A Thermal Mechanism of Ion Formation in MALDI.

An important recent discovery concerning the fundamentals of matrix-assisted laser desorption/ionization (MALDI) is that the abundance of each ion appearing in a spectrum is fixed, regardless of the experimental condition, when an effective temperature associated with the spectrum is fixed. We describe this phenomenon and the thermal picture for the ion formation in MALDI derived from it. Accepting that matrix-to-analyte proton transfer is in quasi-equilibrium as supported by experimental data, the above thermal determination occurs because the primary (matrix) ion formation processes are thermally governed. We propose that the abundances of the primary ions are limited by the autoprotolysis-recombination process regardless of how they are initially produced. Finally, we note that primary ion formation, secondary (analyte) ion formation, and their dissociations occur sequentially while the effective temperature of the matrix plume falls steadily due to cooling associated with expansion.

Formation of gas-phase peptide ions and their dissociation in MALDI: insights from kinetic and ion yield studies.

Insights on mechanisms for the generation of gas-phase peptide ions and their dissociation in matrix-assisted laser desorption ionization (MALDI) gained from the kinetic and ion yield studies are presented. Even though the time-resolved photodissociation technique was initially used to determine the dissociation kinetics of peptide ions and their effective temperature, it was replaced by a simpler method utilizing dissociation yields from in-source decay (ISD) and post-source decay (PSD). The ion yields for a matrix and a peptide were measured by repeatedly irradiating a region on a sample and collecting ion signals until the sample in the region was completely depleted. Matrix- and peptide-derived gas-phase cations were found to be generated by pre-formed ion emission or by ion-pair emission followed by anion loss, but not by laser-induced ionization. The total number of ions, that is, matrix plus peptide, was found to be equal to the number of ions emitted from a pure matrix. A matrix plume was found to cool as it expanded, from around 800-1,000 K to 400-500 K. Dissociation of peptide ions along b/y channels was found to occur statistically, that is, following RRKM behavior. Small critical energy (E0 = 0.6-0.7 eV) and highly negative critical entropy (ΔS(‡) = -30 to -25 eu) suggested that the transition structure was stabilized by multiple intramolecular interactions.

Spectral reproducibility and quantification of peptides in MALDI of samples prepared by micro-spotting.

Previously, we reported that MALDI spectra of peptides became reproducible when temperature was kept constant. Linear calibration curves derived from such spectral data could be used for quantification. Homogeneity of samples was one of the requirements. Among the three popular matrices used in peptide MALDI [i.e., α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and sinapinic acid (SA)], homogeneous samples could be prepared by conventional means only for CHCA. In this work, we showed that sample preparation by micro-spotting improved the homogeneity for all three cases.

Dual track time-of-flight mass spectrometry for peptide quantification with matrix-assisted laser desorption/ionization.

RATIONALE: Previously, we reported a method (Anal. Chem. 2012, 84, 10332) for peptide quantification based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). In the method, the peptide-to-matrix ion abundance ratio was utilized. Implementation of the method with a commercial MALDI-TOF can be somewhat inconvenient because matrix-derived ions are routinely deflected away to avoid detector saturation. A solution for this inconvenience is required. METHODS: We installed a detector to acquire the TOF spectrum of the ions thrown away to avoid detector saturation. By sending the matrix- and peptide-derived ions along two different tracks and detecting them with different detectors, the inconvenience mentioned above could be avoided. RESULTS: Excellent linearity of the calibration curves obtained by the dual track TOF spectrometry is demonstrated. The method also allows for the acquisition of the tandem mass spectrum of a selected peptide, which can be useful for its identification. CONCLUSIONS: We devised the dual track MALDI-TOF MS method to avoid detector saturation and demonstrated that the quantification and identification of peptides can be performed simultaneously.

Matrix suppression as a guideline for reliable quantification of peptides by matrix-assisted laser desorption ionization.

We propose to divide matrix suppression in matrix-assisted laser desorption ionization into two parts, normal and anomalous. In quantification of peptides, the normal effect can be accounted for by constructing the calibration curve in the form of peptide-to-matrix ion abundance ratio versus concentration. The anomalous effect forbids reliable quantification and is noticeable when matrix suppression is larger than 70%. With this 70% rule, matrix suppression becomes a guideline for reliable quantification, rather than a nuisance. A peptide in a complex mixture can be quantified even in the presence of large amounts of contaminants, as long as matrix suppression is below 70%. The theoretical basis for the quantification method using a peptide as an internal standard is presented together with its weaknesses. A systematic method to improve quantification of high concentration analytes has also been developed.

