Investigation of metal attachment to polystyrenes in matrix-assisted laser desorption ionization

Cationization is essential to the matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of a variety of synthetic polymers. This work studied polystyrene cationization in MALDI using the salts of eight different metals. It was found that only the salts of silver, copper, and palladium produced good metal—polystyrene cation signals. More interestingly, it was observed that MALDI could also produce metal-rich cluster cations and that the presence of polystyrenes tended to suppress formation of the metal-rich cluster cations. Based on these results and others, we propose that polystyrene cationization may proceed through gas-phase metal attachment reactions under the conditions used. With this argument, we were able to better explain a reported experimental observation that showed a strong cation concentration effect on measured molecular weight distributions of polystyrenes.     

A study of gas-phase cationization in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A specially constructed split sample probe was used to unequivocally demonstrate that gas-phase cationization occurs within the desorption plume during a matrix-assisted laser desorption/ionization experiment. Two separate samples were prepared for analysis: on side A, a mixture of poly(ethylene glycol) (PEG) 1500 analyte and 2,5-dihydroxybenzoic acid (DHB) matrix, and on side B a mixture of DHB matrix and lithium hydroxide (LiOH), the cationization reagent. Analysis of the data showed that when the ionization laser was focused on the split (so that both sides were illuminated), Li(+)-cationized PEG peaks were observed. Since the PEG analyte did not come into contact with Li(+) in either the solution or solid phase, the only possibility for the observed cationization was a reaction in the gas phase. Due to the difficulty in completely removing the adventitious cations (Na(+) and K(+)) present in DHB and on sample surfaces, gas-phase cationization could not be demonstrated to be either the only or most important mechanism operating in the MALDI experiment.     

Characterization of humic substances by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and laser desorption/ionization (LDI-)TOFMS have been used to characterize Suwannee River humic substances, obtained from the International Humic Substances Society (IHSS), and Armadale soil fulvic acid (ASFA). An array of MALDI matrices were tested for use with humic substances, including alpha-cyano-4-hydroxycinammic acid (CHCA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), 2,5-dihydroxybenzoic acid (DHBA), sinapinic acid, dithranol and norharmane. DHBA yielded the best results, exhibiting superior ionization efficiency, low noise, broad applicability to the analytes of interest, and most importantly producing an abundance of high mass ions, the highest observed being m/z 1848. A number of sample preparation modes were investigated; the overlayer method improved sample/matrix homogeneity and hence shot-to-shot reproducibility. The choice of the matrix, mass ratio of analyte to matrix, and the sample preparation protocol, were found to be the most critical factors governing the quality of the mass spectra. Matrix suppression was greatly enhanced by ensuring good mixing of matrix and analyte in the solid phase, proper optimization of the matrix/analyte ratio, and optimizing delayed extraction to ensure complete matrix-analyte reaction in the plume before ions are moved to the flight tube. A number of common features, in particular specific ions which could not be attributed to the matrices or to contaminants, were present in the spectra of all the humic substances, regardless of origin or operational definition. Additionally, a prominent repeating pattern of peaks separated by 55, 114 and 169 Da was clearly observed in both LDI and MALDI, suggesting that the humic compounds studied here may have quasi-polymeric or oligomeric features.     

Investigation of the mechanism of intracluster proton transfer from sinapinic acid to biomolecular analytes

Experiments have been performed to elucidate the mechanism of proton transfer in ternary clusters containing the matrix-assisted laser-desorption ionization (MALDI) matrix sinapinic acid, nonchromophoric analytes (proline, methionine, and prolylmethionine), and argon. To investigate the mechanism of intracluster proton transfer, ionizing laser power studies were performed at 266 and 355 nm. Baseline studies show that two photons are required at both wavelengths for the formation of sinapinic acid radical cations from sinapinic acid/argon clusters. Studies of the ternary sinapinic acid/biomolecule/argon clusters show that, in all cases, the photon dependence for protonation of the biomolecule is the same as that for formation of the sinapinic acid radical cation. Furthermore, the slopes of the power plots are generally between 1.5 and 2.0, consistent with a two photon ionization process. No evidence of negative ion formation is detected in the negative ion mass spectra. The combined results are consistent with a mechanism of biomolecular intracluster protonation via proton transfer from the photoionized sinapinic acid radical cation. Wavelength dependent trends in matrix and analyte fragment ion formation in conventional MALDI mass spectra and the cluster proton transfer mass spectra were noted. The possible contribution of cluster proton transfer to the analyte protonation mechanism in conventional MALDI is discussed.     

