Matrix suppression and analyte suppression effects of quaternary ammonium salts in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: an investigation of suppression mechanism

In the matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS) analysis of some quaternary ammonium salts (QASs), very clean spectra of the quaternary ammonium ions were recorded with a strong matrix suppression effect (MSE). The QASs also showed a considerable analyte suppression effect (ASE). It was demonstrated that the MSE and ASE of the QASs can be explained well by the cluster ionization model. According to this model, MALDI ions are formed from charged matrix/analyte clusters. Various analyte ions and matrix ions might coexist in the cluster, and they will compete for the limited number of net charges available. If enough quaternary ammonium ions are present in the cluster, they will take away the net charges, thus resulting in the MSE and ASE. Our results also suggest that ‘the cluster ionization model’ is not in conflict with ‘the theory of ionization via secondary gas‐phase reactions’. The initial MALDI ions produced from charged matrix/analyte clusters will collide with other molecules or ions in the MALDI plume. Depending on the properties of the initial ions and the composition of the MALDI plume, secondary gas‐phase reactions might result from these collisions. The final ions observed are the combined results of ‘cluster ionization’ and ‘ionization via secondary gas‐phase reactions’. Copyright © 2009 John Wiley & Sons, Ltd.     

Compelling Evidence for Lucky Survivor and Gas Phase Protonation: The Unified MALDI Analyte Protonation Mechanism

This work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model. Experimental differentiation between the predicted mechanisms becomes possible by the use of deuterated matrix esters as MALDI matrices, which are stable under typical sample preparation conditions and generate deuteronated reagent ions, including the deuterated and deuteronated free matrix acid, only upon laser irradiation in the MALDI process. While the generation of deuteronated analyte ions proves the gas phase protonation model, the detection of protonated analytes by application of deuterated matrix compounds without acidic hydrogens proves the survival of analytes precharged from solution in accordance with the predictions from the Lucky Survivor model. The observed ratio of the two analyte ionization processes depends on the applied experimental parameters as well as the nature of analyte and matrix. Increasing laser fluences and lower matrix proton affinities favor gas phase protonation, whereas more quantitative analyte protonation in solution and intramolecular ion stabilization leads to more Lucky Survivors. The presented results allow for a deeper understanding of the fundamental processes causing analyte ionization in MALDI and may alleviate future efforts for increasing the analyte ion yield.     

Gas-phase oxidation and reduction of some 1-(2-benzothiazolyl)-3,5-diphenyl formazans. Complex formation with transition metals under laser desorption ionization

Five differently substituted 1-(2-benzothiazolyl)-3,5-diphenyl formazans were studied by laser desorption ionization (LDI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry. The best explanation of the results is that the formazan molecules are photoionized to molecular radical ions, which then further react by ion-molecule reactions. Supporting this proposal was the abundant formation of [M − H]⁺ ions under LDI. These ions are not observed at all under either electron or chemical ionization. Under MALDI, the extent of the oxidation process is clearly dependent on the ability of the matrix to act as a reducing agent. With transition metals the formazans formed singly charged 1:2 metal:formazan complexes. The most stable electronic configuration of the complex determined the oxidation state of the metal regardless of its initial oxidation state. In some cases, this process also demanded a gas-phase reduction of the formazan. The ionization efficiency and affinity for complex formation depended on the substituent at the 3-phenyl group; both were increased by an electron donating substituent. The formazans were also tested as potential matrices for MALDI. Reasonable results were observed for several groups of compounds; however, only the piperazine ligands produced higher quality spectra with formazans than with common commercial matrices.     

Matrix-assisted laser desorption/ionization (MALDI) mechanism revisited

Although matrix-assisted laser desorption/ionization (MALDI) was developed more than a decade ago and broad applications have been successfully demonstrated, detailed mechanism of MALDI is still not well understood. Two major models; namely photochemical ionization (PI) and cluster ionization (CI) mechanisms have been proposed to explain many of experimental results. With the photochemical ionization model, analyte ions are considered to be produced from a protonation or deprotonation process involving an analyte molecule colliding with a matrix ion in the gas phase. With the cluster ionization model, charged particles are desorbed with a strong photoabsorption by matrix molecules. Analyte ions are subsequently produced by desolvation of matrix from cluster ions. Nevertheless, many observations still cannot be explained by these two models. In this work, we consider a pseudo proton transfer process during crystallization as a primary mechanism for producing analyte ions in MALDI. We propose an energy transfer induced disproportionation (ETID) model to explain the observation of an equal amount of positive and negative ions produced in MALDI for large biomolecules. Some experimental results are used for comparisons of various models.     