Why do the abundances of ions generated by MALDI look thermally determined?

In a previous study (J. Mass Spectrom. 48, 299-305, 2013), we observed that the abundance of each ion in a matrix-assisted laser desorption ionization (MALDI) spectrum looked thermally determined. To find out the explanation for the phenomenon, we estimated the ionization efficiency and the reaction quotient (QA) for the autoprotolysis of matrix, M + M → [M + H](+) + [M - H](-), from the temperature-controlled laser desorption ionization spectra of α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). We also evaluated the equilibrium constants (KA) for the autoprotolysis at various temperatures by quantum chemical calculation. Primary ion formation via various thermal models followed by autoprotolysis-recombination was compatible with the observations. The upper limit of the effective temperature of the plume where autoprotolysis-recombination occurs was estimated by equating QA with the calculated equilibrium constant. Figure ᅟ

Efficient methods to generate reproducible mass spectra in matrix-assisted laser desorption ionization of peptides.

In our previous matrix-assisted laser desorption ionization (MALDI) studies of peptides, we found that their mass spectra were virtually determined by the effective temperature in the early matrix plume, Tearly, when samples were rather homogeneous. This empirical rule allowed acquisition of quantitatively reproducible spectra. A difficulty in utilizing this rule was the complicated spectral treatment needed to get Tearly. In this work, we found another empirical rule that the total number of particles hitting the detector, or TIC, was a good measure of the spectral temperature and, hence, selection of spectra with the same TIC resulted in reproducible spectra. We also succeeded in obtaining reproducible spectra throughout a measurement by controlling TIC near a preset value through feedback adjustment of laser pulse energy. Both TIC selection and TIC control substantially reduced the shot-to-shot spectral variation in a spot, spot-to-spot variation in a sample, and even sample-to-sample variation in MALDI using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as matrix. Based on the utilization of acquired data, TIC control was more efficient than TIC selection by an order of magnitude. Both techniques produced calibration curves with excellent linearity, suggesting their utility in quantification of peptides.

Quantitative reproducibility of mass spectra in matrix-assisted laser desorption ionization and unraveling of the mechanism for gas-phase peptide ion formation.

In a previous study on matrix-assisted laser desorption ionization (MALDI) of peptides using α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix, we found that the patterns of single-shot spectra obtained under different experimental conditions became similar upon temperature selection. In this paper, we report that absolute ion abundances are also similar in temperature-selected MALDI spectra, even when laser fluence is varied. The result that has been obtained using CHCA and 2,5-dihydroxybenzoic acid as matrices is in disagreement with the hypothesis of laser-induced ionization of matrix as the mechanism for primary ion formation in MALDI. We also report that the total number of ions in such a spectrum is unaffected by the identity, concentration and number of analytes, i.e. it is the same as that in the spectrum of pure matrix. We propose that the generation of gas-phase ions in MALDI can be explained in terms of two thermal reactions, i.e. the autoprotolysis of matrix molecules and the matrix-to-analyte proton transfer, both of which are in quasi-equilibrium in the early matrix plume.

A simple method for quantification of peptides and proteins by matrix-assisted laser desorption ionization mass spectrometry.

Even though matrix-assisted laser desorption ionization (MALDI) is a powerful technique for mass spectrometry of peptides and proteins, it is not quite useful for their quantification that is one of the outstanding problems in quantitative proteomics. The main difficulty lies in the poor reproducibility of MALDI spectra. In this work, a simple method to circumvent this problem has been developed. The method is based on a previous observation that the reaction quotient for the matrix-to-peptide proton transfer evaluated in temperature-selected MALDI was nearly constant regardless of the peptide concentration in the solid sample. This implied a direct proportionality between the relative abundance of an analyte ion in a temperature-selected MALDI spectrum and the concentration of the corresponding neutral in the solid sample. This relation has been confirmed by calibration curves obtained for some peptides. Another characteristic of the relation is that it holds even when other analytes are present. This has been demonstrated for mixtures containing peptides and proteins. This and the fact that the method does not require the addition of internal standards allow rapid and inexpensive quantification of any analyte amenable to MALDI.

Reproducibility of temperature-selected mass spectra in matrix-assisted laser desorption ionization of peptides.