The cationization of synthetic polymers in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Investigations of the salt‐to‐analyte ratio

Matrix‐assisted laser desorption/ionization (MALDI) is a soft ionization technique that when used to analyze synthetic polymer analytes often requires the addition of a metal cationization agent (herein termed the “salt”). The choice of both the matrix and the cationization agent needs to be taken into account when considering the polymer under study; different polymers have shown different affinities toward different cationization agents, and their selectivity can change as the matrix changes. Salt‐to‐analyte ratio (S/A) plots are used in this work to investigate the effect of the quantity of cationization agent employed in the analysis of a poly (methylmethacrylate) (PMMA) analyte with different MALDI matrices. The point at which analyte signal stops increasing with the added cationization agent is termed the “cation saturation point,” and it was found to occur around a S/A of 1. When the analyte signal after this point remains constant, it is termed an “ideal case.” The “non‐ideal case” occurs when the analyte signal decreases after the cation saturation point. The amount of matrix present (measured as the matrix‐to‐analyte molar ratio, M/A) and the use of different counterions for the salt are also found to affect the intensity of the analyte signal. In non‐ideal cases, changes in the counterion or an increase in the M/A are found to increase the analyte signal, often converting an initially observed non‐ideal case into an ideal case. Several experiments attempting to uncover the reason for observation of the non‐ideal S/A behavior are also described.     

Three-layer matrix/sample preparation method for MALDI MS analysis of low nanomolar protein samples

A robust and sensitive sample preparation method is presented for matrix-assisted laser desorption ionization (MALDI) mass spectrometric analysis of low nanomolar concentrations of proteins containing high amounts of common salts and buffers. This method involves the production of densely packed sub-micrometer matrix crystals by depositing a matrix solution on top of a matrix seed-layer prepared on a MALDI target. A sub-microliter aliquot of analyte solution is then directly added to the top of the matrix crystals to form a thin-layer. alpha-Cyano-4-hydroxycinnamic acid (4-HCCA) is used as matrix and demonstrated to give better performance than other commonly used matrices, such as 2,5-dihydroxybenzoic acid (DHB), 2-(4-hydroxy-phenylazo) benzoic acid (HABA), or sinapinic acid. This three-layer method is shown to be superior to the other MALDI sample preparation methods, particularly for handling low nanomolar protein solutions containing salts and buffers.     

Gas-phase basicities of the isomeric dihydroxybenzoic acids and gas-phase acidities of their radical cations

The thermochemical acid/base properties of the six dihydroxybenzoic acids (x,y-DHB) as prototypical matrices used in matrix-assisted laser desorption/ionization (MALDI) have been investigated. The ground-state gas-phase basicities (GB) of the six DHB isomers and the gas-phase acidities (deltaG acid) of the corresponding radical cations ([x,y-DHB]*+) have been determined by Fourier-transform ion cyclotron resonance mass spectrometry employing the thermokinetic method. The gas-phase basicities vary from 814 kJ mol-1 for the least basic isomer, 3,5-DHB, to 831 kJ mol-1 for the most basic isomer, 2,4-DHB. The obtained gas-phase acidities of the corresponding radical cations vary from 815 kJ mol-1 for the most acidic species, 3,4-DHB, to 858 kJ mol-1 for the least acidic one, 2,5-DHB. The results indicate that ground-state proton transfer from the matrix radical cations to the analyte may play a role in the ionization process of MALDI, whereas proton transfer from protonated matrix molecules can be excluded.     

