Matrix Deposition Method Research Articles

Developing a novel strategy for fabricating matrix film to assess the distribution of potassium perfluorooctanic sulfonate by matrix-assisted laser desorption/ionization mass spectrometry imaging

Matrix deposition plays a critical role in image quality of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). To improve the ionization efficiency and overcome the limitation of traditional matrix deposition methods in the face of difficult-to-sublimate or difficult-to-dissolve matrix, covalent organic frameworks (COFs) named COF-DhaTab was successfully synthesized and firstly used as matrix film. It was fabricated by imprinting of sieved COF-DhaTab powder on the surface of a double-sided adhesive tape. Outstanding reproducibility and uniformity of COF-DhaTab film were demonstrated by relative standard deviation (RSD) within 8.37% and 7.71% from dot-to-dot and plate-to-plate, respectively. With the introduction of double-sided adhesive tape, water contact angle (WCA) of COF-DhaTab film increased from 55° to 141°, resulting in significant suppression of analyte diffusion. Moreover, the intensity of potassium perfluorooctanic sulfonate (PFOS, C8F17SO3−, m/z 498.93) was 9.3 × 105, more than six hundred times higher than that using DHB matrix. This enhancement was attributed to the rough surface and multiple branches of the synthesized COF-DhaTab. To verify the ability of COF-DhaTab film as substrate, the spatial distribution of PFOS in zebrafish, rat liver and kidney tissues was explored. Superior imaging capability was displayed with high-spatial resolution and reliable location distribution. These results not only demonstrate the outstanding ability of COF-DhaTab as matrix for MALDI-MS and MALDI-MSI, but also provide a facile approach for fabrication of novel matrix films for MALDI-MSI.

Single-filament imaging mass spectrometry lipidomics in Arthrospira platensis.

Elucidating intra-organismal biochemical and lipid organization in photosynthetic biological cell factories of filamentous cyanobacteria, such as Arthrospira platensis (Spirulina), is important for tracking physiological response mechanisms during growth. Little is known about the filaments' biochemical organization and cellular structure and no label-free imaging techniques exist that provide molecular mapping. We applied ultrahigh-resolution mass spectrometry (MS) with matrix-assisted laser desorption ionization (MALDI) imaging to immobilized Spirulina filaments to investigate the localization of lipids across distinct physiological regions. We optimized matrix selection and deposition methods with the goal of facilitating high spatial, and intra-filament, resolution using untargeted multivariate statistical spectral deconvolution across MS pixels. Our results demonstrate an improved two-step matrix application with an optimized procedure for intra-organismal lipid profiling to improve analyte sensitivity and achieve higher spatial resolution. We evaluate several conventional matrices, namely 2,5-dihydroxybenzoic acid (DHB), superDHB (sDHB), 1,5-diaminonaphthalene (DAN), and a 50:50 mix of DHB and sDHB, and compare delineation and pixel-based elucidation of intra-filament lipidomics. We identified a total of 1626 features that could be putatively assigned a lipid-like formula based on database query and 46 unique features, with associated lipid assignments that were significantly distinct in their intra-filament location. MALDI imaging MS with untargeted statistical spectral deconvolution was used to visualize intra-filament lipidomics organization in Spirulina filaments. Improvements in matrix deposition, including sequential sublimation and pneumatic spraying, increased signal abundance at high spatial resolution and allowed for identification of distinct lipid composition regions. This work outlines a methodology that may be used for micro-ecological untargeted molecular phenotyping.

