Sucrose cryo‐protection facilitates imaging of whole eye sections by MALDI mass spectrometry

Sucrose is used as a cryo‐preservation agent on large mammalian eyes post formalin fixation and is shown to reduce freezing artefacts allowing the collection of 12‐µm thick sections from these large aqueous samples. The suitability of this technique for use in MALDI imaging experiments is demonstrated by the acquisition of the first images of lipid distributions within whole sagittal porcine eye sections. Copyright © 2012 John Wiley & Sons, Ltd.     

Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections

MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of molecular species in tissue specimens. Numerous methodological improvements have been made to optimize tissue section preparation and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the number of signals observed. We systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize experimental variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and number of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystallization behavior as well as their ability to preserve tissue histology were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma biopsy.     

Alkali-hydroxide-doped matrices for structural characterization of neutral underivatized oligosaccharides by MALDI time-of-flight mass spectrometry

We report new approaches using alkali-hydroxide-doped matrices to facilitate structural characterization of neutral underivatized oligosaccharides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS. The approaches involved pretreatment of the analytes with NaOH or LiOH in aqueous solution, followed by mixing them with MALDI matrices prior to MS analysis. It was found that for open-ended neutral underivatized oligosaccharides partial alkaline degradation occurred upon laser desorption and ionization of the hydroxide-pretreated analytes in 2,5-dihydroxybenzoic acid (DHBA). The effect intensified when nonacidic compounds such as 2,4,6-trihydroxyacetophenone (THAP) and 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) were used as matrix. The degradation allowed facile identification of the reducing end residue of the analyte and facilitated its structural characterization by postsource decay TOF-MS. Applying the same technique using matrices composed of LiOH and THAP or AMT led to the production of singly as well as multiple lithiated ions of oligosaccharides containing hexoses with free 3-OH groups. Extensive lithiation through multiple hydrogen-lithium exchanges up to 6 Li atoms was observed for maltoheptaose, beta-cyclodextrin, and dextran 1500. Such a 'lithium tagging' technique makes it possible to differentiate positional isomers of milk-neutral oligosaccharides, lacto-N-difucohexaose I and II (LNDFH-I and LNDFH-II), without the need of chemical derivatization or tandem MS analysis.     

Orthogonal organic and aqueous‐based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous‐based buffer for tissue section preparation before matrix‐assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water–acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14‐day mouse fetus whole‐body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on‐tissue MALDI analysis compared with solely conventional organic rinsing. Copyright © 2013 John Wiley & Sons, Ltd.     

High-resolution MALDI imaging mass spectrometry allows localization of peptide distributions at cellular length scales in pituitary tissue sections

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been used to determine peptide distributions directly from rat, mouse and human pituitary tissue sections. Since these organs are small (10²–10³ μm) the spatial resolution of IMS is a key issue in molecular imaging of pituitary tissue sections. Here we show that high-resolution IMS allows localization of neuropeptide distributions within different cell clusters of a single organ of a pituitary tissue section. The sample preparation protocol does not result in analyte redistribution and is therefore applicable to IMS experiments at cellular length scales. The stigmatic imaging mass spectrometer used in this study produces selected-ion-count images with pixel sizes of 500 nm and a resolving power of 4 μm, yielding superior spatial detail compared to images obtained in microprobe imaging experiments. Furthermore, we show that with imaging mass spectrometry a distinction can be made between different mammalian tissue sections based on differences in the amino acid sequence of neuropeptides with the same function. This example demonstrates the power of IMS for label-free molecular imaging at relevant biological length scales.     

Small molecule analysis by MALDI mass spectrometry

This review focuses on the application of matrix assisted laser desorption/ionization (MALDI) mass spectrometry to the characterization of molecules in the low mass range (<1500 Da). Despite its reputation to the contrary, MALDI is a powerful technique to provide both qualitative and quantitative determination of low molecular weight compounds. Several approaches to minimize interference via sample preparation and matrix selection are discussed, as well as coupling of MALDI to liquid and planar chromatographic techniques to extend its range of applicability.     

Compound and metabolite distribution measured by MALDI mass spectrometric imaging in whole-body tissue sections

The determination of the compound distribution in laboratory animal tissue in early development is a standard process in pharmaceutical research. While this information is traditionally obtained by means of whole-body autoradiography using radiolabeled compounds, this technology does not distinguish between metabolites and parent compound. The technique described in this article, termed matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging, can fill this gap by simultaneously measuring compound and multiple metabolites distributed in whole-body tissue sections, using non-labeled compounds.     