Internal energies of analyte ions generated from different matrix-assisted laser desorption/ionization matrices

A transfer of energy into the internal modes of the matrix and analyte is expected to occur during matrix-assisted laser desorption/ioniziation (MALDI) processes. Both the physical and thermochemical properties of the MALDI matrix used can influence the ion internal energy and analyte ion fragmentation. Here we report the effect of several MALDI matrices on the relative internal energy of the 2'-deoxyadenylyl-(3',5')-2'-deoxyguanosine (AG) anion. Relative internal energies were probed by low-energy collision-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer. Sublimation temperatures of the matrices under study were also determined and found to lie between 409 and 455 K. Analyte ion internal and initial kinetic energies did not correlate with matrix sublimation temperatures. In contrast, a strong correlation between the relative internal energy of the analyte anions and the gas-phase basicity of the matrix anions was found. These results suggest that gas-phase proton transfer reactions play an important role in MALDI analyte ion formation and influence their internal energy and fragmentation behavior. Copyright 2000 John Wiley & Sons, Ltd.     

Solvent-free MALDI investigation of the cationization of linear polyethers with alkali metals

The MALDI technique with solvent-free sample preparation has been applied to evaluate relative gas-phase affinities of polyether chain polymers with alkali metal cations. The study is performed on poly(ethylene glycol) and poly(propylene glycol) polymers of different lengths (PEG600, PEG1000, PPG425, PPG750) and the alkali metal cations Li(+), Na(+), K(+), and Cs(+). The experiments show that the lattice energy of the alkali metal salts employed as cation precursors can have a strong influence on the outcome of conventional MALDI measurements. With the solvent-free method, these crystal binding effects can be made negligible by combining in the same sample alkali metal salts with different counterions. The recorded MALDI spectra show that the polyether-cation aggregation efficiencies decrease systematically with growing cation size. This cation size selectivity is considerably enhanced for the polymers with the shorter chains, which can be attributed to the reduced ability of the polymer to build a coordination shell around the larger cations. The steric effects introduced by the side CH3 group of propylene glycol with respect to ethylene glycol also enhance the preference for cationization of the polymer by the smaller cations. These observations correct some qualitative trends derived from previous studies, which did not account for lattice energy effects of the cation precursors.     

5-Ethyl-2-mercaptothiazole as matrix for matrix-assisted laser desorption/ionization of a broad spectrum of analytes in positive and negative ion mode

A preliminary investigation of the use of 5-ethyl-2-mercaptothiazole as matrix in matrix-assisted laser desorption/ionization (MALDI) of a broad spectrum of analytes is reported. The analytes studied are substance P, insulin, beta-cyclodextrin, triacylglycerols of coconut oil and polypropylene glycol 2000 (PPG 2000). In the positive ion mass spectra of the matrix/analyte combinations, the formation of [M + H]+ and [M + cation]+ species were observed and compared with those obtained by using well-established matrices such as alpha-cyano-4-hydroxycinnamic acid, genticic acid, sinapinic acid and dithranol. In addition, the usefulness of this new matrix for MALDI in negative ion mode is also described using substance P and beta-cyclodextrin as examples.     

Direct surface analysis of fungal species by matrix-assisted laser desorption/ionization mass spectrometry

In this study various methods of sample preparation and matrices were investigated to determine optimum collection and analysis criteria for fungal analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Intact spores and/or hyphae of Aspergillus niger, Rhizopus oryzae, Trichoderma reesei and Phanerochaete chrysosporium were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The fungal samples were applied to the MALDI sample target as untreated, sonicated, or acid/heat treated samples, or blotted directly from the fungal culture with double-stick tape. Ferulic acid or sinapinic acid matrix solution was layered over the dried samples and analyzed by MALDI-MS. Statistical analysis showed that simply using double-stick tape to collect and transfer to a MALDI sample plate typically worked as well as the other preparation methods, and required the least sample handling.     

Characterization of mesogen-jacketed liquid crystalline polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

For synthetic polymers, a proper sample preparation method is essential for successful characterization by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this work, six synthetic mesogen-jacketed liquid crystalline polymers (MJLCPs) with different main-chain, spacer and mesogenic units were investigated by MALDI-TOF mass spectrometry. Several factors that affect the analysis of these polymers were examined. These factors include matrices used, cationization salts used, the concentration of polymers, and the ratio of sample to matrix. After testing different conditions, we found a suitable sample preparation method for these six polymers. The number average molecular weight (M(n)), weight average molecular weight (M(w)) and polydispersity (PD) were calculated using data obtained in the linear mode. The end groups of the polymers were proposed using data obtained in reflectron mode. Copyright 2000 John Wiley & Sons, Ltd.     