Matrix-assisted laser desorption ionization of peptides was investigated using α-cyano-4-hydroxycinnamic acid as the matrix. In each experiment, a set of mass spectra was collected by repetitive irradiation of a spot on a sample. Even though shot-to-shot variation in spectral pattern was significant, it was reproducible for different spots and samples. Each spectrum was tagged with the temperature in the early plume (T(early)) estimated through kinetic analysis of the peptide ion survival probability. T(early) decreased as the shot continued because the thermal conduction got more efficient as the sample got thinner. From each spectral set collected under various experimental conditions, a spectrum tagged with a particular T(early) was selected. Then, patterns of the spectra thus selected were the same. The reaction quotient for the matrix-to-peptide proton transfer determined at a specified T(early) was independent of the sample composition, indicating quasi-thermal equilibrium for this reaction. Furthermore, the van't Hoff plots were linear, also indicating quasi-thermal equilibrium. This, together with the thermal kinetics for the fragmentation of peptide and matrix ions, is responsible for the reproducibility of the mass spectral pattern at a specified T(early).

Degree of ionization in MALDI of peptides: thermal explanation for the gas-phase ion formation.

Degree of ionization (DI) in matrix-assisted laser desorption ionization (MALDI) was measured for five peptides using α-cyano-4-hydroxycinnanmic acid (CHCA) as the matrix. DIs were low 10(-4) for peptides and 10(-7) for CHCA. Total number of ions (i.e., peptide plus matrix) was the same regardless of peptides and their concentration, setting the number of gas-phase ions generated from a pure matrix as the upper limit to that of peptide ions. Positively charged cluster ions were too weak to support the ion formation via such ions. The total number of gas-phase ions generated by MALDI, and that from pure CHCA, was unaffected by the laser pulse energy, invalidating laser-induced ionization of matrix molecules as the mechanism for the primary ion formation. Instead, the excitation of matrix by laser is simply a way of supplying thermal energy to the sample. Accepting strong Coulomb attraction felt by cations in a solid sample, we propose three hypotheses for gas-phase peptide ion formation. In Hypothesis 1, they originate from the dielectrically screened peptide ions in the sample. In Hypothesis 2, the preformed peptide ions are released as part of neutral ion pairs, which generate gas-phase peptide ions via reaction with matrix-derived cations. In Hypothesis 3, neutral peptides released by ablation get protonated via reaction with matrix-derived cations.

The Jahn-Teller effect in CH3Cl+(X̃2E): a combined high-resolution experimental measurement and ab initio theoretical study.

The energy levels of CH(3)Cl(+)X̃(2)E showing strong spin-vibronic coupling effect (Jahn-Teller effect) have been measured up to 3500 cm(-1) above the ground vibrational state using one-photon zero-kinetic energy photoelectron and mass-analyzed threshold ionization spectroscopic method. Theoretical calculations have been also performed to calculate the spin-vibronic energy levels using a diabatic model and ab initio adiabatic potential energy surfaces (PESs). In the theoretical calculations the diabatic potential energy surfaces are expanded by the Taylor expansions up to the fourth-order including the multimode vibronic interactions. The calculated spin-orbit energy splitting (224.6 cm(-1)) for the ground vibrational state is in good agreement with the experimental data (219 ± 3 cm(-1)), which indicates that the Jahn-Teller and the spin-orbit coupling have been properly described in the theoretical model near the zero-point energy level. Based on the assignments predicted by the theoretical calculations, the experimentally measured energy levels were fitted to those from the diabatic model by optimizing the main spectroscopic parameters. The PESs from the ab initio calculations at the level of CASPT2/vq(t)z were thus compared with those calculated from the experimentally determined spectroscopic parameters. The theoretical diagonal elements in the diabatic potential matrix are in good agreement with those determined using the experimental data, however, the theoretical off-diagonal elements appreciably deviate from those determined using the experimental data for geometric points far away from the conical intersections. It is also concluded that the JT effect in CH(3)Cl(+) mainly arises from the linear coupling and the mode coupling between the CH(3) deform (υ(5)) and CH(3) rock (υ(6)) vibrations. The mode couplings between the symmetric C-Cl stretching vibration υ(3) with υ(5) and υ(6) are also important to understand the spin-vibronic structure of the molecule.

Ion yields for some salts in MALDI: mechanism for the gas-phase ion formation from preformed ions.

