Mass Spectral Imaging Research Articles

Sphingolipidomics: a valuable tool for understanding the roles of sphingolipids in biology and disease

The sphingolipidome is the portion of the lipidome that encompasses all sphingoid bases and their derivatives. Whereas the most studied sphingoid base is sphingosine [(2S,3R,4E)-2-aminooctadecene-1,3-diol], mammals have dozens of structural variants, and hundreds of additional types have been found in other eukaryotic organisms and some bacteria and viruses. Multiplying these figures by the N-acyl-derivatives ("ceramides") and the more than 500 phospho- and glyco- headgroups places the number of discrete molecular species in the tens of thousands or higher. Structure-specific, quantitative information about a growing fraction of the sphingolipidome can now be obtained using various types of chromatography coupled with tandem mass spectrometry, and application of these methods is producing many surprises regarding sphingolipid structure, metabolism, and function. Such large data sets can be difficult to interpret, but the development of tools that display results from genomic and lipidomic studies in a pathway relational, nodal, context can make it easier for investigators to deal with this complexity.

Direct Profiling and Imaging of Epicuticular Waxes on Arabidopsis thaliana by Laser Desorption/Ionization Mass Spectrometry Using Silver Colloid as a Matrix

Colloidal silver laser desorption/ionization (LDI) mass spectrometry (MS) was employed to directly profile and image epicuticular wax metabolites on a variety of different surfaces of Arabidopsis thaliana leaves and flowers. Major cuticular wax compounds, such as very long-chain fatty acids, alcohols, alkanes, and ketones, were successfully detected as silver adduct ions. The surface metabolites of different flower organs (carpels, petals, and sepals) were profiled for the first time at a spatial resolution of approximately 100 microm. In addition, mass spectral profiles and images were collected from wild type and a mutant strain, which carried alleles that affect the surface constituents of this organism. One of these mutant alleles (cer2-2) is in a gene whose biochemical functionality is still unclear, although its effect on normal epicuticular wax deposition was the characteristic that led to its original identification. Variations of wax products between different spatial locations for wild type and for a mutant strain were investigated by normalizing the ion intensities to a reference peak ([(107)Ag + (109)Ag](+)). The spatially resolved surface metabolite profiling data of this mutant has provided new insights into the complexity of epicuticular wax deposition at the cellular-resolution scale. This MS-based metabolite imaging technology has the potential to provide valuable data for dissecting metabolism in multicellular organism at the level of single cells.

Three Dimensional Mapping of Neuropeptides and Lipids in Crustacean Brain by Mass Spectral Imaging

Imaging mass spectrometry is emerging as a powerful tool that has been applied extensively for the localization of proteins, peptides, pharmaceutical compounds, metabolites, and lipids in biological tissues. In this article, a three-dimensional mass spectral imaging (3D MSI) technique was developed to examine distribution patterns of multiple neuropeptide families and lipids in the brain of the crab Cancer borealis. Different matrix/solvent combinations were compared for preferential extraction and detection of neuropeptides and lipids. Combined with morphological information, the distribution of numerous neuropeptides throughout the 3D structure of brain was determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Different localization patterns were observed for different neuropeptide families, and isoforms displaying unique distribution patterns that were distinct from the common family distribution trends were also detected. In addition, multiple lipids were identified and mapped from brain tissue slices. To confirm their identities, MS/MS fragmentation was performed. Different lipid species displayed distinct localization patterns, suggesting their potential different functional roles in the nervous system.

A New Dynamic in Mass Spectral Imaging of Single Biological Cells

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has unique capabilities in the area of high-resolution mass spectrometric imaging of biological samples. The technique offers parallel detection of native and non-native molecules at physiological concentrations with potentially submicrometer spatial resolution. Recent advances in SIMS technology have been focused on generating new ion sources that can in turn be used to eject more intact molecular and biological characteristic species from a sample. The introduction of polyatomic ion beams, particularly C60, for TOF-SIMS analysis has created a whole new application of molecular depth profiling and 3D molecular imaging. However, such analyses, particularly at high lateral resolution, are severely hampered by the accompanying mass spectrometry associated with current TOF-SIMS instruments. Hence, we have developed an instrument that overcomes many of the drawbacks of current TOF-SIMS spectrometers by removing the need to pulse the primary ion beam. The instrument samples the secondary ions using a buncher that feeds into a specially designed time-of-flight analyzer. We have validated this new instrumental concept by analyzing a number of biological samples generating 2D and 3D images showing molecular localization on a subcellular scale, over a practical time frame, while maintaining high mass resolution. We also demonstrate large area mapping and the MS/MS capability of the instrument.