Independent assessment of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sample preparation quality: A novel statistical approach for quality scoring

Preparation of samples according to an optimized method is crucial for accurate determination of polymer sample characteristics by Matrix-Assisted Laser Desorption Ionization (MALDI) analysis. Sample preparation conditions such as matrix choice, cationization agent, deposition technique or even the deposition volume should be chosen to suit the sample of interest. Many sample preparation protocols have been developed and employed, yet finding the optimal sample preparation protocol remains a challenge. Because an objective comparison between the results of diverse protocols is not possible, “gut-feeling” or “good enough” is often decisive in the search for an optimum. This implies that sub-optimal protocols are used, leading to a loss of mass spectral information quality. To address this problem a novel analytical strategy based on MALDI imaging and statistical data processing was developed in which eight parameters were formulated to objectively quantify the quality of sample deposition and optimal MALDI matrix composition and finally sum up to an overall quality score of the sample deposition. These parameters can be established in a fully automated way using commercially available mass spectrometry imaging instruments without any hardware adjustments. With the newly developed analytical strategy the highest quality MALDI spots were selected, resulting in more reproducible and more valuable spectra for PEG in a variety of matrices. Moreover, our method enables an objective comparison of sample preparation protocols for any analyte and opens up new fields of investigation by presenting MALDI performance data in a clear and concise way.     

Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices

A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vitamins was investigated. The ionic liquid matrices are composed of equimolar combinations of classical MALDI matrices (sinapinic acid, alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices.     

Mercaptobenzothiazoles: A new class of matrices for laser desorption ionization mass spectrometry

We report 2-mercaptobenzothiazole (MBT) and its analogs as a class of new matrices for matrix-assisted laser desorption mass spectrometry (MALDI-MS) at 337 nm. MBT has been used successfully for the desorption of proteins up to 100,000 u. A comparison with sinapinic acid and α-cyano-4-hydroxycinnamic acid indicates that MBT provides the same level of sensitivity and resolution, but offers the advantage of higher tolerance to sample contaminants such as ionic detergents. 5-Chloro-2-mercaptobenzothiazole (CMBT), an analog of MBT, has been found not only effective for the analysis of peptides, low-mass proteins, and glycolipids, but also superior to conventional matrices for the analysis of muropeptides and at least some oligosaccharides. CMBT also exhibits excellent experimental reproducibility of MALDI-MS results owing to the homogeneous crystallization of the analyte/matrix mixture over the entire sample surface area. Finally, all five mercaptobenzothiazoles studied in this work are shown to be well suited for synthetic polymer analysis.     

Solid state nuclear magnetic resonance as a tool to explore solvent-free MALDI samples

Solid-state Nuclear magnetic resonance (NMR) was used here to explore structural characteristics of samples to be subjected to matrix-assisted laser desorption/ionization (MALDI) and prepared without the use of any solvent. The analytical systems scrutinized in NMR were mixtures of a 2,5-dihydroxybenzoic acid (2,5-DHB) matrix and caesium fluoride (CsF), used as the cationization agent in synthetic polymer MALDI mass analysis, at different molar ratios (1:1, 5:1, and 10:1). Complementary information could be obtained from ¹³C, ¹³³Cs, and ¹⁹F NMR spectra. Grinding the matrix together with the salt in the solid state was shown to induce a strong modification in the molecular organization within the MALDI sample. The evidenced mechano-induced reactions allow strong interactions between the matrix and the cation, up to the formation of a salt, and only occur in the presence of some water molecules. Addition of a poly(ethylene oxide) polymer as the analyte did not further modify the observed molecular organizations. Although relative matrix and salt concentrations in the scrutinized samples were unusual for MALDI analysis, mass spectra of good quality could be obtained and revealed that cation attachment on polymers during the MALDI process is not a matrix-independent event since a lower ionization efficiency was obtained from highly organized solid samples, mostly consisting of 2,5-DHB caesium salt species.     

