Mass Spectrometry Imaging Techniques Enabling Visualization of Lipid Isomers in Biological Tissues.

This Feature focuses on a review of recent developments in mass spectrometry imaging (MSI) of lipid isomers in biological tissues. The tandem MS techniques utilizing online and offline chemical derivatization procedures, ion activation techniques such as ozone-induced dissociation (OzID), ultraviolet photodissociation (UVPD), or electron-induced dissociation (EID), and other techniques such as coupling of ion mobility with MSI are discussed. The importance of resolving lipid isomers in diseases is highlighted.

Metal Oxide Laser Ionization Mass Spectrometry Imaging of Fatty Acids and Their Double Bond Positional Isomers.

We present a novel combination of a metal oxide laser ionization mass spectrometry imaging (MOLI MSI) technique with off-line lipid derivatization by ozone for the detection of fatty acids (FA) and their carbon-carbon double bond (C═C) positional isomers in biological tissues. MOLI MSI experiments were realized with CeO2 and TiO2 nanopowders using a vacuum matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer in the negative mode. The catalytic properties of these metal oxides allow FA cleavage from phospholipids under UV laser irradiation. At the same time, fragile ozonides produced at the sites of unsaturation decomposed, yielding four diagnostic ions specific for the C═C positions. Advantageously, two MOLI MSI runs from a single tissue sprayed with the metal oxide suspension were performed. The first run prior to ozone derivatization revealed the distribution of FAs, while the second run after the reaction with ozone offered additional information about FA C═C isomers. The developed procedure was demonstrated on MSI of a normal mouse brain and human colorectal cancer tissues uncovering the differential distribution of FAs down to the isomer level. Compared to the histological analysis, MOLI MSI showed the distinct distribution of specific FAs in different functional parts of the brain and in healthy and cancer tissues pointing toward its biological relevance. The developed technique can be directly adopted by laboratories with MALDI TOF analyzers and help in the understanding of the local FA metabolism in tissues.

Infrared Laser Desorption of Intact Nanoparticles for Digital Tissue Imaging.

We report a new technique for the digital mapping of biomarkers in tissues based on desorption and counting intact gold nanoparticle (Au NP) tags using infrared laser ablation single-particle inductively coupled plasma mass spectrometry (IR LA SP ICP MS). In contrast to conventional UV laser ablation, Au NPs are not disintegrated during the desorption process due to their low absorption at 2940 nm. A mass spectrometer detects up to 83% of Au NPs. The technique is demonstrated on mapping a proliferation marker, nuclear protein Ki-67, in three-dimensional (3D) aggregates of colorectal carcinoma cells, and the results are compared with confocal fluorescence microscopy and UV LA ICP MS. Precise counting of 20 nm Au NPs with a single-particle detection limit in each pixel by the new approach generates sharp distribution maps of a specific biomarker in the tissue. Advantageously, the desorption of Au NPs from regions outside the tissue is strongly suppressed. The developed methodology promises multiplex mapping of low-abundant biomarkers in numerous biological and medical applications using multielemental mass spectrometers.

Metal Ionization in Sub-atmospheric Pressure MALDI Interface: A New Tool for Mass Spectrometry of Volatile Organic Compounds.

A novel approach for the analysis of volatile organic compounds (VOCs) based on chemical ionization by Au+ ions has been proposed. The ionization is carried out in a commercially available dual sub-atmospheric pressure MALDI/ESI interface without any modifications. The Au+ ions are generated by laser ablation of a gold nanolayer with the MALDI laser, and VOCs are infused via the ESI capillary. The ultrahigh resolving power and sub-ppm mass accuracy of the employed mass spectrometer allow straightforward identification of the formed ion-molecule complexes and other products of Au+ interactions with VOCs in the gas phase. The performance of the technique is demonstrated on the analysis of various classes of organic molecules, namely, alkanes, alkenes, alcohols, aldehydes, ketones, aromatic compounds, carboxylic acids, ethers, or organosulfur compounds, expanding the portfolio of currently available methods for the analysis of VOCs such as secondary electrospray ionization, proton-transfer reaction, and selected ion flow tube mass spectrometry.

Ozonization of Tissue Sections for MALDI MS Imaging of Carbon-Carbon Double Bond Positional Isomers of Phospholipids.