Unusual Analyte-Matrix Adduct Ions and Mechanism of Their Formation in MALDI TOF MS of Benzene-1,3,5-Tricarboxamide and Urea Compounds

Analyte-matrix adducts are normally absent under typical matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) conditions. Interestingly, though, in the analysis of several types of organic compounds synthesized in our laboratory, analyte-matrix adduct ion peaks were always recorded when common MALDI matrices such as 4-hydroxy-α-cyanocinnamic acid (CHCA) were used. These compounds are mainly those with a benzene-1,3,5-tricarboxamide (BTA) or urea moiety, which are important building blocks to make new functional supramolecular materials. The possible mechanism of the adduct formation was investigated. A shared feature of the compounds studied is that they can form intermolecular hydrogen bonding with matrices like CHCA. The intermolecular hydrogen bonding will make the association between analyte ions and matrix molecules stronger. As a result, the analyte ions and matrix molecules in MALDI clusters will become more difficult to be separated from each other. Furthermore, it was found that analyte ions were mainly adducted with matrix salts, which is probably due to the much lower volatility of the salts compared with that of their corresponding matrix acids. It seems that the analyte-matrix adduct formation for our compounds are caused by the incomplete evaporation of matrix molecules from the MALDI clusters because of the combined effects of enhanced intermolecular interaction between analyte-matrix and of the low volatility of matrix salts. Based on these findings, strategies to suppress the analyte-matrix adduction are briefly discussed. In return, the positive results of using these strategies support the proposed mechanism of the analyte-matrix adduct formation.

Gas-phase ionization/desolvation processes and their effect on protein charge state distribution under matrix-assisted laser desorption/ionization conditions

The charge state distribution of proteins was studied as a function of experimental conditions, to improve the understanding of the matrix-assisted laser desorption/ionization (MALDI) mechanisms. The relative abundances of the multiply-charged ions appear to be a function of the matrix chosen, the laser fluence and the matrix-to-analyte molar ratio. A correlation is found between the matrix proton affinity and the yield of singly- versus multiply-charged ions. These results are in good agreement with a model in which gas-phase intracluster reactions play a significant role in analyte ion formation. A new model for endothermic desolvation processes in ultraviolet/MALDI is presented and discussed. It is based upon the existence of highly-charged precursor clusters and, complementary to the ion survivor model of Karas et al., assumes that two energy-dependent processes exist: (i) a soft desolvation involving consecutive losses of neutral matrix molecules, leading to a multiply-charged analyte and (ii) hard desolvation leading to a low charge state analyte, by consecutive losses of charged matrix molecules. These desolvations pathways are discussed in terms of kinetically limited processes. The efficiency of the two competitive desolvation processes seems related to the internal energy carried away by clusters during ablation.     

In-source H/D exchange and ion-molecule reactions using matrix assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry with pulsed collision and reaction gases

Controlled in-source ion-molecule reactions are performed for the first time in an external matrix assisted laser desorption ionization (MALDI) source of a Fourier transform ion cyclotron resonance mass spectrometer. The MALDI source with a hexapole ion guide that was originally designed to incorporate pulsed gas to collisionally cool ions (Baykut, G.; Jertz, R.; Witt, M. Rapid Commun. Mass Spectrom. 2000, 14, 1238-1247) has been modified to allow the study of in-source ion-molecule reactions. Upon laser desorption, a reaction gas was introduced through a second inlet and allowed to interact with the MALDI-generated ions trapped in the hexapole ion guide. Performing ion-molecule reactions in the high pressure range of the ion source prior to analysis in the ion cyclotron resonance (ICR) cell allows to maintain the ultra high vacuum in the cell which is crucial for high mass resolution measurements. In addition, due to the reaction gas pressure in the hexapole product ion formation is much faster than would be otherwise possible in the ICR cell. H/D exchange reactions with different peptides are investigated, as are proton-bound complex formations. A typical experimental sequence would be ion accumulation in the hexapole ion guide from multiple laser shots, addition of cooling gas during ion formation, addition of reaction gas, varied time delays for the ion-molecule reactions, and transmission of the product ions into the ICR cell for mass analysis. In this MALDI source H/D exchange reactions for different protonated peptides are investigated, as well as proton-bound complex formations with the reaction gas triethylamine. Amino acid sequence, structural flexibility and folding state of the peptides can be seen to play a part in the reactivity of such ions.     