Using calibration approaches to compensate for remaining matrix effects in quantitative liquid chromatography/electrospray ionization multistage mass spectrometric analysis of phytoestrogens in aqueous environmental samples

Signal suppression is a common problem in quantitative liquid chromatography/electrospray ionization multistage mass spectrometric (LC/ESI-MS(n)) analysis in environment samples, especially in highly loaded wastewater samples with highly complex matrix. Optimization of sample preparation and improvement of chromatographic separation are prerequisite to improve reproducibility and selectivity. Matrix components are reduced if not eliminated by optimization of sample preparation steps. However, extensive sample preparation may be time-consuming and risk the significant loss of some trace analytes. The best way to further compensate matrix effects is the use of an internal standard for each analyte. However, in a multi-component analysis, finding appropriate internal standards for every analyte is often difficult. In this present study, a more practical alternative option was sought. Matrix effects were assessed using the post-extraction addition method. By comparison of three different calibration approaches, it was found that matrix-matched calibration combined with one internal standard provides a satisfactory method for compensating for any residual matrix effects on all the analytes. Validating experiments on different sewage treatment plant (STP) influent samples analyzing for a range of phytoestrogens showed that this calibration method provided satisfactory results with concentration ratio 96.1-105.7% compared to those by standard addition.     

Charge state distribution shifting of protein ions observed in matrix-assisted laser desorption ionization mass spectrometry

By using a new sample preparation method for matrix-assisted laser desorption ionization, a significant shift to lower mass-to-charge values can be obtained for many protein samples. The sample preparation technique involves the creation of a thin film of protein-doped -cyano-4-hydroxycinnamic acid (CHCA) matrix formed in the presence of glycerol on top of a previously deposited pad of CHCA matrix. The higher charge states were not observed if the laser power was significantly above the threshold needed to produce protein molecular ions. Similar spectra were observed when samples were prepared in the presence of urea. The phenomenon was specific for the CHCA matrix because no effects were observed when sinapinic acid (3,5-dimethoxy-4-hydroxy-trans-cinnamic acid) and 2-(4-hydroxyphenylazo) benzoic acid matrices were used with the new sample preparation method.     

A statistical design of experiments for optimizing the MALDI-TOF-MS sample preparation of polymers. An application in the assessment of the thermo-mechanical degradation mechanisms of poly (ethylene terephthalate)

The sample preparation procedure for MALDI-TOF MS of polymers is addressed in this study by the application of a statistical Design of Experiments (DoE). Industrial poly (ethylene terephthalate) (PET) was chosen as model polymer. Different experimental settings (levels) for matrixes, analyte/matrix proportions and concentrations of cationization agent were considered. The quality parameters used for the analysis were signal-to-noise ratio and resolution. A closer inspection of the statistical results provided the study not only with the best combination of factors for the MALDI sample preparation, but also with a better understanding of the influence of the different factors, individually or in combination, to the signal. The application of DoE for the improvement of the MALDI measure of PET stated that the best combination of factors and levels was the following: matrix (dithranol), proportion analyte/matrix/cationization agent (1/15/1, V/V/V), and concentration of cationization agent (2 g L(-1)). In a second part, multiple processing by means of successive injection cycles was used to simulate the thermo-mechanical degradation effects on the oligomeric distribution of PET under mechanical recycling. The application of MALDI-TOF-MS showed that thermo-mechanical degradation primarily affected initially predominant cyclic species. Several degradation mechanisms were proposed, remarking intramolecular transesterification and hydrolysis. The ether links of the glycol unit in PET were shown to act as potential reaction sites, driving the main reactions of degradation.     

Proton transfer reactions of matrix-assisted laser desorption/ionization matrix monomers and dimers

The gas-phase basicities of monomeric and dimeric deprotonated ferulic and sinapic acids, common matrix-assisted laser desorption/ionization (MALDI) matrices, were determined. A new bracketing method based on structure-reactivity correlations was developed for deriving gas-phase basicities from reaction efficiencies. The matrix dimer anions were found to be significantly less basic than the monomer anions, by about 115 kJ/mol. The low basicity of the dimer anion can qualitatively be explained by resonance stabilization. The energies for proton transfer from dimers to monomers are therefore about 1.2 eV lower than for proton transfer between monomers. For the MALDI process, proton transfer reactions involving matrix dimers provide a low energy pathway for matrix and analyte ion formation.