Visualizing the differential distribution of carbon-carbon double bond (C═C db) positional isomers of unsaturated phospholipids (PL) in tissue sections by use of refined matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) technologies offers a high promise to deeper understand PL metabolism and isomer-specific functions in health and disease. Here we introduce an on-tissue ozonization protocol that enables a particular straightforward derivatization of unsaturated lipids in tissue sections. Collision-induced dissociation (CID) of MALDI-generated ozonide ions (with yields in the several ten percent range) produced the Criegee fragment ion pairs, which are indicative of C═C db position(s). We used our technique for visualizing the differential distribution of Δ9 and Δ11 isomers of phosphatidylcholines in mouse brain and in human colon samples with the desorption laser spot size 15 µm, emphasizing the potential of the technique to expose local isomer-specific metabolism of PLs.

Drug Penetration Analysis in 3D Cell Cultures Using Fiducial-Based Semiautomatic Coregistration of MALDI MSI and Immunofluorescence Images.

In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and laser scanning confocal microscopy (LSCM). MALDI MSI was chosen to detect the distribution of the drug of interest, while fluorescence immunohistochemistry (IHC) followed by LSCM was used to localize the cells featuring specific markers of viability, proliferation, apoptosis and metastasis. The overlay of the mass spectrometry (MS) and IHC spheroid images, typically without any morphological features, required fiducial-based coregistration. The MALDI MSI protocol was optimized in terms of fiducial composition and antigen epitope preservation to allow MALDI MSI to be performed and directly followed by IHC analysis on exactly the same spheroid section. Once MS and IHC images were coregistered, the quantification of the MS and IHC signals was performed by an algorithm evaluating signal intensities along equidistant layers from the spheroid boundary to its center. This accurate colocalization of MS and IHC signals showed limited penetration of the clinically tested drug perifosine into spheroids during a 24 h period, revealing the fraction of proliferating and promigratory/proinvasive cells present in the perifosine-free areas, decrease of their abundance in the perifosine-positive regions, and distinguishing between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure.

Quantitative Assessment of Anti-Cancer Drug Efficacy From Coregistered Mass Spectrometry and Fluorescence Microscopy Images of Multicellular Tumor Spheroids.

Spheroids-three-dimensional aggregates of cells grown from a cancer cell line-represent a model of living tissue for chemotherapy investigation. Distribution of chemotherapeutics in spheroid sections was determined using the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Proliferating or apoptotic cells were immunohistochemically labeled and visualized by laser scanning confocal fluorescence microscopy (LSCM). Drug efficacy was evaluated by comparing coregistered MALDI MSI and LSCM data of drug-treated spheroids with LSCM only data of untreated control spheroids. We developed a fiducial-based workflow for coregistration of low-resolution MALDI MS with high-resolution LSCM images. To allow comparison of drug and cell distribution between the drug-treated and untreated spheroids of different shapes or diameters, we introduced a common diffusion-related coordinate, the distance from the spheroid boundary. In a procedure referred to as "peeling", we correlated average drug distribution at a certain distance with the average reduction in the affected cells between the untreated and the treated spheroids. This novel approach makes it possible to differentiate between peripheral cells that died due to therapy and the innermost cells which died naturally. Two novel algorithms-for MALDI MS image denoising and for weighting of MALDI MSI and LSCM data by the presence of cell nuclei-are also presented.

A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer's disease drug target ß-secretase.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of ß-secretase (BACE1), a key enzyme of the progression of Alzheimer's disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method's applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research. Graphical abstract ᅟ.

Wedelolactone Acts as Proteasome Inhibitor in Breast Cancer Cells.

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

Direct Analysis of Gold Nanoparticles from Dried Droplets Using Substrate-Assisted Laser Desorption Single Particle-ICPMS.

Single particle inductively coupled plasma mass spectrometry (SP-ICPMS) has been generally accepted as a powerful tool in the field of nanoanalysis. The method has usually been restricted to direct nanoparticle (NP) introduction using nebulization or microdroplet generation systems. In this work, AuNPs are introduced into ICPMS by substrate-assisted laser desorption (SALD) directly from a suitable absorbing plastic surface using a commercial ablation cell for the first time. In SALD, desorption of individual NPs is mediated using a frequency-quintupled Nd:YAG laser (213 nm) operated at a rather low laser fluence. Conditions including laser fluence, laser beam scan rate, and carrier gas flow rate were optimized in order to gain the highest AuNP transport efficiency and avoid AuNP disintegration within the laser irradiation. The method was demonstrated on a well-characterized reference material, 56 nm AuNPs with a transport efficiency of 61% and commercially available 86 nm AuNPs. Feasibility of our technique for NP detection and characterization is discussed here, and the results are compared with an established technique, nebulizer SP-ICPMS.