Mammalian Oocyte Analysis by MALDI MSI with Wet-Interface Matrix Deposition Technique

Oocytes are a special kind of biological material. Here, the individual variability of a single cell is important. It means that the opportunity to obtain information about the lipid content from the analysis of a single cell is significant. In our study, we present a method for lipid analysis based on the MALDI-based mass spectrometry imaging (MSI) approach. Our attention was paid to the sample preparation optimization with the aid of a wet-interface matrix deposition system (matrix spraying). Technical considerations of the sample preparation process, such as the number of matrix layers and the position of the spraying nozzle during the matrix deposition, are presented in the article. Additionally, we checked if changing the 2,5-dihydroxybenzoic acid (DHB) and 9-Aminoacridine (9AA) matrix concentration and their solvent composition may improve the analysis. Moreover, the comparison of paraformaldehyde-fixed versus nonfixed cell analysis was performed. We hope that our approach will be helpful for those working on lipid analyses in extraordinary material such as a single oocyte. Our study may also offer clues for anybody interested in single-cell analysis with the aid of MALDI mass spectrometry imaging and the wet-interface matrix deposition method.

An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue.

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time‐of‐flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal‐to‐noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice‐cold EtOH+H2O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 μg/mm2. Including a heat‐induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.

A Matrix Sublimation Device with an Integrated Solvent Nebulizer for MALDI-MSI.

The current matrix deposition methods in MALDI-mass spectrometry imaging (MALDI-MSI) face technical problems related to the inhomogeneous distribution of crystals and the low analyte extraction and cocrystallization efficiency. In this work, an integrated matrix sublimation device with synchronous solvent nebulization was developed for MALDI-MSI. Droplets of solvents were directly introduced into the chamber of the sublimator by using a miniaturized ultrasonic nebulizer unit. The synchronous and asynchronous working modes of solvent nebulization and matrix sublimation were systematically investigated. Imaging of both protein and small metabolite distributions in mouse brain tissue sections was successfully performed using the developed matrix deposition device. The sensitivity and quality of the images were clearly improved in synchronous mode compared with those of the conventional spray and sublimation methods. These results demonstrate that the integrated device with both solvent nebulization and matrix sublimation is a useful tool in MALDI-MSI applications.

MALDI-MS imaging of lipids in corn using a flexible ultrasonic spraying device as matrix deposition method

Matrix application is a critical step in matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) analysis. Here, we describe a low cost, small size, and portable ultrasonic spraying device for various matrix deposition that is ideal for applying to obtain micron-sized (about 1–15 μm) cocrystallisation. The ultrasonic spray method was systematically compared with mainstream matrix application approaches based on optimization experiments. In the approach, ethanol was used as the solvent, and the spray direction was perpendicular to the MALDI sample slide. Seven different matrices were compared and we found that 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) is a more suitable matrix for the detection of triacylglycerol (TAGs) and phosphatidylcholines (PCs) than other matrices. The DCTB matrix solution with concentrations of 5 mg mL−1 was applied on the surfaces of corn sections through ultrasonic spraying. The corn sections were then analysed through MALDI-MS in positive ion mode. MALDI-MSI allowed the simultaneous detection of multiple lipids including 16 TAGs and PCs in corn. The results demonstrated that the ultrasonic spraying device enables the deposition of MALDI matrix solution and provides the advantages of homogeneous micron-sized matrix crystallisation and satisfactory reproducibility.

Identification and imaging of indole-3-carboxamide cannabinoids in hair using matrix-assisted laser-desorption/ionization mass spectrometry

