Contrast-enhanced differential mobility-desorption electrospray ionization-mass spectrometry imaging of biological tissues.

Mass spectrometry imaging (MSI) performed under ambient conditions is a convenient and information-rich method that allows for the comprehensive mapping of chemical species throughout biological tissues with typical spatial resolution in the 40-200 µm range. Ambient MSI methods such as desorption electrospray ionization (DESI) eliminate necessary sample preparation but suffer from lower spatial resolution than laser-based and vacuum techniques. In order to take advantage of the benefits of ambient imaging and to compensate for the somewhat limited spatial resolution, a secondary orthogonal separation nested in the imaging scheme was implemented for more selective discernment of tissue features in the spectral domain. Differential mobility spectrometry (DMS), an ion mobility-based separation that selectively transmits ions based on their high-to-low electric field mobility differences, can significantly reduce background chemical interferences, allowing for increased peak capacity. In this work, DESI DM-MSI experiments on biological tissue samples such as sea algae and mouse brain tissue sections were conducted using fixed DMS compensation voltages that selectively transferred one or a class of targeted compounds. By reducing chemical noise, the signal-to-noise ratio was improved 10-fold and the image contrast was doubled, effectively increasing image quality.

omniSpect: an open MATLAB-based tool for visualization and analysis of matrix-assisted laser desorption/ionization and desorption electrospray ionization mass spectrometry images.

We present omniSpect, an open source web- and MATLAB-based software tool for both desorption electrospray ionization (DESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) that performs computationally intensive functions on a remote server. These functions include converting data from a variety of file formats into a common format easily manipulated in MATLAB, transforming time-series mass spectra into mass spectrometry images based on a probe spatial raster path, and multivariate analysis. OmniSpect provides an extensible suite of tools to meet the computational requirements needed for visualizing open and proprietary format MSI data.

Enhanced direct ambient analysis by differential mobility-filtered desorption electrospray ionization-mass spectrometry.

Desorption electrospray ionization (DESI) is rapidly becoming established as one of the most powerful ionization techniques allowing direct surface analysis by mass spectrometry (MS) in the ambient environment. DESI provides a significant number of unique analytical capabilities for a broad range of applications, both quantitative and qualitative in nature including biological tissue imaging, pharmaceutical quality control, in vivo analysis, proteomics, metabolomics, forensics, and explosives detection. Despite its growing adoption as a powerful high throughput analysis tool, DESI-MS analysis at trace levels often suffers from background chemical interferences generated during the electrospray ionization processes. In order to improve sensitivity and selectivity, a differential mobility (DM) ion separation cell was successfully interfaced to a custom-built DESI ion source. This new hybrid platform can be operated in two modes: the "DM-off" mode for standard DESI analysis and "DM-on mode" where DESI-generated ions are detected after discrimination by the differential mobility cell. The performance of the DESI-DM-MS platform was tested with several samples typically amenable to DESI analysis, including counterfeit pharmaceuticals and binary mixtures of isobaric chemicals of importance in the pharmaceutical and food industries. In the DM-on mode, DESI-MS signal-to-noise ratios were improved by 70-190% when compared to the DM-off mode. Also, the addition of the DM cell enabled selective in-source ion activation of specific DESI-generated precursor ions, providing tandem MS-like spectra in a single stage mass spectrometer.

Small molecule ambient mass spectrometry imaging by infrared laser ablation metastable-induced chemical ionization.

Presented here is a novel ambient ion source termed infrared laser ablation metastable-induced chemical ionization (IR-LAMICI). IR-LAMICI integrates IR laser ablation and direct analysis in real time (DART)-type metastable-induced chemical ionization for open air mass spectrometry (MS) ionization. The ion generation in the IR-LAMICI source is a two step process. First, IR laser pulses impinge the sample surface ablating surface material. Second, a portion of ablated material reacts with the metastable reactive plume facilitating gas-phase chemical ionization of analyte molecules generating protonated or deprotonated species in positive and negative ion modes, respectively. The successful coupling of IR-laser ablation with metastable-induced chemical ionization resulted in an ambient plasma-based spatially resolved small molecule imaging platform for mass spectrometry (MS). The analytical capabilities of IR-LAMICI are explored by imaging pharmaceutical tablets, screening counterfeit drugs, and probing algal tissue surfaces for natural products. The resolution of a chemical image is determined by the crater size produced with each laser pulse but not by the size of the metastable gas jet. The detection limits for an active pharmaceutical ingredient (acetaminophen) using the IR-LAMICI source is calculated to be low picograms. Furthermore, three-dimensional computational fluid dynamic simulations showed improvements in the IR-LAMICI ion source are possible.

Microplasma discharge ionization source for ambient mass spectrometry.