Preformed ion emission is the main assumption in one of the prevailing theories for peptide and protein ion formation in matrix-assisted laser desorption ionization (MALDI). Since salts are in preformed ion forms in the matrix-analyte mixture, they are ideal systems to study the characteristics of preformed ion emission. In this work, a reliable method to measure the ion yield (IY) in MALDI was developed and used for a solid salt benzyltriphenylphosphonium chloride and two room-temperature ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate and trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)phosphinate. IY for the matrix (α-cyano-4-hydroxycinnamic acid, CHCA) was also measured. Taking 1 pmol salts in 25 nmol CHCA as examples, IYs for three salts were similar, (4-8) × 10(-4), and those for CHCA were (0.8-1.2) × 10(-7). Even though IYs for the salts and CHCA remained virtually constant at low analyte concentration, they decreased as the salt concentrations increased. Two models, Model 1 and Model 2, were proposed to explain low IYs for the salts and the concentration dependences. Both models are based on the fact that the ion-pair formation equilibrium is highly shifted toward the neutral ion pair. In Model 1, the gas-phase analyte cations were proposed to originate from the same cations in the solid that were dielectrically screened from counter anions by matrix neutrals. In Model 2, preformed ions were assumed to be released from the solid sample in the form of neutral ion pairs and the anions in the ion pairs were assumed to be eliminated via reactions with matrix-derived cations.

Expansion cooling in the matrix plume is under-recognized in MALDI mass spectrometry.

Time-of-flight (TOF) mass spectra for a peptide (Y(6)) were obtained by utilizing matrix-assisted infrared laser desorption ionization (IR-MALDI) with glycerol as the matrix and by ultraviolet MALDI with α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB). Collisional activation during ion extraction and exothermicity in the gas-phase proton transfer were found to be unimportant as the driving forces for in-source (ISD) and post-source (PSD) decays, indicating that the thermal energy acquired during photo-ablation is responsible for their occurrence. The temperatures of [Y(6) + H](+) in the 'early' and 'late' matrix plumes were estimated by the kinetic analysis of the ISD and PSD yields, respectively. The order of the temperatures was glycerol < DHB ≈ SA < CHCA in the early plume and glycerol < DHB < SA < CHCA in the late plume. For each matrix, the temperature in the late plume was lower than in the early plume by 300-400 K, which was attributed to expansion cooling. The model (thermalization followed by expansion cooling) proposed to explain the occurrence of both rapid ISD and slow PSD is not only in sharp contrast with but also mutually exclusive with the prevailing explanation that the exothermicity in proton transfer and in-plume collisional activation are the driving forces for ion fragmentation in MALDI. The model also explains why MALDI is more successful for mass spectrometry of labile molecules than other desorption techniques that do not utilize a matrix. Factors affecting the plume temperature are also discussed.

Dissociation kinetics of singly protonated leucine enkephalin investigated by time-resolved photodissociation tandem mass spectrometry.

The yields of post-source decay (PSD) and time-resolved photodissociation (PD) at 193 and 266 nm were measured for singly protonated leucine enkephalin ([YGGFL + H](+)), a benchmark in the study of peptide ion dissociation, by using tandem time-of-flight mass spectrometry. The peptide ion was generated by matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as the matrix. The critical energy (E(0)) and entropy (DeltaS(++) at 1000 K) for the dissociation were determined by Rice-Ramsperger-Kassel-Marcus fit of the experimental data. MALDI was done for a mixture of YGGFL and Y(6) and the plume temperature determined by the kinetic analysis of [Y(6) + H](+) data were used to improve the precision of E(0) and DeltaS(++) for [YGGFL + H](+). E(0) and DeltaS(++) thus determined (E(0) = 0.67 +/- 0.08 eV, DeltaS(++) = -24.4 +/- 3.2 eu with 1 eu = 4.184 J K(-1)mol(-1)) were significantly different from those determined by blackbody infrared radiative dissociation (BIRD) (E(0) = 1.10 eV, DeltaS(++) = -14.9 eu), and by surface-induced dissociation (SID) (E(0) = 1.13 eV, DeltaS(double dagger) = -10.3 eu). Analysis of the present experimental data with the SID kinetics (and BIRD kinetics also) led to an unrealistic situation where not only PSD and PD but also MALDI-TOF signals could not be detected. As an explanation for the discrepancy, it was suggested that transition-state switching occurs from an energy bottleneck (SID/BIRD) to an entropy bottleneck (PSD/PD) as the internal energy increases.

Photodissociation spectroscopy of CD3I+ generated by mass-analyzed threshold ionization for structure determination.

A method is devised better to resolve the subbands of the ground vibronic band in the mass-analyzed threshold ionization (MATI) spectrum of CD(3)I. By selective photodissociation of CD(3)I(+) in these subbands, high-resolution spectra for the A(2)A(1)<--X(2)E(3/2) transition are recorded. Spectral analysis confirms our previous suggestion that these subbands are due to cations in different rotational K states; this demonstrates the capability of MATI to generate rovibronically selected ion beams. By using the rotational constants of CH(3)I(+) and CD(3)I(+) obtained by spectral analysis, the zero-point-level geometries of the cations in the X(2)E(3/2) and A(2)A(1) states are determined. To the best of our knowledge, this is the first time that the capability of MATI-PD to determine the geometry of a gas-phase polyatomic cation in an excited electronic state is demonstrated.