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. Copyright © 2013 John Wiley & Sons, Ltd.     

Laserspray ionization (LSI) ion mobility spectrometry (IMS) mass spectrometry

A simple device is described for desolvation of highly charged matrix/analyte clusters produced by laser ablation leading to multiply charged ions that are analyzed by ion mobility spectrometry-mass spectrometry. Thus, for example, highly charged ions of ubiquitin and lysozyme are cleanly separated in the gas phase according to size and mass (shape and molecular weight) as well as charge using Tri-Wave ion mobility technology coupled to mass spectrometry. This contribution confirms the mechanistic argument that desolvation is necessary to produce multiply charged matrix-assisted laser desorption/ionization (MALDI) ions and points to how these ions can be routinely formed on any atmospheric pressure mass spectrometer.     

Online Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry with In situ Mixing

Online matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is reported with in situ matrix/analyte aerosol mixing. The analyte and matrix were dissolved in separate solvents and pneumatically atomized into particles. Continuous flows of analyte particles and matrix droplets in nitrogen were mixed in a tee. The resulting particles were sampled using a nozzle, focused into a beam with an aerodynamic lens system, and vertically deposited continuously on a movable target. The matrix/analyte mixture was desorbed/ionized using a 266 nanometers pulsed laser at an incident angle of 45°. The nascent ions were analyzed using reflectron TOF MS. The performance of online MALDI-TOF MS was evaluated by the analysis of palmityl palmitate with the lithium salt of 2, 4-dihydroxybenzoic acid as the matrix. Strong and stable MALDI signals of palmityl palmitate were obtained. The matrix solvent mixture and the analyte concentration were optimized and the results demonstrate the development of an alternative for online MALDI analysis.     

Using fluorescence dyes as a tool for analyzing the MALDI process

In a recent paper (Setz, P. D.; Knochenmuss, R. Phys. Chem. A2005, 109, 4030-4037) energy-transfer from excited matrix molecules to fluorescent traps was used to study the role of pooling reactions for the ionization processes in matrix-assisted laser desorption ionization (MALDI) using 2,5-dihydroxybenzoic acid as matrix. Exciton trapping was shown to interfere with matrix ionization. These investigations were extended to analyze the influence of fluorescent traps on both matrix and analyte ions for alpha-cyano-4-hydroxycinnamic acid and further matrices. A strong influence of the fluorescent traps on both matrix and analyte ionization was revealed, depending on the matrix:trap ratio, and manifested itself differently for low and high mass analytes. The observations are rationalized by the intermediate formation of a "hot spot" due to an efficient conversion of electronic excitation energy to vibronic energy within the fluorescent traps. This process favors the desorption and ionization of small vaporizable analytes and compromises the cluster ablation process needed for larger analyte ions.     

Production and fragmentation of multiply charged ions in 'electron-free' matrix-assisted laser desorption/ionization

An unusually large fraction of multiply charged ions is observed in 'electron-free' matrix-assisted laser desorption/ionization (MALDI). Here we investigate how the yield of multiply charged ions depends on experimental parameters in MALDI. It is found to increase if measures are taken to limit the number of electrons in the plume, for example, by using non-metallic MALDI targets or low laser pulse energies. The ionization energy of the matrix is another important parameter that affects the yield of multiply charged ions: matrices with high ionization energies lead to greater intensities of multiply charged ions. It is furthermore proposed that some of the fragment ions observed in MALDI are due to reactions of analyte with electrons in the plume. The possibility of electron capture dissociation of multiply charged ions produced by MALDI is shown.     

Oxidation of ferrocene derivatives in desorption/lonization on porous silicon.