In this paper, we demonstrate the first use of a microplasma ionization source for ambient mass spectrometry. This device is a robust, easy-to-operate microhollow discharge that enables ambient direct analysis of gaseous, liquid, and solid-phase samples with minimum requirements in terms of operating power and high purity gas consumption. The initial performance of the microplasma device has been evaluated by ionizing samples containing dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methyl salicylate, caffeine, l-leucine, l-histidine, loratadine, ibuprofen, acetaminophen, acetylsalicylic acid, and cocaine in various forms. These molecules are diverse in nature, but almost all have relatively high proton affinities. Thus, the major species observed in all obtained mass spectra corresponded to protonated molecules. Though these microplasmas are known to produce significant densities of metastable species and electrons with mean energies greater than several electronvolt, minimal fragmentation was observed. Background spectra showed prominent signals corresponding to H(+)(H(2)O)(2) ions and a distinct lack of H(3)O(+). Small water cluster ions are likely the dominant proton transfer agents, giving rise to mass spectral data very similar to that obtained using other plasma-based ambient ionization techniques. The simplicity, low cost, low power, low rate of gas consumption, and possibility of being batch-fabricated, makes these microplasma devices attractive candidates as ion sources for miniaturized mass spectrometry and other field detection applications.

Deblurring molecular images using desorption electrospray ionization mass spectrometry.

Traditional imaging techniques for studying the spatial distribution of biological molecules such as proteins, metabolites, and lipids, require the a priori selection of a handful of target molecules. Imaging mass spectrometry provides a means to analyze thousands of molecules at a time within a tissue sample, adding spatial detail to proteomic, metabolomic, and lipidomic studies. Compared to traditional microscopic images, mass spectrometric images have reduced spatial resolution and require a destructive acquisition process. In order to increase spatial detail, we propose a constrained acquisition path and signal degradation model enabling the use of a general image deblurring algorithm. Our analysis shows the potential of this approach and supports prior observations that the effect of the sprayer focuses on a central region much smaller than the extent of the spray.

Desorption electrospray/metastable-induced ionization: a flexible multimode ambient ion generation technique.

Presented here is a novel multimode ambient ion source termed desorption electrospray/metastable-induced ionization (DEMI), which integrates the benefits and circumvents some of the limitations of desorption electrospray ionization (DESI, polarity range limited) and direct analysis in real time (DART)-type metastable-induced chemical ionization (MICI, molecular weight limited). This ion source allows three unique operation modes, each with unique capabilities, including spray (DESI-like)-only, MICI-only, and DEMI (multimode), and can be thus operated in each of these modes allowing the detection of a wider range of analytes of interest. Ion source operation in the MICI-only mode is particularly well suited for the analysis of low-polarity, low-molecular weight compounds in powdered, solid, or dissolved samples. Operation of the ion source in spray-only mode shows superior performance for the analysis of high-molecular weight, high-polarity compounds over the MICI-only mode. Heating the nebulizer gas in spray-only mode allows improved analyte solubility in the spray solvent, enabling up to an order of magnitude improvement in sensitivity. Perhaps the most appealing mode of operation of the ion source is the DEMI mode which allows the simultaneous detection of compounds within a much broader range of polarities and molecular weights than each of the individual modes. For drug quality screening and counterfeit detection applications, operation in the DEMI mode results in the generation of both protonated and sodiated analytes. The observation of such complementary ionic species facilitates compound identification when investigating unknowns.

Beta electron-assisted direct chemical ionization (BADCI) probe for ambient mass spectrometry.

A new low activity (63)Ni ionization technique, beta electron-assisted direct chemical ionization (BADCI) is reported and applied to the analysis of active ingredients in solid pharmaceutical tablets without sample preparation.

Combining two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy, imaging desorption electrospray ionization mass spectrometry, and direct analysis in real-time mass spectrometry for the integral investigation of counterfeit pharmaceuticals.

During the past decade, there has been a marked increase in the number of reported cases involving counterfeit medicines in developing and developed countries. Particularly, artesunate-based antimalarial drugs have been targeted, because of their high demand and cost. Counterfeit antimalarials can cause death and can contribute to the growing problem of drug resistance, particularly in southeast Asia. In this study, the complementarity of two-dimensional diffusion-ordered (1)H nuclear magnetic resonance spectroscopy (2D DOSY (1)H NMR) with direct analysis in real-time mass spectrometry (DART MS) and desorption electrospray ionization mass spectrometry (DESI MS) was assessed for pharmaceutical forensic purposes. Fourteen different artesunate tablets, representative of what can be purchased from informal sources in southeast Asia, were investigated with these techniques. The expected active pharmaceutical ingredient was detected in only five formulations via both nuclear magnetic resonance (NMR) and mass spectrometry (MS) methods. Common organic excipients such as sucrose, lactose, stearate, dextrin, and starch were also detected. The graphical representation of DOSY (1)H NMR results proved very useful for establishing similarities among groups of samples, enabling counterfeit drug "chemotyping". In addition to bulk- and surface-average analyses, spatially resolved information on the surface composition of counterfeit and genuine antimalarial formulations was obtained using DESI MS that was performed in the imaging mode, which enabled one to visualize the homogeneity of both genuine and counterfeit drug samples. Overall, this study suggests that 2D DOSY (1)H NMR, combined with ambient MS, comprises a powerful suite of instrumental analysis methodologies for the integral characterization of counterfeit antimalarials.

Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed.

Organism surfaces represent signaling sites for attraction of allies and defense against enemies. However, our understanding of these signals has been impeded by methodological limitations that have precluded direct fine-scale evaluation of compounds on native surfaces. Here, we asked whether natural products from the red macroalga Callophycus serratus act in surface-mediated defense against pathogenic microbes. Bromophycolides and callophycoic acids from algal extracts inhibited growth of Lindra thalassiae, a marine fungal pathogen, and represent the largest group of algal antifungal chemical defenses reported to date. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging revealed that surface-associated bromophycolides were found exclusively in association with distinct surface patches at concentrations sufficient for fungal inhibition; DESI-MS also indicated the presence of bromophycolides within internal algal tissue. This is among the first examples of natural product imaging on biological surfaces, suggesting the importance of secondary metabolites in localized ecological interactions, and illustrating the potential of DESI-MS in understanding chemically-mediated biological processes.

Noncovalent protein tetramers and pentamers with "n" charges yield monomers with n/4 and n/5 charges.

In recent years mass spectrometry based techniques have emerged as structural biology tools for the characterization of macromolecular, noncovalent assemblies. Many of these efforts involve preservation of intact protein complexes within the mass spectrometer, providing molecular weight measurements that allow the determination of subunit stoichiometry and real-time monitoring of protein interactions. Attempts have been made to further elucidate subunit architecture through the dissociation of subunits from the intact complex by colliding it into inert gas atoms such as argon or xenon. Unfortunately, the amount of structural information that can be derived from such strategies is limited by the nearly ubiquitous ejection of a single, unfolded subunit. Here, we present results from the gas-phase dissociation of protein-protein complexes upon collision into a surface. Dissociation of a series of tetrameric and pentameric proteins demonstrate that alternative subunit fragments, not observed through multiple collisions with gas atoms, can be generated through surface collision. Evidence is presented for the retention of individual subunit structure, and in some cases, retention of noncovalent interactions between subunits and ligands. We attribute these differences to the rapid large energy input of ion-surface collisions, which leads to the dissociation of subunits prior to the unfolding of individual monomers.

Surface-induced dissociation shows potential to be more informative than collision-induced dissociation for structural studies of large systems.

The ability to preserve noncovalent, macromolecular assemblies intact in the gas phase has paved the way for mass spectrometry to characterize ions of increasing size and become a powerful tool in the field of structural biology. Tandem mass spectrometry experiments have the potential to expand the capabilities of this technique through the gas-phase dissociation of macromolecular complexes, but collisions with small gas atoms currently provide very limited fragmentation. One alternative for dissociating large ions is to collide them into a surface, a more massive target. Here, we demonstrate the ability and benefit of fragmenting large protein complexes and inorganic salt clusters by surface-induced dissociation (SID), which provides more extensive fragmentation of these systems and shows promise as an activation method for ions of increasing size.

Surface-induced dissociation of peptides and protein complexes in a quadrupole/time-of-flight mass spectrometer.

A novel in-line surface-induced dissociation (SID) device was designed and implemented in a commercial QTOF instrument (Waters/Micromass QTOF II). This new setup allows efficient SID for a broad range of molecules. It also allows direct comparison with conventional collision-induced dissociation (CID) on the same instrument, taking advantage of the characteristics of QTOF instrumentation, including extended mass range, improved sensitivity, and better resolution compared with quadrupole analyzers and ion traps. Various peptides and a noncovalent protein complex have been electrosprayed and analyzed with the new SID setup. Here we present SID of leucine enkephalin, fibrinopeptide A, melittin, insulin chain-B, and a noncovalent protein complex from wheat, heat shock protein 16.9. The SID spectra were also compared to CID spectra. With the SID setup installed, ion transmission proved to be efficient. SID fragmentation patterns of peptides are, in general, similar to CID, with differences in the relative intensities of some peaks such as immonium ions, backbone cleavage b- versus y-type ions, and y- versus y-NH3 ions, suggesting enhanced accessibility to high-energy/secondary fragmentation channels with SID. Furthermore, these results demonstrate that the in-line SID setup is a valid substitute for CID, with potential advantages for activation of singly/multiply charged peptides and larger species such as noncovalent protein complexes.

Symmetrical gas-phase dissociation of noncovalent protein complexes via surface collisions.

Previous gas-phase dissociation experiments of protein-protein complexes have resulted in product ion distributions that are asymmetric by charge and mass, providing limited insight into the chemical nature of subunit organization and interaction. In these experiments, a symmetric charge distribution results from an "energy sudden" collision of protein-protein complexes with a surface, indicating that it may be possible to probe the suboligomeric structure of noncovalent complexes in the gas phase. It is proposed that energy sudden surface activation of cytochrome C homodimers results in dissociation without significant unfolding of one of the monomeric subunits. Previously proposed mechanisms for the dissociation of protein-protein complexes are discussed in the context of these results. These experiments demonstrate the potential to preserve the structural details of subunit interaction within a protein-protein complex and help elucidate the asymmetric nature of macromolecular dissociation in the gas phase.