configuration of the electrode plates, (b) configuration of the matrix on the metal surface, (c) configuration of the suspended matrix, (d) picture of the DHB/SS, and (e) picture of the suspended DHB.

diaphragm. The laser beam examined the sample along a path that was about 14° from the surface normal. The sample probes were installed from the side of the sample electrode and fixed during experiments. Because the laser beam was fixed, only one sampling position was examined, and a fresh sample probe was always used whenever a new sampling position was preferable. The selected beam was focused by a fused-silica lens (f ) 150 mm) to a spot of roughly 83 µm in diameter (full width at 10% maximum) at the sample surface. A flat top beam structure with 4-6 small hotspots distributed near the edge was obtained at the sample surface, as examined by a laser beam profiler (WinCamD, DataRay Inc. Boulder Creek, CA). The laser beam size and energy for fluence calculation corresponded to the value at the ion source region. All the matrix compounds were obtained commercially (Sigma-Aldrich Co., St. Louis, MO) and were used without further purification. To prepare thick DHB on SS substrates (DHB/SS), saturated DHB in 50% aqueous acetonitrile was deposited onto the probes by the conventional dried-droplet method. The probes were preheated to 70 °C to allow rapid solution drying and were then further vacuum-dried. The procedure was repeated until a matrix thickness of 100-300 µm was obtained. SA and THAP were prepared via a similar manner except that they were dissolved in 67% aqueous acetonitrile. The thin DHB film on SS substrates was prepared by dropping 0.5 µL of 1/10 saturated DHB solution (in 50% aqueous acetonitrile) on the probes. After drying, 1 µL of acetone was applied to the probe to recrystallize the sample rapidly, resulting in a homogeneous thin DHB layer. To prepare suspended DHB, the saturated DHB solution was carefully deposited near the hole of the substrate. The droplet was pulled by using a pipet tip to form an aqueous film covering the hole and dried in ambient conditions before further vacuum-drying. The suspended DHB sample was normally obtained by repeating the procedure three to four times, and the resultant crystal thickness was 500-800 µm. Suspended SA and THAP were not available because their crystal wafers were too fragile to be held on the hollow sample electrode.

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J. Phys. Chem. B, Vol. 114, No. 33, 2010 10855 TABLE 1: Appearance Threshold Fluences of Electrons and Ions from Various Samples threshold fluence (mJ/cm2)a sample DHB SA/SS empty SS THAP/SS

Figure 2. Threshold photoelectron intensity of various samples as a function of laser fluence. The criterion for valid signals was S/N > 20 for photoelectron (S/N > 3 for ions), as shown by the shaded areas. O, suspended DHB; b, DHB on SS; 2, SA on SS; 0, empty SS; (, THAP on SS.

The signal-to-noise ratio (S/N) was used in the statistical analyses. An S/N ratio above 20 was defined as the criterion used to select valid photoelectrons because a transient signal from the laser cavity appeared at the same time, so the reported threshold fluences were slightly overestimated. The criterion for ion appearance threshold was S/N > 3 because there was no electronic noise picked up at longer flight times. To compare the rate of signal decline, the “half-life time” method used by Quist and co-workers32 was conducted. It defines the number of laser shots that reduces the value of the fitting curve of the signal by half and is symbolized as N1/2 in the present work. No photoelectron and ion signal was observed when an empty hollow substrate was used. Unless otherwise mentioned, the threshold values in this work represent the threshold fluence for the appearance of photoelectrons. The theoretical part examined trends in changing ionization potentials of oligomeric clusters (DHB)n with increasing size and changing structure, using quantum-chemical DFT calculations. Geometry optimization and calculations of normal vibration frequencies of all structures were carried out using the Gaussian-03 program within the B3LYP approximation with the standard 6-31G* basis set. Total energies of the optimized structures were recalculated (single-point) with the extended 6-311++G** basis set. Results Mass Spectrometric Details. For all three matrices used in this work, photoelectrons began to appear at much lower laser fluences than were required for producing ions. The threshold behaviors of DHB, SA, and THAP were distinct from each other, as shown in Figure 2. The appearance of photoelectrons from the empty SS substrate was also presented for comparison. With DHB crystals, a clear photoelectron signal started at roughly 0.03-0.05 mJ/cm2, and the intensity increased rapidly with the increase of laser fluence. The difference between the suspended DHB and DHB/SS was within the experimental uncertainty because the values approached the detection limit of the laser energy detector. In the cases of SA and THAP, the threshold fluences were 0.41 and 8.39 mJ/cm2, respectively, and the signal also increased sharply with fluence. Table 1 lists the photoelectron threshold fluence of various samples. The corresponding appearance thresholds of ions are also reported in Table 1, but most of these values were recorded with a large laser beam area because the maximum achievable laser energy with the small laser beam area was intangible to produce ions, except with SA. Increasing the laser beam area was effective