Analysis of erlotinib and its metabolites in rat tissue sections by MALDI quadrupole time-of-flight mass spectrometry

A qualitative and quantitative analysis of erlotinib (RO0508231) and its metabolites was carried out on rat tissue sections from liver, spleen and muscle. Following oral administration at a dose of 5 mg/kg, samples were analyzed by matrix-assisted laser desorption ionization (MALDI) with mass spectrometry (MS) using an orthogonal quadrupole time-of-flight instrument. The parent compound was detected in all tissues analyzed. The metabolites following drug O-dealkylation could also be detected in liver sections. Sinapinic acid (SA) matrix combined with the dried-droplet method resulted in better conditions for our analysis on tissues. Drug quantitation was investigated by the standard addition method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on the tissue extracts. The presence of the parent compound and of its O-demethylated metabolites was confirmed in all tissue types and their absolute amounts calculated. In liver the intact drug was found to be 3.76 ng/mg tissue, while in spleen and muscle 6- and 30-fold lower values, respectively, were estimated. These results were compared with drug quantitation obtained by whole-body autoradiography, which was found to be similar. The potential for direct quantitation on tissue sections in the presence of an internal standard was also investigated using MALDI-MS. The use of alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix resulted in better linearity for the calibration curves obtained with reference solutions of the drug when compared to SA, but on tissue samples no reliable quantitative analysis was possible owing to the large variability in the signal response. MS imaging experiments using MALDI in MS/MS mode allowed visualizing the distribution of the parent compound in liver and spleen tissues. By calculating the ratio between the total ion intensities of MS images for liver and spleen sections, a value of 6 : 1 was found, which is in good agreement with the quantitative data obtained by LC-MS/MS analysis.     

MALDI imaging mass spectrometry of lipids by adding lithium salts to the matrix solution

Mass spectrometry imaging of lipids using MALDI–TOF/TOF mass spectrometers is of growing interest for chemical mapping of organic compounds at the surface of tissue sections. Many efforts have been devoted to the best matrix choice and deposition technique. Nevertheless, the identification of lipid species desorbed from tissue sections remains problematic. It is now well-known that protonated, sodium- and potassium-cationized lipids are detected from biological samples, thus complicating the data analysis. A new sample preparation method is proposed, involving the use of lithium salts in the matrix solution in order to simplify the mass spectra with only lithium-cationized molecules instead of a mixture of various cationized species. Five different lithium salts were tested. Among them, lithium trifluoroacetate and lithium iodide merged the different lipid adducts into one single lithium-cationized species. An optimized sample preparation protocol demonstrated that the lithium trifluoroacetate salt slightly increased desorption of phosphatidylcholines. Mass spectrometry images acquired on rat brain tissue sections by adding lithium trifluoroacetate showed the best results in terms of image contrast. Moreover, more structurally relevant fragments were generated by tandem mass spectrometry when analyzing lithium-cationized species.     

Rapid fingerprinting of red wines by MALDI mass spectrometry

Here we report a simple and fast method for wine fingerprinting based on direct matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis of different red wine samples, useful for batch-to-batch analysis and for the detection of key compounds even in trace amounts which may vary from vintage to vintage, and from one treatment to another one. A series of 20 samples from different wines were subjected to MALDI mass spectrometry. We found that 2,5-dihydroxybenzoic acid is far superior with respect to all the matrices tested To the best of our knowledge this is the first application of an effective wine profiling not limited to detection of anthocyanins. More than 80 molecular species were detected. Moreover, qualitative and quantitative differences were observed, owing to the nature and relative abundance of different chemical compounds among the wines.     

Matrix-dependent cationization in MALDI mass spectrometry

The matrix dependence in cationization processes, the competition between cationization and protonation and the question of whether gas-phase cation transfer or attachment of free cations dominates in matrix-assisted laser desorption/ionization mass spectrometry were studied. Two different sample preparation methods were employed, the dried-droplet sample preparation and a mixture of solid matrix, analyte and salt. The latter ensures that the formation of cation adducts takes place in the gas phase. By monitoring the suppression of matrix signals for different matrices, it was found that matrices with high gas-phase metal ion binding energies require high analyte concentrations for matrix suppression to occur. By comparing the mass spectra obtained using sinapinic acid or sinapinic methyl ester as a matrix, a correlation between cationization and deprotonation of matrix molecules was found. It is also demonstrated that attachment of free gas-phase cations, rather than cation transfer from the cationized matrix, is the predominant process in cationization.     

Uncovering matrix effects on lipid analyses in MALDI imaging mass spectrometry experiments

The specific matrix used in matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) can have an effect on the molecules ionized from a tissue sample. The sensitivity for distinct classes of biomolecules can vary when employing different MALDI matrices. Here, we compare the intensities of various lipid subclasses measured by Fourier transform ion cyclotron resonance (FT‐ICR) IMS of murine liver tissue when using 9‐aminoacridine (9AA), 5‐chloro‐2‐mercaptobenzothiazole (CMBT), 1,5‐diaminonaphthalene (DAN), 2,5‐Dihydroxyacetophenone (DHA), and 2,5‐dihydroxybenzoic acid (DHB). Principal component analysis and receiver operating characteristic curve analysis revealed significant matrix effects on the relative signal intensities observed for different lipid subclasses and adducts. Comparison of spectral profiles and quantitative assessment of the number and intensity of species from each lipid subclass showed that each matrix produces unique lipid signals. In positive ion mode, matrix application methods played a role in the MALDI analysis for different cationic species. Comparisons of different methods for the application of DHA showed a significant increase in the intensity of sodiated and potassiated analytes when using an aerosol sprayer. In negative ion mode, lipid profiles generated using DAN were significantly different than all other matrices tested. This difference was found to be driven by modification of phosphatidylcholines during ionization that enables them to be detected in negative ion mode. These modified phosphatidylcholines are isomeric with common phosphatidylethanolamines confounding MALDI IMS analysis when using DAN. These results show an experimental basis of MALDI analyses when analyzing lipids from tissue and allow for more informed selection of MALDI matrices when performing lipid IMS experiments.     

