Next-generation pathology practices with mass spectrometry imaging.

Mass spectrometry imaging (MSI) is a powerful technique that reveals the spatial distribution of various molecules in biological samples, and it is widely used in pathology-related research. In this review, we summarize common MSI techniques, including matrix-assisted laser desorption/ionization and desorption electrospray ionization MSI, and their applications in pathological research, including disease diagnosis, microbiology, and drug discovery. We also describe the improvements of MSI, focusing on the accumulation of imaging data sets, expansion of chemical coverage, and identification of biological significant molecules, that have prompted the evolution of MSI to meet the requirements of pathology practices. Overall, this review details the applications and improvements of MSI techniques, demonstrating the potential of integrating MSI techniques into next-generation pathology practices.

Toward understanding the ionization mechanism of matrix-assisted ionization using mass spectrometry experiment and theory.

RATIONALE: Matrix-assisted ionization (MAI) mass spectrometry does not require voltages, a laser beam, or added heat to initiate ionization, but it is strongly dependent on the choice of matrix and the vacuum conditions. High charge state distributions of nonvolatile analyte ions produced by MAI suggest that the ionization mechanism may be similar to that of electrospray ionization (ESI), but different from matrix-assisted laser desorption/ionization (MALDI). While significant information is available for MAI using mass spectrometers operating at atmospheric and intermediate pressure, little is known about the mechanism at high vacuum. METHODS: Eleven MAI matrices were studied on a high-vacuum time-of-flight (TOF) mass spectrometer using a 266 nm pulsed laser beam under otherwise typical MALDI conditions. Detailed comparisons with the commonly used MALDI matrices and theoretical prediction were made for 3-nitrobenzonitrile (3-NBN), which is the only MAI matrix that works well in high vacuum when irradiated with a laser. RESULTS: Screening of MAI matrices with good absorption at 266 nm but with various degrees of volatility and laser energies suggests that volatility and absorption at the laser wavelength may be necessary, but not sufficient, criteria to explain the formation of multiply charged analyte ions. 3-NBN produces intact, highly charged ions of nonvolatile analytes in high-vacuum TOF with the use of a laser, demonstrating that ESI-like ions can be produced in high vacuum. Theoretical calculations and mass spectra suggest that thermally induced proton transfer, which is the major ionization mechanism in MALDI, is not important with the 3-NBN matrix at 266 nm laser wavelength. 3-NBN:analyte crystal morphology is, however, important in ion generation in high vacuum. CONCLUSIONS: The 3-NBN MAI matrix produces intact, highly charged ions of nonvolatile compounds in high-vacuum TOF mass spectrometers with the aid of ablation and/or heating by laser irradiation, and shows a different ionization mechanism from that of typical MALDI matrices.

Utilizing AgNPt-SALDI to Classify Edible Oils by Multivariate Statistics of Triacylglycerol Profile.

Edible oils are valuable sources of nutrients, and their classification is necessary to ensure high quality, which is essential to food safety. This study reports the establishment of a rapid and straightforward SALDI-TOF MS platform used to detect triacylglycerol (TAG) in various edible oils. Silver nanoplates (AgNPts) were used to optimize the SALDI samples for high sensitivity and reproducibility of TAG signals. TAG fingerprints were combined with multivariate statistics to identify the critical features of edible oil discrimination. Eleven various edible oils were discriminated using principal component analysis (PCA). The results suggested the creation of a robust platform that can examine food adulteration and food fraud, potentially ensuring high-quality foods and agricultural products.

Toward the Rational Design of Universal Dual Polarity Matrix for MALDI Mass Spectrometry.

A series of novel anthranilic acid derivatives I-IV, of which COOH-NH2 (I) and COOH-NHMe (IV) are endowed with acid and base bifunctionality, were designed and synthesized for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry applications in dual polarity molecular imaging of biological samples, particularly for lipids. The heat of protonation, deprotonation, and proton transfer reaction as well as the capability of analyzing biomolecules in both positive and negative ion modes for I-IV were systematically investigated under standard 355 nm laser excitation. The results indicate correlation between dual polarity and acid-base property. Further, COOH-NHMe (IV) showed a unique performance and was successfully applied as the matrix for MALDI-TOF mass spectrometry imaging (MSI) for studying the mouse brain. Our results demonstrate the superiority of COOH-NHMe (IV) in detecting more lipid and protein species compared to commercially available matrices. Moreover, MALDI-TOF MSI results were obtained for lipid distributions, making COOH-NHMe (IV) a potential next generation universal matrix.