Different kinds of new synthetic cannabinoids (SCs) have been continuously developed to evade drug monitoring. Segmental hair analysis offers a longer period for retrospective drug detection compared with blood or urine. In this study, matrix-assisted laser-desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometric imaging (MALDI-FT ICR MSI) was developed for direct identification and imaging of synthetic indole-3-carboxamide cannabinoids in hair samples using the positive ion mode. The target SCs include N-(adamantan-1-yl)-1-pentyl-1H-indole-3-carboxamide (APICA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-AB-PICA), N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (ADB-PINACA) and N-(1-amino -3,3-dimethyl-1-oxobutan-2-yl)-1-(5-fluoropentyl)-1H-indole-3-carboxamide (5F-ADBICA). The MALDI-MS and MS/MS were first performed on the scraped hair soaked in a mixture of the four SCs after matrix sublimation. This method may provide a detection power for SCs to the 0.1 ng level per 2 cm hair. Target cannabinoids were identified by MS1 and MS2. Matrix deposition methods including airbrush sprayer and sublimation were compared. The method was then applied in revealing the spatial distribution of APICA and 5F-ADBICA in real hair samples from two drug abusers by comparing MS1 and MS2 spectra. The metabolites of APICA and 5F-ADBICA were also presumed to be present in the positive hair samples. Furthermore, a comprehensive comparison between a MALDI-FT ICR MS and a MALDI time-of-flight–MS instrument was performed in detection-sensitivity and specificity for positive real samples. The proposed method provides a powerful tool for drug supervision and forensic medicine analysis in a wide time window, and the sample amount required was also decreased.

Improvement of Sensitivity and Reproducibility for Imaging of Endogenous Metabolites by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) is a powerful technique to visualize the distributions of biomolecules without any labeling. In MALDI-MSI experiments, the choice of matrix deposition method is important for acquiring favorable MSI data with high sensitivity and high reproducibility. Generally, manual or automated spray-coating and automated sublimation methods are used, but these methods have some drawbacks with respect to detection sensitivity, spatial resolution, and data reproducibility. Herein, we present an optimized matrix deposition method of sublimation coupled with recrystallization using 9-aminoacridine (9-AA) as a matrix capable of ionizing endogenous metabolites. The matrix recrystallization process after sublimation was optimized for the solvent concentration and reaction temperature for matrix-metabolite co-crystallization. This optimized method showed excellent reproducibility and spatial resolution compared to the automatic spray-coating method. Furthermore, the recrystallization step after sublimation remarkably improved the detectability of metabolites, including amino acids, nucleotide derivatives, and lipids, compared with the conventional sublimation method. To date, there have been no other reports of 9-AA-based sublimation combined with recrystallization. The present method provides an easy, sensitive, and reproducible matrix deposition method for MALDI-MSI of endogenous metabolites. Graphical Abstract.

Insights into the MALDI Process after Matrix Deposition by Sublimation Using 3D ToF-SIMS Imaging.

Imaging mass spectrometry (IMS) has become a powerful tool to characterize the spatial distribution of biomolecules in thin tissue sections. In the case of matrix-assisted laser desorption ionization (MALDI) IMS, homogeneous matrix deposition is critical to produce high-quality ion images, and sublimation in particular has shown to be an excellent matrix deposition method for the imaging of lipids. Matrix deposition by sublimation is, however, a completely solvent-free system, which ought to prevent the mixing of matrix and analytes thought to be necessary for successful MALDI. Using 3D time-of-flight secondary ion imaging mass spectrometry, we have studied the matrix-tissue interface in 3D with high resolution to understand the MALDI process of lipids after matrix deposition by sublimation. There is a strong indication that diffusion is the process by which lipids migrate from the tissue to the matrix layer. We show that triacylglycerols and phospholipids have a delayed migratory trend as compared to diacylglycerols and monoacylglycerols, which is dependent on time and matrix thickness. Additional experiments show that a pure lipid's capacity to migrate into the matrix is dependent on its fluidity at room temperature. Furthermore, it is shown that cholesterol can only migrate in the presence of a (fluid) lipid and appears to fluidize lipids, which could explain its colocalization with the diacylglycerols and monoacylglycerols in the matrix.

Methodologies for the airbrush application of MALDI matrices

There is still a need to develop reliable and robust matrix deposition methods for matrix-assisted laser desorption/ionisation mass spectrometry that are applicable to a range of matrices, solvents and analyte types. This paper presents a robust methodology for the airbrush application of matrices along with the implications of varying the set-up and airbrush parameters. A small number of organic analytes and metal salts are analysed in both positive and negative ion modes to exemplify this methodology. In the analyses with the airbrush deposited matrices, performance was enhanced when compared to standard pipette deposition with the need for a search for sweat spots greatly diminished due to the increase homogeneity of the matrix surface and resultant analyte spots. As expected, the graphite matrices were shown to specifically outperform the organic matrices in negative ion mode.