suspended thick crystal/SS thin crystal/SS

electron

ion

0.03 0.05 0.45 0.41 0.56 8.39

21.0c 21.0c 20.5c 16.5b 138.4c 25.1c

a All numbers are calibrated with beam size and laser energy at ion source position. b Result obtained using the same laser beam size as for electron detection. c Scaled values with respect to footnote b using relative values obtained with large laser beam size (roughly 210 µm in diameter at the sample surface).

for ion production, but this introduced irregular laser beam profiles with many hotspots. As a consequence, the threshold fluences of ions reported in Table 1 can be compared only qualitatively with the result of the photoelectron. The photoelectron signal declined when the same sample position was subjected to continuous laser irradiation. The rates of decline were faster with higher laser fluence, which was different from the result of biomolecular ions reported previously.33 Figure 3a-c shows the changes of photoelectron signal of various samples as a function of the number of laser shots at three laser fluences. The data from the suspended DHB and DHB/SS samples qualitatively overlaid each other in most of the fluence range. When utilizing 3.74, 0.75, and 0.15 mJ/cm2, the N1/2 of DHB/SS was 10, 17 and above 50, respectively. The corresponding N1/2 of suspended DHB became 14, 50, and above 50, respectively. For SA, the N1/2 was 22 for 3.74 mJ/cm2, and no apparent decay was observed for 0.75 mJ/cm2. In general, higher fluence produced a stronger photoelectron intensity but a faster decay rate. Notably, no decay of the electron signal from the empty SS substrate was observed at any laser fluence. Increasing the laser fluence increased the photoelectron intensity. Figure 4 shows the results obtained by averaging the signal of the first five laser shots of suspended DHB, DHB/SS, and SA/SS on a logarithmic scale. The results from suspended DHB and DHB/SS were similar. The SA/SS delivered photoelectrons at significantly higher laser fluence but increased with a stronger dependency with fluence than DHB. The saturation of the signal in the high fluence range may be due to an overly large electron distribution above the detector area. Apart from the data points that represented no detectable signal and saturation, the linear fit to the suspended DHB, DHB/SS, and SA/SS data gave slopes of roughly 1.0, 1.2, and 1.7, respectively. Theoretical Structure and Energetic Predictions. We examined trends in the IPs for three elementary structural types of (DHB)n cluster, when n varies in the range n ) 1-8, as shown in Figure 5: Type A: quasi-linear chains that imitate the chain structure with dimeric units (DHB)2 in the crystalline DHB (structure A in Figure 5). Type B: quasi-planar cyclic structures consisting of monomeric units that are connected by H-bonds between ortho-OH groups and the O atoms of carbonyl groups (structure B in Figure 5). Type C: three-dimensional cyclic structures from the monomeric units connected by H-bonds between the OH group and an O atom of carbonyl groups of the neighbor moieties (structure C in Figure 5). They have been obtained over several steps. Initially, the geometry of the (HCOOH)n formic acid oligomers

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Figure 3. Intensity evolutions of photoelectrons over 50 laser shots with laser fluences of (a) 3.74, (b) 0.75, and (c) 0.15 mJ/cm2 at fixed sample positions. O, suspended DHB; b, DHB on SS; 2, SA on SS; and 0, empty SS.

Figure 4. Photoelectron intensity with various sample conditions as a function of laser fluence (logarithmic scale). The slopes of best fit were 1.2, 1.0, and 1.7 for DHB on SS, suspended DHB, and SA on SS, respectively. O, suspended DHB; b, DHB on SS; and 2, SA on SS.

