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.     

CHCA-modified Au nanoparticles for laser desorption ionization mass spectrometric analysis of peptides

Gold nanoparticles (AuNPs) have been studied as a potential solid-state matrix for laser desorption/ionization mass spectrometry (LDI-MS) but the efficiency in ionization remains low. In this report, AuNPs are capped by a self-assembled monolayer of cysteamine and modified with α-cyano-4-hydroxycinnanic acid (CHCA) for effective MALDI measurements. CHCA-terminated AuNPs offer marked improvement on peptide ionization compared with citrate-capped or cysteamine-capped AuNPs. The coating also effectively suppresses formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra. Addition of glycerol and citric acid to the peptide/AuNPs sample further improves the performance of these AuNPs for LDI-MS analysis. Glycerol appears to enhance the dispersion of AuNPs in sample spots, increasing the sample ionization and shot-to-shot reproducibility, while citric acid serves as an external proton donor, providing high production of protonated analyte ions and reducing fragmentation of peptides on the nanoparticle-based surface. Optimal ratios of citric acid, glycerol, and AuNPs in sample solution have been systematically studied. A more than 10-fold increase for desorption ionization of peptides can be achieved by combining 5% glycerol and 20 mM citric acid with the CHCA-terminated AuNPs. The applicability of the CHCA-AuNPs for LDI-MS analysis of protein digests has also been demonstrated. This work shows the potential of AuNPs for SALDI-MS analysis, and the improvement with chemical functionalization, controlled dispersion, and use of an effective proton donor.     

Ionization mechanism of oligonucleotides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The ionization of nucleosides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was systematically investigated using adenine (A), thymine (T), guanine (G) and cytosine (C) with several common matrices. Experimental results of the protonation and deprotonation of the bases of A, T, G and C in the matrices 2,5-dihydroxybenzoic acid (2,5-DHB), alpha-cyano-4-hydroxycinnamic acid (alpha-CHCA) and 3-hydroxypicolinic acid (3-HPA) provide an insight into the ionization mechanism of oligonucleotides in MALDI. It was found that the low ion signal from DNA in poly-G in MALDI as reported in earlier work could be attributed to the fact that the base of G is difficult to ionize. Our results suggest that the ionization of DNA in MALDI is dominated by the protonation and deprotonation of bases and it is basically independent of the backbone of DNA. Both the protonation and deprotonation are strongly structure dependent. The protonation is dominated by pre-protonation before laser ablation, while the deprotonation is controlled by the thermal reaction.     

Functionalized nanoparticles for liquid atmospheric pressure matrix-assisted laser desorption/ionization peptide analysis

Nanoparticles for the extraction of peptides and subsequent analysis using atmospheric pressure matrix-assisted laser desorption/ionization (APMALDI) have been evaluated. The atmospheric pressure source allows for particles to be directly introduced in the liquid matrix, minimizing sample loss and analysis time. Described in this work are two sample preparation procedures for liquid APMALDI analysis: a C18 functionalized silica nanoparticle for hydrophobic extractions, and an aptamer functionalized magnetite core nanoparticle for rapid, affinity extractions. The C18 particles provide a non-selective support for rapid profiling applications, while the aptamer particles are directed towards reducing the complexity in biological samples. The aptamer functionalized particles provide a more selective analyte-nanoparticle interaction whereby the tertiary structure of the analyte becomes more critical to the extraction. In both cases, the liquid APMALDI matrix provides a support for ionization, and acts as the releasing agent for the analyte-particle interaction. Additionally, analyte enrichment was possible due to the large surface-to-volume ratio of the particles. The experiments conducted with functionalized nanoparticles, in an atmospheric pressure liquid matrix, present a basis for further methodologies and utilities of silica nanoparticles to be developed.     

Au@SiO2 core-shell nanoparticles for laser desorption/ionization time of flight mass spectrometry

In matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the analysis capability, especially for small molecules, is often compromised by the addition of organic matrices due to the existence of background signals. Herein we report a new detection method on the utility of core-shell nanoparticles (CSNPs) as energy transfer structure in LDI-TOF-MS. The LDI-TOF-MS based on gold-silica core-shell nanoparticles with ultrathin silica shell of 2-4 nm (Au@utSiO(2) CSNPs) was effectively applied to the analysis of many compounds, especially for small functional molecules and polymers, which was more promising than MALDI-TOF-MS.     