Assessing the Potential of Metal-Assisted Imaging Mass Spectrometry in Cancer Research.

In the last decade, imaging mass spectrometry (IMS) has been the primary tool for biomolecular imaging. While it is possible to map a wide range of biomolecules using matrix-assisted laser desorption/ionization IMS ranging from high-molecular-weight proteins to small metabolites, more often than not only the most abundant easily ionisable species are detected. To better understand complex diseases such as cancer more specific and sensitive methods need to be developed to enable the detection of lower abundance molecules but also molecules that have yet to be imaged by IMS. In recent years, a big shift has occurred in the imaging community from developing wide reaching methods to developing targeted ones which increases sensitivity through the use of more specific sample preparations. This has been primarily marked by the advent of solvent-free matrix deposition methods for polar lipids, chemical derivatization for hormones and metabolites, and the use of alternative ionization agents for neutral lipids. In this chapter, we discuss two of the latest sample preparations which exploit the use of alternative ionization agents to enable the detection of certain classes of neutral lipids along with free fatty acids by high-sensitivity IMS as demonstrated within our lab.

Electric Field-Assisted Matrix Coating Method Enhances the Detection of Small Molecule Metabolites for Mass Spectrometry Imaging.

Small molecule metabolites (SMMs, typically <500 Da) are important cellular constituents closely associated with tumor development and progression. However, in situ label-free detection of tissue SMMs has been limited due to interference from matrix and/or low sensitivity. Herein, we develop an electric field-assisted scanning-spraying (EFASS) matrix coating system to deposit matrix on tissue with crystal sizes of <10 μm, followed by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in negative ion mode. A comparison with other matrix deposition methods (i.e., airbrush and sublimation) using common matrixes (i.e., N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC), 9-aminoacridine (9-AA), 2,5-dihydroxybenoic acid (DHB)) indicated that the EFASS system could effectively enhance detection sensitivity and the number of tissue SMMs detected. MSI of five gastric cancer tissues coated with NEDC by the EFASS system demonstrated that significantly increased levels of fatty acids (i.e., palmitic acid and oleic acid) and nucleosides monophosphate (i.e., uridine monophosphate, adenosine monophosphate, and guanosine monophosphate) and significantly decreased levels of nucleosides (i.e., inosine, guanosine, and uridine) and N-acetylneuraminic acid were observed in cancerous areas.

Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry.

For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.

Inkjet-printed microelectrodes on PDMS as biosensors for functionalized microfluidic systems.

Microfluidic systems based on polydimethylsiloxane (PDMS) have gained popularity in recent years. However, microelectrode patterning on PDMS to form biosensors in microchannels remains a worldwide technical issue due to the hydrophobicity of PDMS and its weak adhesion to metals. In this study, an additive technique using inkjet-printed silver nanoparticles to form microelectrodes on PDMS is presented. (3-Mercaptopropyl)trimethoxysilane (MPTMS) was used to modify the surface of PDMS to improve its surface wettability and its adhesion to silver. The modified surface of PDMS is rendered relatively hydrophilic, which is beneficial for the silver droplets to disperse and thus effectively avoids the coalescence of adjacent droplets. Additionally, a multilevel matrix deposition (MMD) method is used to further avoid the coalescence and yield a homogeneous pattern on the MPTMS-modified PDMS. A surface wettability comparison and an adhesion test were conducted. The resulting silver pattern exhibited good uniformity, conductivity and excellent adhesion to PDMS. A three-electrode electrochemical biosensor was fabricated successfully using this method and sealed in a PDMS microchannel, forming a lab-on-a-chip glucose biosensing system.

