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.

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.

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.

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.

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 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.

Lactic Acidosis Interferes With Toxicity of Perifosine to Colorectal Cancer Spheroids: Multimodal Imaging Analysis.

Colorectal cancer (CRC) is a disease with constantly increasing incidence and high mortality. The treatment efficacy could be curtailed by drug resistance resulting from poor drug penetration into tumor tissue and the tumor-specific microenvironment, such as hypoxia and acidosis. Furthermore, CRC tumors can be exposed to different pH depending on the position in the intestinal tract. CRC tumors often share upregulation of the Akt signaling pathway. In this study, we investigated the role of external pH in control of cytotoxicity of perifosine, the Akt signaling pathway inhibitor, to CRC cells using 2D and 3D tumor models. In 3D settings, we employed an innovative strategy for simultaneous detection of spatial drug distribution and biological markers of proliferation/apoptosis using a combination of mass spectrometry imaging and immunohistochemistry. In 3D conditions, low and heterogeneous penetration of perifosine into the inner parts of the spheroids was observed. The depth of penetration depended on the treatment duration but not on the external pH. However, pH alteration in the tumor microenvironment affected the distribution of proliferation- and apoptosis-specific markers in the perifosine-treated spheroid. Accurate co-registration of perifosine distribution and biological response in the same spheroid section revealed dynamic changes in apoptotic and proliferative markers occurring not only in the perifosine-exposed cells, but also in the perifosine-free regions. Cytotoxicity of perifosine to both 2D and 3D cultures decreased in an acidic environment below pH 6.7. External pH affects cytotoxicity of the other Akt inhibitor, MK-2206, in a similar way. Our innovative approach for accurate determination of drug efficiency in 3D tumor tissue revealed that cytotoxicity of Akt inhibitors to CRC cells is strongly dependent on pH of the tumor microenvironment. Therefore, the effect of pH should be considered during the design and pre-clinical/clinical testing of the Akt-targeted cancer therapy.

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.

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.

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.

MALDI MS Imaging at Acquisition Rates Exceeding 100 Pixels per Second.

The practicality of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) applied to molecular imaging of biological tissues is limited by the analysis speed. Typically, a relatively low speed of stop-and-go micromotion of XY stages is considered as a factor substantially reducing the rate with which fresh sample material can be supplied to the laser spot. The sample scan rate in our laboratory-built high-throughput imaging TOF mass spectrometer was significantly improved through the use of a galvanometer-based optical scanner performing fast laser spot repositioning on a target plate. The optical system incorporated into the ion source of our MALDI TOF mass spectrometer allowed focusing the laser beam via a modified grid into a 10-µm round spot. This permitted the acquisition of high-resolution MS images with a well-defined pixel size at acquisition rates exceeding 100 pixel/s. The influence of selected parameters on the total MS imaging time is discussed. The new scanning technique was employed to display the distribution of an antitumor agent in 3D colorectal adenocarcinoma cell aggregates; a single MS image comprising 100 × 100 pixels with 10-µm lateral resolution was recorded in approximately 70 s. Graphical Abstract.

Anti-cancer effects of wedelolactone: interactions with copper and subcellular localization.

Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu2+ in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu2+ build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu2+. The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu2+ and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretreated with neocuproine, implying that the complex of WL and Cu2+ is formed inside the cells. WL caused a two-fold increase in the lysosomal level of copper as well as copper-dependent lysosome membrane permeabilization. On the other hand, the protective effects of overexpression of thioredoxin 1 imply that WL exerts the main oxidative impact inside the nucleus. The interactions of WL with copper may be essential for therapeutic performance and selectivity against cancer cells, taking into account that a number of cancer types, including breast cancer, exhibit increased intratumoral copper levels or altered copper distribution.

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.

Thin-layer chromatography combined with diode laser thermal vaporization inductively coupled plasma mass spectrometry for the determination of selenomethionine and selenocysteine in algae and yeast.

In this work we present a simple and cost-effective approach for the determination of selenium species in algae and yeast biomass, based on a combination of thin-layer chromatography (TLC) with diode laser thermal vaporization inductively coupled plasma mass spectrometry (DLTV ICP MS). Extraction of freeze-dried biomass was performed in 4M methanesulphonic acid and the selenium species were vaporized from cellulose TLC plates employing a continuous-wave infrared diode laser with power up to 4 W using a simple laboratory-built apparatus. Selenomethionine and selenocysteine were quantified with limits of detection 3 µg L-1 in a Se-enriched microalgae Chlorella vulgaris and yeast certified reference material SELM-1. Results delivered by TLC-DLTV ICP MS were consistent with those obtained by a routine coupling of high-performance liquid chromatography (HPLC) to ICP MS. In addition, the TLC approach is capable of analyzing extract containing even undiluted crude hydrolysates that could damage HPLC columns.

LC coupled to ESI, MALDI and ICP MS - A multiple hyphenation for metalloproteomic studies.

A new multiple detection arrangement for liquid chromatography (LC) that supplements conventional electrospray ionization (ESI) mass spectrometry (MS) detection with two complementary detection techniques, matrix-assisted laser desorption/ionization (MALDI) MS and substrate-assisted laser desorption inductively coupled plasma (SALD ICP) MS has been developed. The combination of the molecular and elemental detectors in a single separation run is accomplished by utilizing a commercial MALDI target made of conductive plastic. The proposed platform provides a number of benefits in today's metalloproteomic applications, which are demonstrated by analysis of a metallothionein mixture. To maintain metallothionein complexes, separation is carried out at a neutral pH. The effluent is split; a major portion is directed to ESI MS while the remaining 1.8% fraction is deposited onto a plastic MALDI target. Dried droplets are overlaid with MALDI matrix and analysed consecutively by MALDI MS and SALD ICP MS. In the ESI MS spectra, the MT isoform complexes with metals and their stoichiometry are determined; the apoforms are revealed in the MALDI MS spectra. Quantitative determination of metallothionein isoforms is performed via determination of metals in the complexes of the individual protein isoforms using SALD ICP MS.

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.

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.

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.