New Strategy to Preserve Phosphate by Ionic Liquid Matrices in Matrix-Assisted Laser Desorption/Ionization: A Case of Adenosine Nucleotides.

Adenosine -5'-triphosphate (ATP) plays a valuable role in metabolic activity to produce adequate energy in a biosystem. A high ATP/AMP ratio has a correlation with diabetes that induces suppression of AMP-activated protein kinase (AMPK). Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) has outstanding potential in determining the ratio of several types of adenosine phosphates in a sample to rapidly understand the primary energy transfer in metabolism. Although MALDI is viewed as a soft ionization technique for MS analysis, excess photon energy might crack the phosphate bonds leading to misinterpretation of the ATP level. In this work, ionic liquid matrices (ILMs) were employed to reduce fragmentation and increase the detection efficiency during the MALDI process. This study demonstrated for the first time that 2,5-dihydroxybenzoic acid pyridine (DHBP) is one of the most effective matrices for further quantitative analysis of adenosine nucleotides. This systematic screening of ILMs also enhances the fundamental understanding of MALDI.

1000-Fold Preconcentration of Per- and Polyfluorinated Alkyl Substances within 10 Minutes via Electrochemical Aerosol Formation.

We present a simple and efficient method for preconcentrating per- and polyfluorinated alkyl substances (PFAS) in water. Our method was inspired by the sea-spray aerosol enrichment in nature. Gas bubbles in the ocean serve to scavenge surface active material, carrying it to the air-ocean interface, where the bubbles burst and form a sea-spray aerosol. These aerosol particles are enriched in surface-active organic compounds such as free fatty acids and anionic surfactants. In our method, we in situ generate H2 microbubbles by electrochemical water reduction using a porous Ni foam electrode. These H2 bubbles pick up PFAS as they rise through the water column that contains low concentration PFAS. When these bubbles reach the water surface, they burst and produce aerosol droplets that are enriched in PFAS. Using this method, we demonstrated ∼1000-fold preconcentration for ten common PFAS in the concentration range from 1 pM to 1 nM (or ∼0.5 ng/L to 500 ng/L) in 10 min. We also developed a diffusion-limited adsorption model that is in quantitative agreement with the experimental data. In addition, we demonstrated using this method to preconcentrate PFAS in tap water, indicating its potential use for quantitative analysis of PFAS in real-world water samples.

Unprecedented Ionization Processes in Mass Spectrometry Provide Missing Link between ESI and MALDI.

In the field of mass spectrometry, producing intact, highly-charged protein ions from surfaces is a conundrum with significant potential payoff in application areas ranging from biomedical to clinical research. Here, we report on the ability to form intact, highly-charged protein ions on high vacuum time-of-flight mass spectrometers in the linear and reflectron modes achievable using experimental conditions that allow effective matrix removal from both the sample surfaces and from the charged clusters formed by the laser ablation event. The charge states are the highest reported on high vacuum mass spectrometers, yet they remain at only around a third of the highest charge obtained using laser ablation with a suitable matrix at atmospheric pressure. Other than physical instrument modifications, the key to forming abundant and stable highly-charged ions appears to be the volatility of the matrix used. Cumulative results suggest mechanistic links between the ionization process reported here and traditional ionization methods of electrospray ionization and matrix-assisted laser desorption/ionization.

Towards developing a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) compatible tissue expansion protocol.

Mass spectrometry imaging (MSI) visualizes spatial distribution of molecules in a biological tissue. However, compared with traditional microscopy-based imaging, conventional MSI is limited to its spatial resolution, resulting in difficulties in identifying detailed tissue morphological characters, such as lesion boundary or nanoscale structures. On the other hand, expansion microscopy, a tissue expansion method widely used in optical imaging to improve morphological details, has great potential to solve insufficient spatial resolution in mass spectrometry imaging (MSI). However, expansion microscopy was not originally designed for MSI, resulting in problems while combining expansion microscopy and MSI such as expanded sample fragility, vacuum stability and molecule loss during sample preparation. In this research we developed a MALDI MSI compatible expansion protocol by adjusting sample preparation methods during tissue expansion, successfully combining expansion microscopy with MSI. After tissue expansion the expanded sample can be readily applied to MALDI MSI sample preparation and further data acquisition. The MALDI MSI compatible expansion protocol has great potential to be widely applied in MALDI MSI sample preparation to facilitate improvement of MSI spatial resolution.

Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

New insights into the role of marine plastic-gels in microplastic transfer from water to the atmosphere via bubble bursting.

The pervasiveness of microplastics (MPs) in global oceans is raising concerns about their adverse impacts on ecosystems. The mechanistic understanding of MP transport is critical for evaluating its fate, flux, and ecological risks specifically. Currently, bubble bursting is believed to represent an important route for MP transfer from sea surfaces to the atmosphere. However, the detailed mechanisms of the complex physico-chemical interactions between MPs, water composition, and gel particles in the air-sea interface remain unknown. Our results suggested three steps for MP transfer between air-sea phases: (1) MPs incorporating into gel aggregates in the water column; (2) further accumulation of plastic-gel aggregate in the surface layer phase; finally (3) ejection of aggregates from the sea when bubbles of trapped air rise to the surface and burst. The water composition (e.g., high salinity, gel concentration and viscosity) can modulate plastic-gel aggregation and subsequent transport from water to the atmosphere. The possible mechanism may be closely tied to the formation of plastic-gel via cation-linking bridges, thereby enhancing plastic-gel ejection into air. Collectively, this work offers unique insights into the role of marine plastic-gels in determining MP fate and transport, especially at air-sea interfaces. The data also provide a better understanding of the corresponding mechanism that may explain the fates of missing plastics in the ocean.

Rapid detection of perfluorinated sulfonic acids through preconcentration by bubble bursting and surface-assisted laser desorption/ionization.

We developed a preconcentration method in which aerosol droplets containing enriched perfluorinated sulfonic acids (PFSs) are generated through bubble bursting and collected. The droplets were subjected to PFS analysis of perfluorohexane sulfonic acid (PFHxS) and perfluorooctanesulfonic acid (PFOS) through surface-assisted laser desorption/ionization-time-of-flight mass spectrometry; silver nanoplates (AgNPts) were assisting materials. The method was highly efficient, with an approximately three-order magnitude enhancement (5 × 10-13 to 1 × 10-11 M). Ultralow PFS concentrations (0.5 ng/L of PFOS; 0.4 ng/L of PFHxS) were detected in preconcentrated tap water containing PFSs. Our method has potential for rapid real-world PFS detection in water.

Soft Matrix-Assisted Laser Desorption/Ionization for Labile Glycoconjugates.

Since its introduction, matrix-assisted laser desorption/ionization (MALDI) has been widely used for the mass analysis of biomolecules. The "soft ionization" of MALDI enables accurate mass determination of intact biomolecules. However, the ionization and desorption processes of MALDI are not adequately soft as many labile biomolecules, such as glycoconjugates containing sialic acid or the sulfate functional group, easily dissociate into fragments and sometimes, no intact molecules are observed. In this study, we compared the conventional matrix of MALDI, namely 2,5-dihydroxybenzoic acid, to various soft matrices of MALDI-specifically, 5-methoxysalicylic acid, diamond nanoparticle trilayers, HgTe nanostructures, ionic liquid, and droplets of frozen solutions-by using three labile glycoconjugates as analytes: gangliosides, heparin, and pullulan. We demonstrated that droplets of frozen solution are the softest matrices for gangliosides and heparin. In particular, droplets of frozen solution do not generate fragments for gangliosides and can be used to determine the relative abundance of various gangliosides, whereas ionic liquid 2,5-dihydroxybenzoic acid butylamine is the most suitable matrix for pullulan mass analysis. Graphical Abstract.

Fundamental Studies of New Ionization Technologies and Insights from IMS-MS.

Exceptional ion mobility spectrometry mass spectrometry (IMS-MS) developments by von Helden, Jarrold, and Clemmer provided technology that gives a view of chemical/biological compositions previously not achievable. The ionization method of choice used with IMS-MS has been electrospray ionization (ESI). In this special issue contribution, we focus on fundamentals of heretofore unprecedented means for transferring volatile and nonvolatile compounds into gas-phase ions singly and multiply charged. These newer ionization processes frequently lead to different selectivity relative to ESI and, together with IMS-MS, may provide a more comprehensive view of chemical compositions directly from their original environment such as surfaces, e.g., tissue. Similarities of results using solvent- and matrix-assisted ionization are highlighted, as are differences between ESI and the inlet ionization methods, especially with mixtures such as bacterial extracts. Selectivity using different matrices is discussed, as are results which add to our fundamental knowledge of inlet ionization as well as pose additional avenues for inquiry. IMS-MS provides an opportunity for comparison studies relative to ESI and will prove valuable using the new ionization technologies for direct analyses. Our hypothesis is that some ESI-IMS-MS applications will be replaced by the new ionization processes and by understanding mechanistic aspects to aid enhanced source and method developments this will be hastened.