were optimized for n ) 2-6, producing rather complicated three-dimensional “core” rings that were connected by short and strong “internal” hydrogen bonds. Then all “external” C-H bonds were replaced by C-C6H3(OH)2 fragments, and these structures were used as starting ones for the final optimizations of the structures of the C type. The optimized structures of A, B, and C type are depicted for dimer and hexamer clusters (DHB)n. Relative energies of A, B, and C type structures and corresponding values of vertical ionization potentials calculated as the difference of total energies of neutral (DHB)n clusters and their ions (DHB)nb+ are given in Table 2 for n ) 1, 2, 4, 6, and 8, except for the C type dimer and octamer that are unstable upon optimization. One can see from Table 2 that the structures A and C are most favorable in energetic terms, but the tendency of IP of the clusters (DHB)n with increasing n appears to be quite similar for all three elementary structural types, at least at the semiquantitative level; when passing from monomer to octamer, the calculated values of IP decrease monotonically in a hyperbolic manner from ∼8.2 to ∼6.5 eV and can be presumed to decrease even further for larger n. This tendency remains strong when expanding the basis set from 6-31G* to 6-311++G** for more accurate energy prediction, in which the resultant IP values increased systematically by 0.2-0.3 eV with the higher basis set. Moreover, the calculated IP values (overestimated against experiment by a few tenths of an electronvolt) for various structures with the same n are rather close to each other, with differences between them not exceeding 0.2-0.3 eV. Discussion Threshold Fluence of Photoelectrons. The production of photoelectrons is a clear indication of ionization reactions, but

the electron detection is highly sensitive to the transmission loss and the detection method. The transmission loss is due mainly to imperfections of the electric field that fail to guide the electrons to the detector. In the present work, the electron signal changed with the laser irradiation position and the local electric field. Similar photoelectron signal changes were not reported in the past, probably due to the insufficient transmission efficiency of electrons through the long TOF tubes of conventional instruments. Changing the detection method may reduce the transmission loss but may introduce additional experimental uncertainties. The electron-capture reaction by sulfate hexafluoride (SF6) is an indirect but effective method for electron detection, especially if ion-trapping is performed.24,25,31 However, in such a method, the presence of an “effective electron signature” depends on the electron concentration and the electron-capture cross section of SF6. Because the electroncapture cross section is a function of electron energy, the kinetic energy change of photoelectrons ejected from various surface compositions or laser condition may have greatly altered the appearance of SF6-. The threshold fluences of photoelectrons in Table 1 are by far the lowest reported. They are even lower than the result estimated by the values in the works of Dashtiev and co-workers and Frankevich and co-workers.24,25 The only comparable result is the observation obtained using a quartz crystal microbalance by Quist and co-workers,32 where negatively charged particles from ferulic acid were ejected with much lower fluences than that for positive and neutral particles; the negative particles ejected with low fluence were assumed to be photoelectrons by the authors. In our work, the power-law dependence of photoelectrons on laser fluence (Figure 4) is closer to the yield of laser-induce fluorescence of crystalline DHB34 than ions. However, quantitative analyses based on mass spectra should be interpreted with care, and additional evidence is necessary to elucidate the correlation between the fluorescence and the photelectrons. Origin of Photoelectrons. The data from Figures 2-4 clearly indicate that the photoelectrons most likely originate from the matrix. Two pieces of evidence suggest that the matrix-metal hybrid orbital is unlikely to be a significant contributor in the production of photoelectrons. First, the same electron behaviors were observed for suspended DHB and DHB/SS; if metal played an essential role, the results from suspended DHB should be quite distinct from those with DHB/SS. Second, decreasing the thickness of the DHB crystal did not promote the production of electrons (Table 1), a result that contradicts the prediction of a reduced Fermi level near the matrix-metal interface. Indeed, a substantial increase of threshold fluence for thin DHB on SS was observed, whereas the threshold of DHB ions remained

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Figure 5. Stable configurations of A, B, and C types of (DHB)2 and (DHB)6 predicted by density functional theory calculations. A, quasi-linear chain structure; B, quasi-planar cyclic structure; and C, 3D-cyclic structure. Red, gray, and white balls represent oxygen, carbon and hydrogen atoms, respectively.

TABLE 2: Ionization Potentials and Relative Energies of Various Structures of (DHB)n Oligomers Calculated at B3LYP Level with 6-31G* Basis Sets (in eV) A structure, (DHB)n DHB (DHB)2 (DHB)4 (DHB)6 (DHB)8

B

C

Erel

IP

Erel

IP

0 0 0 0

8.24a 7.22 6.76 6.60 6.40

+0.73 +0.32 +0.17 +0.11

7.10 7.00 6.70 6.53

a Observed IP of DHB monomer is 8.05 eV.20 stable.