In matrix-assisted laser desorption/ionization (MALDI), the true molecular structures of some analytes are not represented by the observed ions due to a redox reaction. In earlier reports, electron transfer from analyte to chemical matrix has been proposed for the oxidation of ferrocene derivatives in MALDI. To address such a redox phenomenon in laser desorption/ionization processes, two ferrocene derivatives, FcCH2CH2Fc and FcCH2NMe2 [Fc:(CsHs)Fe(CsH4)], were analyzed by a matrix-free method, desorption/ionization on porous silicon (DIOS). The oxidized species, Fc+CH2NMe2 and FcCH2CH2Fc+, were detected in the DIOS mass spectra. The results suggested that electron transfer from the analytes to the sample target occurs during the ionization process.     

Titanium dioxide anatase as matrix for matrix-assisted laser desorption/ionization analysis of small molecules

The use of inorganic species as assisting materials in matrix-assisted laser desorption/ionization (MALDI) analysis is an alternative approach to avoid interfering matrix ions in the low-mass region of the mass spectra. Reports of the application of inorganic species as matrices in MALDI analysis of small molecules are, however, scarce. Nevertheless, titanium dioxide (TiO(2)) powder has been reported to be a promising matrix medium. In this study we further explore the use of TiO(2) as a matrix for the MALDI analysis of low molecular weight compounds. We present results showing that nanosized TiO(2) anatase and TiO(2) rutile perform better as MALDI matrices than a commercial TiO(2) anatase/rutile mixture. Moreover, when using nanosized TiO(2) anatase as a matrix, high-quality mass spectra can be obtained with strong analyte signals and weak or non-existing matrix interference ions. Furthermore, our results show that the phase type plays an important role in the application of TiO(2) as a MALDI matrix.     

9,10‐Diphenylanthracene as a matrix for MALDI‐MS electron transfer secondary reactions

The most common secondary‐ionization mechanism in positive ion matrix‐assisted laser desorption/ionization (MALDI) involves a proton transfer reaction to ionize the analyte. Peptides and proteins are molecules that have basic (and acidic) sites that make them susceptible to proton transfer. However, non‐polar, aprotic compounds that lack basic sites are more difficult to protonate, and creating charged forms of this type of analyte can pose a problem when conventional MALDI matrices are employed. In this case, forming a radical molecular ion through electron transfer is a viable alternative, and certain matrices may facilitate the process. In this work, we investigate the performance of a newly developed electron‐transfer secondary reaction matrix: 9,10‐diphenylanthracene (9,10‐DPA). The use of 9,10‐DPA as matrix for MALDI analysis has been tested using several model compounds. It appears to promote ionization through electron transfer in a highly efficient manner as compared to other potential matrices. Thermodynamic aspects of the observed electron transfers in secondary‐ionization reactions were also considered, as was the possibility for kinetically controlled/endothermic, electron‐transfer reactions in the MALDI plume. Copyright © 2012 John Wiley & Sons, Ltd.     

Small molecule matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a polymer matrix

We have employed a light-absorbing electrically conductive polymer as a matrix to determine the molecular mass of small organic molecules using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method, which is in contrast to the usual MALDI strategy for matrix selection in which a small molecule matrix is used with a high molecular mass analyte, addresses the problem of matrix interference which limits the usefulness of MALDI-TOF for small molecule analysis. Use of negative ion mode offers advantages for this application. Using this approach, we have obtained clean molecular ion mass spectra of small organic molecules in the mass range 100-300 Da.     

A pitfall of using 2‐[(2E)‐3‐(4‐tert‐butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile as a matrix in MALDI TOF MS: chemical adduction of matrix to analyte amino groups