Monitoring time‐dependent degradation of phospholipids in sectioned tissues by MALDI imaging mass spectrometry

Imaging mass spectrometry (IMS) is useful for visualizing the localization of phospholipids on biological tissue surfaces creating great opportunities for IMS in lipidomic investigations. With advancements in IMS of lipids, there is a demand for large‐scale tissue studies necessitating stable, efficient and well‐defined sample handling procedures. Our work within this article shows the effects of different storage conditions on the phospholipid composition of sectioned tissues from mouse organs. We have taken serial sections from mouse brain, kidney and liver thaw mounted unto ITO‐coated glass slides and stored them under various conditions later analyzing them at fixed time points. A global decrease in phospholipid signal intensity is shown to occur and to be a function of time and temperature. Contrary to the global decrease, oxidized phospholipid and lysophospholipid species are found to increase within 2 h and 24 h, respectively, when mounted sections are kept at ambient room conditions. Imaging experiments reveal that degradation products increase globally across the tissue. Degradation is shown to be inhibited by cold temperatures, with sample integrity maintained up to a week after storage in ?80 °C freezer under N₂ atmosphere. Overall, the results demonstrate a timeline of the effects of lipid degradation specific to sectioned tissues and provide several lipid species which can serve as markers of degradation. Importantly, the timeline demonstrates oxidative sample degradation begins appearing within the normal timescale of IMS sample preparation of lipids (i.e. 1–2 h) and that long‐term degradation is global. Taken together, these results strengthen the notion that standardized procedures are required for phospholipid IMS of large sample sets, or in studies where many serial sections are prepared together but analyzed over time such as in 3‐D IMS reconstruction experiments. Copyright © 2014 John Wiley & Sons, Ltd.     

Direct profiling and imaging of peptides and proteins from mammalian cells and tissue sections by mass spectrometry

Mass spectrometry can be used to map the distribution of targeted compounds in tissue, providing important molecular information in many areas of biological research. Matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) is well suited for the analysis of tissue samples with a spatial resolution of about 30 microm for compounds in a mass range from 1000 to over 50 000 Da. Direct analysis of tissue sections requires spotting or coating of the tissue with a matrix compound typically sinapinic acid or other cinnamic acid analogs. A raster of this sample by the laser beam and subsequent mass analysis of the desorbed ions can record molecular intensities throughout the section. The overall process is illustrated by profiling and imaging of mouse epididymis sections where protein activity changes markedly throughout the section.     

Recent methodological advances in MALDI mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for characterization of large, thermally labile biomolecules. Advantages of this analytical technique are high sensitivity, robustness, high-throughput capacity, and applicability to a wide range of compound classes. For some years, MALDI-MS has also been increasingly used for mass spectrometric imaging as well as in other areas of clinical research. Recently, several new concepts have been presented that have the potential to further advance the performance characteristics of MALDI. Among these innovations are novel matrices with low proton affinities for particularly efficient protonation of analyte molecules, use of wavelength-tunable lasers to achieve optimum excitation conditions, and use of liquid matrices for improved quantification. Instrumental modifications have also made possible MALDI-MS imaging with cellular resolution as well as an efficient generation of multiply charged MALDI ions by use of heated vacuum interfaces. This article reviews these recent innovations and gives the author’s personal outlook of possible future developments.

Decellularization of intact tissue enables MALDI imaging mass spectrometry analysis of the extracellular matrix

Matrix‐assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful molecular mapping technology that offers unbiased visualization of the spatial arrangement of biomolecules in tissue. Although there has been a significant increase in the number of applications employing this technology, the extracellular matrix (ECM) has received little attention, likely because ECM proteins are mostly large, insoluble and heavily cross‐linked. We have developed a new sample preparation approach to enable MALDI IMS analysis of ECM proteins in tissue. Prior to freezing and sectioning, intact tissues are decellularized by incubation in sodium dodecyl sulfate. Decellularization removes the highly abundant, soluble species that dominate a MALDI IMS spectrum while preserving the structural integrity of the ECM. In situ tryptic hydrolysis and imaging of tryptic peptides are then carried out to accommodate the large sizes of ECM proteins. This new approach allows the use of MALDI IMS for identification of spatially specific changes in ECM protein expression and modification in tissue. Copyright © 2015 John Wiley & Sons, Ltd.     

Identification of inorganic and organic microliths in kidney sections by laser microprobe mass spectrometry

Laser microprobe mass spectrometry is used to identify intrarenal microliths; they appear to consist of either oxalate, urate or phosphate. Crystalline and amorphous deposits in rat and human kidney are pin-pointed by the laser beam and their chemical composition determined by mass spectrometry. The method has the potential for wide application in the identification of single organic, inorganic or combination crystals in histological sections.