Erel

IP

transform to A +0.21 6.95 -0.4 6.50 b b

Structure not

the same for all sample conditions. Notably, the power-law relationships of less than second order in Figure 4 imply that the energy-pooling of multiple excitons4 is not critically needed for electron detachment under low laser fluences. Such energypooling processes may become significant only when high laser fluences are used. The theoretical calculation partly resembled our observation that the ejection of photoelectrons from DHB is not an energydemanding process. The IP value of DHB clusters reduced with cluster size, which is similar to the result previously found for one DHB associated with proline clusters.18 For DHB tetramers to octamers, the IP values were well within the summed energy of two 355 or 337 nm laser photons. Although the IP (or work function) of crystalline DHB is currently unavailable, it is expected to be lower than the IP of DHB clusters because it contains near-infinite solvation numbers and three-dimensional architecture. In contrast to the full quantum-chemical method used in this work, an increase in IP with cluster size was previously predicted by Kinsel and co-workers for the solidstate arginine-DHB-water complexes.19 The disagreement between this computational study and our observation is probably due mainly to the distinct system used in these two works or partly to the uncertainty of the point charges model used in the simulation of Kinsel and co-workers. It is known35,36 that the structure of crystalline DHB (with n f ∞) contains chains of dimeric structural units (DHB)2 that have rather strong internal hydrogen bonds between carboxyl groups and are connected with the adjacent dimers by means of weaker H-bonds between hydroxyl groups, localized in the position meta to the carboxyl group (see structures A in Figure 5). For the gaseous clusters (DHB)n, the structural problem is

much more complicated because numerous conformers with various ionization potential can correspond in principle to each value of n, and a systematic search of the most energetically favorable structures between them is computationally prohibitive. Moreover, the cluster structure under conditions of energy “feeding up” in a MALDI experiment can differ from the best structure of a free cluster. One can suppose that the above-mentioned tendency found for the elementary A, B, and C structures of the isolated clusters remains valid for the real crystalline structures of similar and more complicated (DHB)n clusters, such as the irregular matrix structures prepared by fast crystallization methods. Upon ionization of all the clusters under discussion, an electron is removed from the corresponding highest occupied molecular orbitals which, as indicated by Mulliken population analysis, are presented everywhere as a linear combination of occupied π molecular orbitals of all benzene rings in the cluster with minor other contributions. The predicted IP of DHB clusters deviate from the observation by Lin and Knochenmuss20 in which the IP levels off at 7.8 eV. Although an unambiguous IP was determined for the DHB monomer, the increase in possible geometries hindered the precise determination of the ionization threshold for molecular clusters, as proposed by the authors. Scaling the predicted IP of the DHB monomer in the present work to the observation by Lin and Knochenmuss requires a factor of 0.934. However, this scaling factor increases the deviation between the prediction and the observation for clusters. This may occur if the geometry of clusters in the molecular beam was predicted to be energetically less favorable. The abundance of the global minima geometry may be increased if a low molecular beam temperature can be achieved. Contribution of Photoelectrons to MALDI. Our results show that the ionization reaction of MALDI starts under much softer conditions than previously proposed, at least for the ejection of photoelectrons. It is thus important to rationalize the impact of the electron-rich environment on the production of DHB ions, especially when ion generation demands laser fluence more than 2 orders of magnitude higher than electron ejection. One cannot, however, rule out the possible contribution of other parallel or consecutive reaction channels leading to ion production under high laser fluence conditions. Ions may also be released from precharged matrixes and analytes in crystals by laser excitation,1,37 but such reactions require prompt phase

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Figure 6. Qualitative sequential MALDI reactions along the laserfluence coordinate. The shed areas denote the materials that are subjected to phase transition. The reactions started from low fluence persist toward high laser fluences. M represents matrix molecules, underlined species are final products, and dashed arrows represent processes involving extensive ion-molecular reactions. Ions may also be produced by disintegration of a precharged crystal (*) in the high laser fluence range, followed by neutralization with photoelectrons.