Some preliminary matrix-assisted laser desorption/ionization imaging experiments on maternal and fetal sides of human placenta.

An investigation on placenta proteins has been carried out by matrix-assisted laser desorption/ionization (MALDI) ion imaging (II) experiments. This was performed by laser irradiation of the maternal and fetal sides of placenta tissue. To investigate the possible changes in protein profile due to the development of gestational diabetes mellitus (GDM), five placenta samples from GDM patients and five placenta samples from healthy pregnant women were analyzed. An extensive optimization of the tissue slice treatment and of the matrix deposition method was performed. As already observed in MALDI spectra of placenta homogenates, and also in the MALDI-II condition, the most abundant peaks are due to hemoglobin α chain, hemoglobin β chain and hemoglobin γ chain. However, higher molecular weight protein species were detected in the m/z range 20,000-47,000. The species at m/z 30335, m/z 31235 and m/z 32000 show some differences in their abundance in the maternal and fetal sides of the tissue in both classes of subjects under investigation. Comparison with the literature data suggest that they can result from the presence of mitochondrial proteins at tissue level.

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.

Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS): A Challenge for Reliable Quantitative Analyses.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is capable of determining the distribution of hundreds of molecules at once directly from tissue sections. Since tissues are analyzed intact without homogenization, spatial relationships of molecules are preserved. The technology is, therefore, undoubtedly powerful to investigate the molecular complexity of biological processes. However, several technical refinements are essential for full exploitation of MALDI-IMS to dictate dynamics alteration of biomolecules in situ; these include ways to collect tissues, target-specific tissue pretreatment, matrix choice for efficient ionization, and matrix deposition method to improve imaging resolution. Furthermore, for MALDI-IMS to reach its full potential, quantitative property in the IMS should be strengthened. We review the challenges and new approaches for optimal imaging of proteins, lipids and metabolites, highlighting a novel quantitative IMS of energy metabolites in the recent literature.

Recent advances in single-cell MALDI mass spectrometry imaging and potential clinical impact

Single-cell analysis is gaining popularity in the field of mass spectrometry as a method for analyzing protein and peptide content in cells. The spatial resolution of MALDI mass spectrometry (MS) imaging is by a large extent limited by the laser focal diameter and the displacement of analytes during matrix deposition. Owing to recent advancements in both laser optics and matrix deposition methods, spatial resolution on the order of a single eukaryotic cell is now achievable by MALDI MS imaging. Provided adequate instrument sensitivity, a lateral resolution of ˜10 µm is currently attainable with commercial instruments. As a result of these advances, MALDI MS imaging is poised to become a transformative clinical technology. In this article, the crucial steps needed to obtain single-cell resolution are discussed, as well as potential applications to disease research.

EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI-TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix-deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix-deposition strategy for LC-MALDI-TOF/TOF MS using an automated instrument that produces a nebulised matrix "mist" under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5-DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix-deposition strategy with LC-MALDI-TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC-ESI-IT-MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor-stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC-MALDI-TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.

Solvent-Free Matrix Dry-Coating for MALDI Imaging of Phospholipids

A fast and simple, solvent-free matrix deposition protocol was developed for positive ionization mode phospholipid analysis in tissues. Finely ground 2,5-dihydroxybenzoic acid was deposited onto sagittal mouse brain sections using a dry-coating technique, in which solid matrix particles were filtered directly onto the tissue through a 20-microm stainless steel sieve. Phospholipid signals were obtained directly off these sections, allowing acquisition of high-resolution MS images. These images were compared to those from serial sections that were spray-coated with a thin-layer chromatography (TLC) reagent sprayer. Signals obtained from the dry matrix deposition method were comparable to those from spray-coated sections, producing identical localization patterns with a simpler and faster sample preparation with virtually no analyte delocalization. This approach was found to yield highly reproducible results, eliminating much of the variance caused by operator differences, and making it an attractive alternative to the currently used matrix application methods.