Rotational state selection of a CH3I+ ion beam using vacuum ultraviolet-mass-analyzed threshold ionization spectroscopy: characterization using photodissociation spectroscopy.

The A(2)A(1)<--X(2)E(3/2) transition of CH(3)I(+) was investigated by photodissociation (PD) of the cation generated by one-photon mass-analyzed threshold ionization (MATI). Compared to the PD spectrum obtained by excitation of the cation in the main 0-0 band in the MATI spectrum, those obtained by excitation of the cations in the satellite structures showed substantially simplified rotational structures for nondegenerate vibronic bands. Spectral simplification occurred because each satellite consisted mostly of cations with one K quantum number. Spectroscopic constants in the ground vibronic state and in the 2(1)3(5), 2(1)3(8), 3(9), and 3(13) nondegenerate vibrational states in A(2)A(1) were determined via spectral fitting. Also, those in the 2(1)3(n)6(1) (n=1?) degenerate state, which had been reported previously, was improved. The K quantum number in each satellite determined by the present high resolution study was compatible with the prediction by the symmetry selection rule for photoionization. That is, the K quantum number of the ion core in high Rydberg states accessed by one-photon excitation was found to be conserved upon pulsed field ionization. This work demonstrates generation of mass-selected, vibronically selected, and K-selected ion beam by one-photon MATI.

K selection in one-photon mass-analyzed threshold ionization of CH3I and CD3I to the X2E3/2 state cations.

One-photon mass-analyzed threshold ionization (MATI) spectra for the X (2)E(3/2) states of CH(3)I(+) and CD(3)I(+) were measured using vacuum ultraviolet radiation generated by four-wave mixing in Kr. Spin-orbit density functional theory calculations at the B3LYP/aug-cc-pVTZ level and spin-orbit/Jahn-Teller calculations were made to aid vibrational assignment. Each vibrational band consisted of several peaks due to different DeltaK transitions, which could be assigned by using molecular parameters determined in the previous high resolution photodissociation spectroscopic study. Possibility of generating mass-selected, vibronically selected and K-selected ion beam with decent intensity by one-photon MATI was demonstrated. The ionization energies to the X (2)E(3/2) states of CH(3)I(+) and CD(3)I(+) corrected for the rotational contribution were 9.5386+/-0.0006 and 9.5415+/-0.0006 eV, respectively.

One-photon mass-analyzed threshold ionization spectroscopy (MATI) of trans-dichloroethylene (trans-C2H2Cl2): cation structure determination via Franck-Condon fit.

One-photon mass-analyzed threshold ionization (MATI) spectrum of trans-C(2)H(2)Cl(2) was obtained by using vacuum ultraviolet radiation generated by four-wave mixing in Kr. The ionization energy determined from the position of the 0-0 band in the spectrum was 9.6306 +/- 0.0006 eV. Ten vibrational fundamentals for the cation were identified. The spectrum also displayed abundant overtones and combinations, most of which could be assigned adequately by comparing with the quantum chemical results. It was found that channel interaction was not important for this system. The equilibrium geometry of the cation was estimated through the Franck-Condon fit.

One-photon mass-analyzed threshold ionization spectroscopy of 2-chloropropene (2-C3H5Cl) and its vibrational assignment based on the density-functional theory calculations.

A high-quality mass-analyzed threshold ionization (MATI) spectrum of 2-chloropropene, 2-C3H5Cl, is reported. Its ionization energy determined for the first time from the 0-0 band position was 9.5395+/-0.0006 eV. Almost all the peaks in the MATI spectrum could be vibrationally assigned utilizing the frequencies calculated at the B3LYP6-311++G(3df,3pd) level and the Franck-Condon factors calculated with the molecular parameters obtained at the same level. In particular, the observed methyl torsional progression could be reproduced very well through quantum-mechanical calculations using the molecular parameters obtained at this level. Dramatic lowering of the torsional barrier inferred from the experimental data was entirely compatible with the B3LYP6-311++G(3df,3pd) results. The torsional barrier and the internal rotational constant determined by fits to six torsional peaks were 53.6 and 5.20 cm(-1), respectively. A brief discussion at the level of molecular orbital is presented to account for the dramatic lowering of the torsional barrier upon ionization.