MALDI MS and ICP MS detection of a single CE separation record: a tool for metalloproteomics.

In this work, a novel approach based on off-line coupling of a single run of capillary electrophoresis (CE) separation to both matrix-assisted laser desorption/ionization (MALDI) and substrate-assisted laser desorption inductively coupled plasma (SALD ICP) mass spectrometry (MS) is presented. Using a liquid junction and subatmospheric deposition chamber, CE fractions were extracted from a separation capillary and collected as 20-nL droplets on a custom-built polyethylene terephthalate glycol (PETG) target plate coated with a 10-nm gold layer which guaranteed compatibility with both MALDI and SALD ICP techniques. The MALDI matrix solution was then added to the produced spots. After it was dried, the separation record was consecutively analyzed in MALDI MS and ICP MS instruments. Thus, both proteomic and metallomic information was obtained off-line from a single CE run. The concept was demonstrated by the analysis of a mixture of rabbit liver metallothionein isoforms. In an additional study, the droplets representing the archived separation record were alternately mixed with two different MALDI matrices to obtain complementary information on both the apoproteins and their complexes with metals from a single separation run. The presented technique is a viable alternative to online coupling of column separation to electrospray MS and nebulizer ICP MS.

Rapid matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging with scanning desorption laser beam.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) imaging of surfaces and tissues is a rapidly evolving technique having great potential in the field of biosciences. In earlier times, acquisition of a single high-resolution MS image could take days. Despite the recent introduction of high-repetition rate lasers to increase sample throughput of axial TOF MS instruments, obtaining a high-resolution image still requires a few hours. This paper shows that a substantial increase in the throughput of the TOF MS-based tissue imaging can be achieved by incorporating a mirror providing high-speed precision scanning of the laser beam along the sample surface. Equipped with the scanning mirror, a laboratory-built axial MALDI TOF MS instrument utilizing a 4-kHz UV laser recorded a 100 × 100 pixel MS image in ~11 min using 100 laser shots per pixel. This is almost an order of magnitude faster when compared to a modern commercial instrument equipped with 1-kHz laser.

Comparison of Cu+, Ag+, and Au+ Ions as Ionization Agents of Volatile Organic Compounds at Subatmospheric Pressure.

Ionization of volatile organic compounds (VOCs) by coinage metal ions (Cu+, Ag+, and Au+) generated by laser desorption and ionization (LDI) of a metal nanolayer in subatmospheric conditions is explored. The study was performed in a commercial subatmospheric dual MALDI/ESI ion source. Five compounds representing different VOC classes were chosen for a detailed study of the metal ionization mechanism: ethanol, acetone, acetic acid, xylene, and cyclohexane. In the gas phase, ion molecular complexes of all three metal ions were formed, typically with two ligand molecules. The successful detection of the metal complexes with VOCs strongly depended on the applied voltages across the ion source, minimizing the in-source fragmentation. The employed orbital trap with ultrahigh resolving power and sub-parts-per-million mass accuracy allowed unambiguous identification of the formed complexes based on their molecular formulas. The detection limits of the studied compounds in the gas were in the range 0.1-1.4 nmol/L. Compared to Cu+ and Ag+ ions, Au+ ions exhibited the highest reactivity, often complicating spectra by side products of reactions. On the other hand, they also allowed detecting saturated hydrocarbons, which did not produce any signals with Ag+ and Cu+.

Detection of Single Ag Nanoparticles Using Laser Desorption/Ionization Mass Spectrometry.

The detection of a single entity (molecule, cell, particle, etc.) was always a challenging subject. Here we demonstrate the detection of single Ag nanoparticles (NPs) using subatmospheric pressure laser desorption/ionization mass spectrometry (LDI MS). The sample preparation, measurement conditions, generated ions, and limiting experimental factors are discussed here. We detected from 84 to 95% of the deposited 80 nm Ag NPs. The presented LDI MS platform is an alternative to laser ablation inductively coupled plasma mass spectrometry for imaging distribution of individual NPs across the sample surface and has a great potential for multiplexed mapping of low-abundance biomarkers in tissues.

Fate of Gold Nanoparticles in Laser Desorption/Ionization Mass Spectrometry: Toward the Imaging of Individual Nanoparticles.

