preparation-of-homogeneous-maldi-samples-for-quantitative-applications

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

Academia Sinica

This work demonstrates a quantitative interpretation of ion desorption in matrix-assisted laser desorption/ionization (MALDI). The theoretical modeling incorporates transition state theory for the desorption of surface ions, assuming chemical and thermal equilibrium in the solid state prior to desorption. It is distinct from conventional models that assume chemical equilibrium in the gas phase. This solid-state thermodynamic interpretation was used to examine the desorption of pure 2,4,6-trihydroxyacetophenone (THAP) and of angiotensin I mixed with THAP. It successfully described the changes in ion yield with the effective temperature under various laser fluence and initial temperature conditions. The analysis also revealed the key role played by ion concentration in the modeling to provide the best fit of the model to observations. On the other hand, divergence of the ion beam with laser fluence was examined using an imaging detection method, and the signal saturation normally seen at high fluence was appropriately reduced by ion focusing. Simplified but deceptive theoretical interpretations were obtained when the analysis was conducted without adequate calibration of the instrument bias.

Solid-phase thermodynamic interpretation of ion desorption in matrix-assisted laser desorption/ionization.

This investigation concerns the initial chemical reactions that affect the ionization of matrixes in matrix-assisted laser desorption/ionization (MALDI). The study focuses on the relaxations of photon energy that occur on a comparable time scale to that of ionization, in which the available laser energy is shared and the ionization condition is changed. The relaxations include fluorescence, fragmentation, and nonradiative relaxation from the excited state to the ground state. With high absorption cross section and long excited-state lifetime, photoionization of matrix plays an important role if sufficient laser energy is used. Under other conditions, thermal ionization of the molecule in the ground state is predicted to be one of the important reactions. Evidence of change in the branching ratio of initial reactions with the matrix and the excitation wavelength was obtained with α-cyano-4-hydroxycinnamic acid, sinapinic acid, 2,5-dihydroxybenzoic acid, and 2,4,6-trihydroxyacetophenone. These matrixes are studied by obtaining their mixed crystal absorption spectra, fluorescence properties, laser-induced infrared emission, and product ions. The exact ionization pathway depends on the chemical properties of matrixes and the excitation conditions. This concept may explain the diversity of experimental results observed in MALDI experiments, which provides an insight into the ensemble of chemical reactions that govern the generation of ions.

Analysis of initial reactions of MALDI based on chemical properties of matrixes and excitation condition.

Diamond nanoparticles (DNPs) were incorporated into matrix-assisted laser desorption/ionization (MALDI) samples to enhance the sensitivity of the mass spectrometer to carbohydrates. The DNPs optimize the MALDI sample morphology and thermalize the samples for thermally labile compounds because they have a high thermal conductivity, a low extinction coefficient in UV-vis spectral range, and stable chemical properties. The best enhancement effect was achieved when matrix, DNP, and carbohydrate solutions were deposited and vacuum-dried consecutively to form a trilayer sample morphology. It allows the direct identification of underivatized carbohydrates mixed with equal amount of proteins because no increase in the ion abundance of proteins was achieved. For dextran with an average molecular weight of 1500, the trilayer method typically improves the sensitivity by 79- and 7-fold in comparison to the conventional dried-droplet and thin-layer methods, respectively.

Selective enhancement of carbohydrate ion abundances by diamond nanoparticles for mass spectrometric analysis.

One of the reasons that thermally induced reactions are not considered a crucial mechanism in ultraviolet matrix-assisted laser desorption ionization (UV-MALDI) is the low ion-to-neutral ratios. Large ion-to-neutral ratios (10(-4)) have been used to justify the unimportance of thermally induced reactions in UV-MALDI. Recent experimental measurements have shown that the upper limit of the total ion-to-neutral ratio is approximately 10(-7) at a high laser fluence and less than 10(-7) at a low laser fluence. Therefore, reexamining the possible contributions of thermally induced reactions in MALDI may be worthwhile. In this study, the concept of polar fluid was employed to explain the generation of primary ions in MALDI. A simple model, namely thermal proton transfer, was used to estimate the ion-to-neutral ratios in MALDI. We demonstrated that the theoretical calculations of ion-to-neutral ratios exhibit the same trend and similar orders of magnitude compared with those of experimental measurements. Although thermal proton transfer may not generate all of the ions observed in MALDI, the calculations demonstrated that thermally induced reactions play a crucial role in UV-MALDI.

Thermal proton transfer reactions in ultraviolet matrix-assisted laser desorption/ionization.

Although several reaction models have been proposed in the literature to explain matrix-assisted laser desorption/ionization (MALDI), further study is still necessary to explore the important ionization pathways that occur under the high-temperature environment of MALDI. 2,4,6-Trihydroxyacetophenone (THAP) is an ideal compound for evaluating the contribution of thermal energy to an initial reaction with minimum side reactions.