Formation of Metal-Related Ions in Matrix-Assisted Laser Desorption Ionization.

In a study of the metal-related ion generation mechanism in matrix-assisted laser desorption ionization (MALDI), crystals of matrix used in MALDI were grown from matrix- and salt-containing solutions. The intensities of metal ion and metal adducts of the matrix ion obtained from unwashed crystals were higher than those from crystals washed with deionized water, indicating that metal ions and metal adducts of the matrix ions are mainly generated from the surface of crystals. The contributions of preformed metal ions and metal adducts of the matrix ions inside the matrix crystals were minor. Metal adducts of the matrix and analyte ion intensities generated from a mixture of dried matrix, salt, and analyte powders were similar to or higher than those generated from the powder of dried droplet crystals, indicating that the contributions of the preformed metal adducts of the matrix and analyte ions were insignificant. Correlation between metal-related ion intensity fluctuation and protonated ion intensity fluctuation was observed, indicating that the generation mechanism of the metal-related ions is similar to that of the protonated ions. Because the thermally induced proton transfer model effectively describes the generation of the protonated ions, we suggest that metal-related ions are mainly generated from the salt dissolution in the matrix melted by the laser. Graphical Abstract ᅟ.

Theoretical investigation of low detection sensitivity for underivatized carbohydrates in ESI and MALDI.

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mainly generate protonated ions from peptides and proteins but sodiated (or potassiated) ions from carbohydrates. The ion intensities of sodiated (or potassiated) carbohydrates generated by ESI and MALDI are generally lower than those of protonated peptides and proteins. Ab initio calculations and transition state theory were used to investigate the reasons for the low detection sensitivity for underivatized carbohydrates. We used glucose and cellobiose as examples and showed that the low detection sensitivity is partly attributable to the following factors. First, glucose exhibits a low proton affinity. Most protons generated by ESI or MALDI attach to water clusters and matrix molecules. Second, protonated glucose and cellobiose can easily undergo dehydration reactions. Third, the sodiation affinities of glucose and cellobiose are small. Some sodiated glucose and cellobiose dissociate into the sodium cations and neutral carbohydrates during ESI or MALDI process. The increase of detection sensitivity of carbohydrates in mass spectrometry by various methods can be rationalized according to these factors. Copyright © 2016 John Wiley & Sons, Ltd.

Ionization Mechanism of Matrix-Assisted Laser Desorption/Ionization.

In past studies, mistakes in determining the ionization mechanism in matrix-assisted laser desorption/ionization (MALDI) were made because an inappropriate ion-to-neutral ratio was used. The ion-to-neutral ratio of the analyte differs substantially from that of the matrix in MALDI. However, these ratios were not carefully distinguished in previous studies. We begin by describing the properties of ion-to-neutral ratios and reviews early experimental measurements. A discussion of the errors committed in previous theoretical studies and a comparison of recent experimental measurements follow. We then describe a thermal proton transfer model and demonstrate how the model appropriately describes ion-to-neutral ratios and the total ion intensity. Arguments raised to challenge thermal ionization are then discussed. We demonstrate how none of the arguments are valid before concluding that thermal proton transfer must play a crucial role in the ionization process of MALDI.

Ion intensity and thermal proton transfer in ultraviolet matrix-assisted laser desorption/ionization.

The ionization mechanism of ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) was investigated by measuring the total cation intensity (not including sodiated and potasiated ions) as a function of analyte concentration (arginine, histidine, and glycine) in a matrix of 2,4,6-trihydroxyacetophenone (THAP). The total ion intensity increased up to 55 times near the laser fluence threshold as the arginine concentration increased from 0% to 1%. The increases were small for histidine, and a minimal increase occurred for glycine. Time-resolved fluorescence intensity was employed to investigate how analytes affected the energy pooling of the matrix. No detectable energy pooling was observed for pure THAP and THAP/analyte mixtures. The results can be described by using a thermal proton transfer model, which suggested that thermally induced proton transfer is crucial in the primary ion generation in UV-MALDI.