人参皂苷的基质辅助激光解吸质谱研究

利用MALDI-TOFMS测定了八种人参皂苷的分子量, 并分析了西洋参总皂苷的组成。同时, 进行了灵敏度实验, 并探讨了基质及碱金属离子的影响, 证明该方法灵敏度高, 重复性好, 结果准确。是测定极性小分子分子量的有效方法。     

Matrix-assisted laser desorption/ionization mechanism study with dihydroxybenzoic acid isomers as matrices

Desorption and ionization efficiencies of matrix-assisted laser desorption/ionization (MALDI) for various biomolecules with different dihydroxybenzoic acid isomers were studied. No clear relationships were observed between MALDI biomolecule signals vs. gas-phase basicity, proton affinity and ionization potential. This indicates the the gas-phase protonation mechanism is not adequate to explain the observed results.     

Multiplexed screening of cellular uptake of gold nanoparticles using laser desorption/ionization mass spectrometry

Gold nanoparticles (AuNPs) are highly promising candidates as drug delivery agents into cells of interest. We describe for the first time the multiplexed analysis of nanoparticle uptake by cells using mass spectrometry. We demonstrate that the cellular uptake of functionalized gold nanoparticles with cationic or neutral surface ligands can be readily determined using laser desorption/ionization mass spectrometry of cell lysates. The surface ligands have "mass barcodes" that allow different nanoparticles to be simultaneously identified and quantified at levels as low as 30 pmol. Using this method, we find that subtle changes to AuNP surface functionalities can lead to measurable changes in cellular uptake propensities.     

Identification of137Cs in an individual microparticle by laser desorption/ionization ion trap mass spectrometer

The detection of radiocesium in microparticles was performed by using an ion trap mass spectrometer coupled with laser desorption and ionization. Pulsed laser desorbed particle and the resulted ions were analyzed by an ion trap mass analyzer. The presence of radiocesium, especially about¹³⁷Cs, in microparticles was verified by single as well as successive particle analysis. The detection limit was reached to ≈ag/particle level with a signal-to-background ratio of 4. The inhomogeneous distribution of particle size and the irregular shapes of particle limit the quantitative evaluation of¹³⁷Cs concentration in the microparticle. But this high sensitivity allows to monitor directly the radiocesium from small amounts of a microparticle sample.     

Matrix-free infrared soft laser desorption/ionization

Infrared soft laser desorption/ionization was performed using a 2.94 µm Er : YAG laser and a commercial reflectron time-of-flight mass spectrometer. The instrument was modified so that a 337 nm nitrogen laser could be used concurrently with the IR laser to interrogate samples. Matrix-assisted laser desorption/ionization (MALDI), laser desorption/ionization and desorption/ionization on silicon with UV and IR lasers were compared. Various target materials were tested for IR soft desorption ionization, including stainless steel, aluminum, copper, silicon, porous silicon and polyethylene. Silicon surfaces gave the best performance in terms of signal level and low-mass interference. The internal energy resultant of the desorption/ionization was assessed using the easily fragmented vitamin B₁₂ molecule. IR ionization produced more analyte fragmentation than UV-MALDI analysis. Fragmentation from matrix-free IR desorption from silicon was comparable to that from IR-MALDI. The results are interpreted as soft laser desorption and ionization resulting from the absorption of the IR laser energy by the analyte and associated solvent molecules. Copyright © 2004 John Wiley & Sons, Ltd.     

Two-photon ionization thresholds of matrix-assisted laser desorption/ionization matrix clusters

Direct two-photon ionization of the matrix has been considered a likely primary ionization mechanism in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This mechanism requires that the vertical ionization threshold of matrix materials be below twice the laser photon energy. Because dimers and larger aggregates may be numerous in the early stages of the MALDI plume expansion, their ionization thresholds are important as well. We have used two-color two-photon ionization to determine the ionization thresholds of jet cooled clusters of an important matrix, 2,5-dihydroxy benzoic acid (DHB), and mixed clusters with the thermal decomposition product of DHB, hydroquinone. The thresholds of the clusters were reduced by only a few tenths of an eV compared to the monomers, to an apparent limit of 7.82 eV for pure DHB clusters. None of the investigated clusters can be directly ionized by two nitrogen laser photons (7.36 eV), and the ionization efficiency at the thresholds is low.     