Contribution of thermal energy to initial ion production in matrix-assisted laser desorption/ionization observed with 2,4,6-trihydroxyacetophenone.

A comprehensive theoretical calculation that couples space- and velocity-focusing is developed for optimizing the design of a time-of-flight (TOF) mass spectrometer. Conventional designs for ion sources of TOF mass spectrometers deviate from the optimal condition because the velocity- and space-focusing conditions are considered separately for two ions with simplified equations. The result of a reexamination taking into account all essential ions reveals that the conventional ion source design, especially the length of the ion extraction region, results in poor resolving power. The comprehensive calculation demonstrates that the resolving power increases when the length of the extraction region is shorter than that of the conventional ion source. A numerical analysis indicates that the resolving power dramatically increases when the effective extraction potential compensates for the initial kinetic energy spread of ions. With typically used extraction potentials, the newly optimized ion source improves the resolving power by more than two orders of magnitude compared with the conventional design. This new theoretical interpretation can also be used to predict the optimal extraction potential and extraction delay in conventional ion sources to substantially improve the resolving power. This comprehensive calculation method is effective not only for designing new high-resolution instruments but also for optimizing commercial products.

Coupled Space- and Velocity-Focusing in Time-of-Flight Mass Spectrometry-a Comprehensive Theoretical Investigation.

This work demonstrates a method to prepare homogeneous distributions of analytes to improve data reproducibility in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). Natural-air drying processes normally result in unwanted heterogeneous spatial distributions of analytes in MALDI crystals and make quantitative analysis difficult. This study demonstrates that inducing Marangoni flows within drying droplets can significantly reduce the heterogeneity problem. The Marangoni flows are accelerated by changing substrate temperatures to create temperature gradients across droplets. Such hydrodynamic flows are analyzed semi-empirically. Using imaging mass spectrometry, changes of heterogeneity of molecules with the change of substrate temperature during drying processes are demonstrated. The observed heterogeneities of the biomolecules reduce as predicted Marangoni velocities increase. In comparison to conventional methods, drying droplets on a 5 °C substrate while keeping the surroundings at ambient conditions typically reduces the heterogeneity of biomolecular ions by 65%-80%. The observation suggests that decreasing substrate temperature during droplet drying processes is a simple and effective means to reduce analyte heterogeneity for quantitative applications. Graphical Abstract ᅟ.

Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.

Quantitative analysis with mass spectrometry (MS) is important but challenging. Matrix-assisted laser desorption/ionization (MALDI) coupled with time-of-flight (TOF) MS offers superior sensitivity, resolution and speed, but such techniques have numerous disadvantages that hinder quantitative analyses. This review summarizes essential obstacles to analyte quantification with MALDI-TOF MS, including the complex ionization mechanism of MALDI, sensitive characteristics of the applied electric fields and the mass-dependent detection efficiency of ion detectors. General quantitative ionization and desorption interpretations of ion production are described. Important instrument parameters and available methods of MALDI-TOF MS used for quantitative analysis are also reviewed.This article is part of the themed issue 'Quantitative mass spectrometry'.

Critical factors determining the quantification capability of matrix-assisted laser desorption/ionization- time-of-flight mass spectrometry.

Glycoconjugates are ubiquitously present and play a critical role in various biological processes. Due to their low stability and incredibly high degree of structural diversity, the structural characterization of glycan generally requires chemical derivatization and sophisticated instrumentation. Herein, we report a method for complicated glycan characterization in a single assay by employing 2,5-dihydroxybenzoic acid functionalized mercury telluride nanoparticles (HgTe@DHB NPs) as a dual ionization-dissociation element in matrix-assisted laser desorption/ionization mass spectrometry. Using a linear glycan, HgTe@DHB NPs promote laser-induced extensive and intense dissociation of the glycan, superior to HgTe microparticles and other inorganic nanoparticles (TiO2, ZnO, and Mn2O3 NPs). Abundant generation of diagnostic glycosidic (Y-, and B-type ions) and cross-ring cleavage (A-type ions) ions permits unambiguous determination of the composition, sequence, branching, and linkage of labile sialylated glycans. The general utility of this approach was demonstrated by the characterization of labile sialylated glycans and two sets of complicated isomeric glycans. This phenomenon was delineated further by investigating the NP's physico-chemical characteristics, revealing that their nanoscale-dependent thermodynamic properties, including UV absorption, photoelectron release dynamics and thermal energy, were the key to levitate temperature synergistically, thus inducing spontaneous glycan decomposition during the nanoparticle-assisted laser desorption-ionization process. Our results show that this "pseudo-MS/MS" obtained by HgTe@DHB can be beneficial for the analysis of biologically relevant and more complicated carbohydrates, without the need for chemical pre-derivatization and conventional tandem mass spectrometry.

Yin-Hung Lai

Genomic Research Center

Academia Sinica

Preparation of Homogeneous MALDI Samples for Quantitative Applications