transitions to minimize the recombination of precharged ion pairs in crystals, a condition demanding middle to high laser fluences. Indeed, the result in the present work may be adopted to the cluster-ionization/lucky survivor model1,13 to rationalize the source of electrons for rapid neutralization of highly charged positive ions. Thus, attempts to illustrate other ionization mechanisms for DHB should take into consideration the negatively charged environment, or at least an excess of electrons. An apparent example is the suppression of positive ions by neutralization with photoelectrons.13,31 If neutralization occurs in the solid phase, the released energy may increase the surface temperature to accelerate sublimation or thermal ionization reactions. For DHB, neutralization may be reduced using a thin matrix because fewer photoelectrons are ejected from the thinner DHB. This could be an essential reason for the enhancement of positive ions with decreasing matrix thickness reported in literature,30 but further examinations are necessary. As discussed previously,12 electron-capture ionization may be responsible for production of negative ions if the laser fluence is high enough to desorb solid DHB. This scenario is still valid because the DHB morphologies did not show significant changes upon irradiation at the threshold fluence of photoelectrons. Under such conditions, the desorption of the solid matrix was very slow because the increase in the surface temperature by laser excitation was only a few degrees Kelvin, as estimated using the formulation in the literature.38 The lower onset energy for the ejection of neutral molecules as compared to ions was also reported in the literature.38-42 With high laser fluence, the sharp decay of electron intensities as a function of the number of laser shots (Figure 3) is probably due to photochemical decay of the matrixes upon continuous laser excitation (see Supporting Information), which subsequently changes the ionization property.43 The photoelectron decay is quite similar to that of ions in routine analyses, in which intense and continuous ion signals are normally observed only at “sweet spots”. The proton or electron disproportionation likely demands higher energy than direct photoionization; otherwise, such reaction products should possess similar threshold fluences. Thus, a reaction sequence of DHB under various laser fluences can be divided qualitatively into three regions, as summarized in Figure 6. The lowest fluence region induces only photoionization but without appreciable temperature increase. Some of the photoelectrons can be captured by matrix molecules in the upper layer when traveling through the crystal. In the medium

Liu et al. fluence region, the photoionization accelerates and the laser fluence is high enough to increase the temperature for the slow phase transition (sublimation) of the surface layer. Under this condition, negative matrix ions can be released to the gas phase or produced via electron capture of gaseous matrix molecules. As laser fluence further develops, volume phase transition bursts and is accompanied by complicated primary and secondary reactions, including proton/electron disproportionations, ion evaporations, or charge-separation of precharged molecules in crystals. Most reactions occur in the solid phase or the dense environment; especially, photoionization may occur in deep crystal regions due to the low reaction barrier. The major channel of ion reduction is likely the neutralization of positive ions by highly mobile photoelectrons. For SA and THAP, the relative abundances of photoelectrons and ions in the ionization region are different from DHB; thus, the reaction sequence diagram may require considerable revisions. With a reduced extent of electron detachment, charge disproportionations may dominate the ionization pathway. This can be seen from the complicated positive ions produced by DHB in comparison with the simple protonated intact molecules seen in SA and THAP. As a consequence, the investigation of secondary reactions for SA or THAP would be more feasible than that for DHB. Conclusions For DHB, SA, and THAP, the ejection of photoelectrons via photoionization precedes other possible primary ionization reactions. The major source of photoelectrons is likely to be solid matrix molecules with a large cluster size; no evidence of a positive contribution from the metal substrate is obtained. The IP of crystalline DHB may be considerably lower than two UV laser photons because the appearance of photoelectron versus laser fluence showed a relationship of about first order. Theoretical prediction shows that the IP values of DHB tetramers or larger are well below the two photon energy of commonly used lasers. Such photon-to-electron conversion efficiency of crystalline DHB is very high because the ejection of photoelectrons requires less than 1% of the laser fluence for ion ejection. With such a low fluence, the photoionization occurs before material desorption because the estimated surface temperature increase is only a few degrees Kelvin. Although other primary reactions may be also involved during ion ejection, the impact of the electron-rich environment on those reactions should be considered. Thus, the detailed ion ejection mechanism of every matrix may be unique because the laser fluence demanded for ion and electron ejection of every matrix is distinct. It is thus important to further investigate the correlation between electrons and other primary reactions, especially in the high laser fluence region where intense and complex plasma is produced. The thermodynamics properties of solid ions have been studied using THAP in our group and will be reported shortly. In addition, the cold but electron-rich environment produced by low laser fluences may be advantageous for enhancing the ion yield of thermally labile molecules. The associated studies using oligosaccharides are currently under investigation. Acknowledgment. The authors thank Profs. Yuan Tseh Lee and Sheng-Hsian Lin and Mr. Yin-Hung Lai for useful discussions. We are grateful to the Academia Sinica Computing Center for providing essential computing resources. We acknowledge financial support from the Genomics Research Center, Academia Sinica, and the National Science Council of Taiwan (Contract no. NSC 98-2113-M-001-021-MY3).

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