This study focuses on mapping the spatial distribution of Au nanoparticles (NPs) by laser desorption/ionization mass spectrometry imaging (LDI MSI). Laser interaction with NPs and associated phenomena, such as change of shape, melting, migration, and release of Au ions, are explored at the single particle level. Arrays of dried droplets containing low numbers of spatially segregated NPs were reproducibly prepared by automated drop-on-demand piezo-dispensing and analyzed by LDI MSI using an ultrahigh resolution orbital trapping instrument. To enhance the signal from NPs, an in source gas-phase chemical reaction of generated Au ions with xylene was employed. The developed technique allowed the detecting, chemical characterization, and mapping of the spatial distribution of Au NPs; the ion signals were detected from as low as ten 50 nm Au NPs on a pixel. Furthermore, the Au NP melting dynamics under laser irradiation was monitored by correlative atomic force microscopy (AFM) and scanning electron microscopy (SEM). AFM measurements of Au NPs before and after LDI MSI analysis revealed changes in NP shape from a sphere to a half-ellipsoid and total volume reduction of NPs down to 45% of their initial volume.

Diode laser thermal vaporization inductively coupled plasma mass spectrometry.

An approach of sample introduction for inductively coupled mass spectrometry (ICPMS), diode laser thermal vaporization (DLTV) is described. The method allows quantitative determination of metals in submicroliter volumes of liquid samples. Laser power is sufficient to induce pyrolysis of a suitable substrate with the deposited sample leading to aerosol generation. Unlike existing sample introduction systems based on laser ablation, it uses a NIR diode laser rather than an expensive high-energy pulsed laser. For certain elements, this sample introduction technique may serve as an alternative to solution analysis with conventional nebulizers. Using a prearranged calibration set, DLTV ICPMS provides rapid and reproducible sample analysis (RSD ~ 10%). Sample preparation is fast and simple, and the prepared samples can easily be archived and transported. The limits of detection for Co, Ni, Zn, Mo, Cd, Sn, and Pb deposited on the preprinted paper were found to be in the range of 0.4-30 pg. The method was characterized, optimized, and applied to the determination of Co in a drug preparation, Pb in whole blood, and Sn in food samples without any sample pretreatment.

Detection of melamine in infant formula and grain powder by surface-assisted laser desorption/ionization mass spectrometry.

We have developed a method for the determination of melamine (MEL), ammeline (AMN), and ammelide (AMD) by surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs). The major peaks for MEL, AMN, and AMD at m/z 127.07, 128.05, and 129.04 are assigned to the [MEL + H](+), [AMN + H](+), and [AMD + H](+) ions. Because the three tested compounds adsorb weakly onto the surfaces of the Au NPs through Au-N bonding, they can be easily concentrated from complex samples by applying a simple trapping/centrifugation process. The SALDI-MS method provides limits of detection of 5, 10, and 300 nM for MEL, AMN, and AMD, respectively, at a signal-to-noise ratio of 3. The signal variation for 150-shot average spectra of the three analytes within the same spot was 15%, and the batch-to-batch variation was 20%. We have validated the practicality of this approach by the analysis of these three analytes in infant formula and grain powder. This simple and rapid SALDI-MS approach holds great potential for screening of MEL in foods.

Disulfiram increases the efficacy of 5-fluorouracil in organotypic cultures of colorectal carcinoma.

Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.

Combined contactless conductometric, photometric, and fluorimetric single point detector for capillary separation methods.

This work for the first time combines three on-capillary detection methods, namely, capacitively coupled contactless conductometric (C(4)D), photometric (PD), and fluorimetric (FD), in a single (identical) point of detection cell, allowing concurrent measurements at a single point of detection for use in capillary electrophoresis, capillary electrochromatography, and capillary/nanoliquid chromatography. The novel design is based on a standard 6.3 mm i.d. fiber-optic SMA adapter with a drilled opening for the separation capillary to go through, to which two concentrically positioned C(4)D detection electrodes with a detection gap of 7 mm were added on each side acting simultaneously as capillary guides. The optical fibers in the SMA adapter were used for the photometric signal (absorbance), and another optical fiber at a 45 degrees angle to the capillary was applied to collect the emitted light for FD. Light emitting diodes (255 and 470 nm) were used as light sources for the PD and FD detection modes. LOD values were determined under flow-injection conditions to exclude any stacking effects: For the 470 nm LED limits of detection (LODs) for FD and PD were for fluorescein (1 x 10(-8) mol/L) and tartrazine (6 x 10(-6) mol/L), respectively, and the LOD for the C(4)D was for magnesium chloride (5 x 10(-7) mol/L). The advantage of the three different detection signals in a single point is demonstrated in capillary electrophoresis using model mixtures and samples including a mixture of fluorescent and nonfluorescent dyes and common ions, underivatized amino acids, and a fluorescently labeled digest of bovine serum albumin.