2‐[(2E)‐3‐(4‐tert‐Butylphenyl)‐2‐methylprop‐2‐enylidene]malononitrile (DCTB) has been considered as an excellent matrix for matrix‐assisted laser desorption/ionization (MALDI) of many types of synthetic compounds. However, it might provide troublesome results for compounds containing aliphatic primary or secondary amino groups. For these compounds, strong extra ion peaks with a mass difference of 184.1 Da were usually observed, which might falsely indicate the presence of some unknown impurities that were not detected by other matrices. On the basis of the possible mechanisms proposed, these extra ions are the products of nucleophilic reactions between analyte amino groups and DCTB molecules or radical cations. In these reactions, an amino group replaces the dicyanomethylene group of DCTB forming a matrix adduct via a ? C?N‐bond. An aliphatic primary amine could react easily with DCTB and the reaction could start once they are mixed in a MALDI solution. For an aliphatic secondary amine, on the other hand, the reaction most likely occurs in the gas phase. Protonation of amino groups by adding acid seems to be a useful way to stop DCTB adduction for compounds with one single amino group, but not for compounds with multiple amino groups. Unlike aliphatic primary or secondary amines, aliphatic tertiary amines and aromatic amines do not yield DCTB adducts. This is because tertiary amines do not have the required transferrable H‐(N) atom to form an extra ? C?N‐bond, while aromatic amines are not sufficiently nucleophilic to attack DCTB. In view of the possible matrix adduction, care should be taken in MALDI time‐of‐flight mass spectrometry (TOF MS) when DCTB is used as the matrix for compounds containing amino group(s). Copyright © 2010 John Wiley & Sons, Ltd.     

A Mechanism for Ionization of Nonvolatile Compounds in Mass Spectrometry: Considerations from MALDI and Inlet Ionization

Mechanistic arguments relative to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) address observations that predominately singly charged ions are detected. However, recently a matrix assisted laser ablation method, laserspray ionization (LSI), was introduced that can use the same sample preparation and laser as MALDI, but produce highly charged ions from proteins. In MALDI, ions are generated from neutral molecules by the photon energy provided to a matrix, while in LSI ions are produced inside a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass spectrometer. Some LSI matrices also produce highly charged ions with MALDI ion sources operated at intermediate pressure or high vacuum. The operational similarity of LSI to MALDI, and the large difference in charge states observed by these methods, provides information of fundamental importance to proposed ionization mechanisms for LSI and MALDI. Here, we present data suggesting that the prompt and delayed ionization reported for vacuum MALDI are both fast processes relative to producing highly charged ions by LSI. The energy supplied to produce these charged clusters/droplets as well as their size and time available for desolvation are determining factors in the charge states of the ions observed. Further, charged droplets/clusters may be a common link for ionization of nonvolatile compounds by a variety of MS ionization methods, including MALDI and LSI.     

MALDI Mass Spectrometry Imaging of Neuronal Cell Cultures

Mass spectrometry imaging (MSI) provides the ability to detect and identify a broad range of analytes and their spatial distributions from a variety of sample types, including tissue sections. Here we describe an approach for probing neuropeptides from sparse cell cultures using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MSI—at single cell spatial resolution—in both MS and tandem MS modes. Cultures of Aplysia californica neurons are grown on an array of glass beads embedded in a stretchable layer of Parafilm M. As the membrane is stretched, the beads/neurons are separated physically and the separated beads/neurons analyzed via MALDI TOF MS. Compared with direct MS imaging of samples, the stretching procedure enhances analyte extraction and incorporation into the MALDI matrix, with negligible analyte spread between separated beads. MALDI tandem MSI using the stretched imaging approach yields localization maps of both parent and fragment ions from Aplysia pedal peptide, thereby confirming peptide identification. This methodology represents a flexible platform for MSI investigation of a variety of cell cultures, including functioning neuronal networks.     

If the ionization mechanism in fast-atom bombardment involves ion/molecule reactions, what are the reagent ions? The time dependence of fast-atom bombardment mass spectra and parallels to chemical ionization

The evaporation in vacuo of the matrices used and the particle-induced desorption of matrix molecules in fast-atom bombardment (FAB) contribute to a proposed high pressure region above the FAB matrix known as the selvedge region. If the neutral number density is sufficiently high, ions formed upon bombardment may undergo collisions with molecules, yielding matrix-related cluster ions and, in cases when the analyte is desorbed in neutral form, protonated and deprotonated analyte molecules. Similarities with the chemical ionization (CI experiment have been pointed out previously and are further developed here. If FAB is similar to CI, then the response depends on the structures of the reagent ions — those ions that react with gas phase analyte molecules. We consider here the time dependence of positive and negative ion FAB spectra to attempt to identify the reagent ions of FAB. A model is suggested for the FAB ion source which evaluates similarities to a CI source, as well as spatial aspects that are unique to desorption/ionization techniques.