Clustering of polycyclic aromatic hydrocarbons in matrix-assisted laser desorption/ionization and laser desorption mass spectrometry

The desorption/ionization behaviour of polycyclic aromatic hydrocarbons (PAHs) in matrix-assisted laser desorption/ionization (MALDI) and laser desorption (LD) mass spectrometry was studied by the solvent-free sample preparation method. As the understanding of the desorption/ionization mechanism in MALDI is normally hampered by the different ionization and desorption efficiencies of the analytes, this work was focused on the analyses of a homologous series of four hexabenzocoronenes (HBCs) possessing virtually the same ionization efficiency: HBC parent, hexamethyl-hexabenzocoronene (HBC-C1), hexapropyl-hexabenzocoronene (HBC-C3) and hexakis(dodecyl)-hexabenzocoronene (HBC-C12). The different signal intensities obtained in their mass spectra can be related to differences in their desorption efficiencies, which are attributed to the different strengths of the intermolecular interactions between unsubstituted and alkylated HBCs in the solid state. The influence of the aromatic structure of PAHs on their photoionization/desorption probability was investigated. As a model system, an equimolar mixture composed of HBC-C12 and hexakis(dodecyl)-hexaphenylbenzene (HPB-C12) was chosen. The aromatic structures of both molecules and thus their absorption coefficients at the laser wavelength differ substantially and have a huge influence on their photoionization efficiency. The combined effect of laser light absorption and intermolecular interactions on the desorption/ionization behaviour of giant PAHs was further studied by using an equimolar mixture composed of a larger PAH (C(222)H(42)) and its dendritic precursor (C(222)H(150)). This mixture shows the opposite behaviour to that of the former example, because the balance between desorption and ionization efficiency has changed significantly. The present investigation should be of interest for providing a better understanding of MALDI and LD spectra obtained from natural PAH-containing samples, such as heavy oils, asphaltenes or pitches, for which our artificial mixtures represent suitable model systems.     

Reduction of organic dyes in matrix-assisted laser desorption/ionization and desorption/ionization on porous silicon

Reduction of analytes in matrix-assisted laser desorption/ionization (MALDI) often obscures the actual determination of molecular structure. To address the redox reactions in laser desorption/ionization processes, the organic dyes Methylene Blue, Janus Green B, Crystal Violet and Rhodamine B were analyzed by MALDI or by desorption/ionization on porous silicon (DIOS). Susceptibility to reduction in MALDI was dependent on both the reduction potentials of analytes and the molar ratio of analyte to matrix molecules. Addition of Cu(II) ions as an electron scavenger suppressed the reduction of Methylene Blue in MALDI. The results suggested that electron transfer to analytes from the sample target and/or from the matrix contributed to the reduction. In DIOS, the reductions of organic dyes were more prominent than in MALDI, and were not prevented by Cu(II) ion doping, probably due to direct contact of the analytes with silicon which had little electric resistance.     

Silica nanoparticles pre-spotted onto target plate for laser desorption/ionization mass spectrometry analyses of peptides

We report on the simple deposition of Stöber silica nanoparticles (SiO₂ NPs) on conventional MALDI target plate for high throughput laser desorption/ionization mass spectrometry (LDI-MS) analyses of peptide mixtures with sensitivity in the femtomolar range. This low-cost easily prepared material allowed straightforward LDI experiments by deposition of the studied samples directly onto a pre-spotted MALDI plate. This analytical strategy can be performed in any laboratory equipped with a MALDI-TOF instrument. All key benefits of organic matrix-free technologies were satisfied while maintaining a high level of detection performances (sensitivity and reproducibility/repeatability). In particular, sample preparation was simple and detection in the low mass range was not hampered by matrix ions. Imaging studies were undertaken to query sample dispersion into the inert SiO₂ NPs and to help into the search of the best experimental conditions producing homogeneous analyte distribution within the deposit. In contrast to commercial disposable LDI targets designed for single use and requiring an adaptor such as NALDI™, the proposed SiO₂ NPs pre-spotting on a MALDI target plate allowed very easily switching between MALDI and LDI experiments. They can be conducted either simultaneously (positions with an organic matrix or SiO₂ NPs) or in the row (support prepared in advance, stored and washed after use). The overall cost and versatility of the methodology made it very attractive to MALDI users in many domains (peptidomics, proteomics, metabolomics).     

Competing ion decomposition channels in matrix-assisted laser desorption ionization

We gauged the internal energy transfer for two dissociative ion decomposition channels in matrix-assisted laser desorption ionization (MALDI) using the benzyltriphenylphosphonium (BTP) thermometer ion [PhCH 2PPh 3] (+). Common MALDI matrixes [alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), and 2,5-dihydroxycinnamic acid (DHB)] were studied with nitrogen laser (4 ns pulse length) and mode-locked 3 x omega Nd:YAG laser (22 ps pulse length) excitation. Despite the higher fluence required to initiate fragmentation, BTP ions indicated lower internal energy transfer with the picosecond laser in all three matrixes. These differences can be rationalized in terms of phase explosion induced by the nanosecond laser vs a stress-confinement-driven desorption mechanism for the picosecond laser. For the two ion production channels of the BTP thermometer ion, breaking a single bond can result in the formation of benzyl/tropylium ions, F1, or triphenylphosphine ions, F2. In SA and DHB, as well as in CHCA at low fluence levels, the efficiency of these channels (expressed by the branching ratio I F1/ I F2) is moderately in favor of producing tropylium ions, 1 < I F1/ I F2 < 6. As the laser fluence is increased, for CHCA, there is a dramatic shift in favor of the tropylium ion production, with I F1/ I F2 approximately 30 for the nanosecond and the picosecond laser, respectively. This change is correlated with the sudden increase in the BTP internal energies in CHCA in the same laser fluence range. The large changes observed in internal energy deposition for CHCA with laser fluence can account for its ability to induce fragmentation in peptides more readily than SA and DHB.     

Internal energy build-up in matrix-assisted laser desorption/ionization

This paper reports detailed studies on the internal energy of ions formed in matrix-assisted laser desorption/ionization (MALDI) using delayed extraction MALDI-time-of-flight (TOF) and atmospheric pressure (AP) MALDI mass spectrometric (MS) methods. We use benzylpyridinium cations as internal energy probes. Our study reveals three distinct contributions to internal energy build-up in vacuum-MALDI (classical MALDI-TOF), each having different effects on ion fragmentation. Some fragments are formed before ion extraction (i.e. no more than 100 ns after the laser impact), and they are therefore well resolved and recorded as sharp signals in the MALDI-TOFMS scan. This prompt fragmentation can have two origins: (i) in-plume thermal activation, presumably always present, and (ii) in-plume chemical activation, in the course of reactions with hydrogen radicals. In addition to early internal energy build-up associated with these well-resolved promptly formed fragments, a broad peak slightly offset to higher masses could be detected corresponding to fragments formed after the extraction has started. This second signal corresponds to a third source of internal energy in MALDI ions, (iii) the extraction-induced collisional activation of the ions with the neutral components of the plume. These three contributions are difficult to quantify in vacuum-MALDI, because of the combined influence of several parameters (nature of the matrix, spot-to-spot variability, total laser exposure, delay time, acceleration voltage) on extraction-induced fragmentation. AP-MALDI, on the other hand, has two advantages for comparative studies of analyte fragmentation. First, extraction-induced fragmentation is absent, and only the contributions of early plume activation remain. Second, the reproducibility is far better than in vacuum-MALDI. AP-MALDI is therefore expected to shed new light on the early steps of the MALDI process.     

Coffee-ring effects in laser desorption/ionization mass spectrometry

This report focuses on the heterogeneous distribution of small molecules (e.g. metabolites) within dry deposits of suspensions and solutions of inorganic and organic compounds with implications for chemical analysis of small molecules by laser desorption/ionization (LDI) mass spectrometry (MS). Taking advantage of the imaging capabilities of a modern mass spectrometer, we have investigated the occurrence of “coffee rings” in matrix-assisted laser desorption/ionization (MALDI) and surface-assisted laser desorption/ionization (SALDI) sample spots. It is seen that the “coffee-ring effect” in MALDI/SALDI samples can be both beneficial and disadvantageous. For example, formation of the coffee rings gives rise to heterogeneous distribution of analytes and matrices, thus compromising analytical performance and reproducibility of the mass spectrometric analysis. On the other hand, the coffee-ring effect can also be advantageous because it enables partial separation of analytes from some of the interfering molecules present in the sample. We report a “hidden coffee-ring effect” where under certain conditions the sample/matrix deposit appears relatively homogeneous when inspected by optical microscopy. Even in such cases, hidden coffee rings can still be found by implementing the MALDI-MS imaging technique. We have also found that to some extent, the coffee-ring effect can be suppressed during SALDI sample preparation.