Laser Ablation Coupled to a Flowing Atmospheric Pressure Afterglow for Ambient Mass Spectral Imaging

A plasma-based ambient desorption/ionization mass spectrometry (ADI-MS) source was used to perform molecular mass spectral imaging. A small amount of sample material was ablated by focusing 266 nm laser light onto a spot. The resulting aerosol was transferred by a nitrogen stream to the flowing afterglow of a helium atmospheric pressure glow discharge ionization source; the ionized sample material was analyzed by a Leco Unique time-of-flight mass spectrometer. Two-dimensional mass spectral images were generated by scanning the laser beam across a sample surface. The total analysis time for a 6 mm (2) surface, which is limited by the washout of the ablation chamber, was less than 30 min. With this technique, a spatial resolution of approximately 20 microm has been achieved. Additionally, the laser ablation configuration was used to obtain depth information of over 2 mm with a resolution of approximately 40 microm. The combination of laser ablation with the flowing atmospheric pressure afterglow source was used to analyze several sample surfaces for a wide variety of analytes and with high sensitivity (LOD of 5 fmol for caffeine).

Mass Spectral Imaging of Glycophospholipids, Cholesterol, and Glycophorin A in Model Cell Membranes

Time of flight secondary ion mass spectrometry (ToF-SIMS) and the Langmuir-Blodgett (LB) technique have been used to create and analyze reproducible membrane mimics of the inner and outer leaflets of a cellular membrane to investigate lipid-protein and lipid-lipid interactions. Films composed of phospholipids, cholesterol and an integral membrane protein were utilized. The results show the outer membrane leaflet mimic (DPPC/cholesterol/glycophorin A LB film) consisting of a single homogeneous phase whereas the inner membrane leaflet mimic (DPPE/cholesterol/glycophorin A LB film) displays heterogeneity in the form of two separate phases. A DPPE/cholesterol phase and a glycophorin A phase. This points to differences in membrane domain formation based upon the different chemical composition of the leaflets of a cell membrane. The reliability of the measurements was enhanced by establishing the influence of the matrix effect upon the measurement and by utlilizing PCA to enhance the contrast of the images.

Automated MALDI Matrix Deposition Method with Inkjet Printing for Imaging Mass Spectrometry

Careful matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is critical for producing reproducible analyte ion signals. Traditional methods for matrix deposition are often considered an art rather than a science, with significant sample-to-sample variability. Here we report an automated method for matrix deposition, employing a desktop inkjet printer (<$200) with 5760 x 1440 dpi resolution and a six-channel piezoelectric head that delivers 3 pL/drop. The inkjet printer tray, designed to hold CDs and DVDs, was modified to hold microscope slides. Empty ink cartridges were filled with MALDI matrix solutions, including DHB in methanol/water (70:30) at concentrations up to 40 mg/mL. Various samples (including rat brain tissue sections and standards of small drug molecules) were prepared using three deposition methods (electrospray, airbrush, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed that matrix crystals were formed evenly across the sample. There was minimal background signal after storing the matrix in the cartridges over a 6-month period. Overall, the mass spectral images gathered from inkjet-printed tissue specimens were of better quality and more reproducible than from specimens prepared by the electrospray and airbrush methods.

Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging

Common organic matrix-assisted laser desorption/ionization (MALDI) matrices, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and alpha-cyano-4-hydroxycinnamic acid, were found to undergo sublimation without decomposition under conditions of reduced pressure and elevated temperature. This solid to vapor-phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temperature, and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid standards robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcrystalline morphology of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible and eliminated the potential for spreading of analytes arising from solvent deposition during matrix application.

Heat-Exchanger Deposits in an Inverted Steam-Assisted Gravity Drainage Operation. Part 2. Organic Acid Analysis by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

As a complement to the bulk analyses presented in part 1 (Jennings, D. W.; Shaikh, A. Energy Fuels 2007, 21, 176−184), we report detailed compositional analysis of the organic acids from heat-exchanger deposits and produced water from an inverted steam-assisted gravity drainage (SAGD) bitumen production process. The current analyses were performed by ultrahigh-resolution negative-ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry [(−) ESI FT-ICR MS] and reveal distributions of molecular weight, heteroatom content, aromaticity, and carbon number for several thousand organic acids in each sample. High mass accuracy combined with Kendrick mass defect analysis allows for assignment of a unique elemental composition, CcHhNnOoSs, to each mass spectral peak. Three-dimensional mass spectral images generated from the molecular formulas reveal compositional differences that may be rationalized on the basis of sample processing. In particular, we report lower overall heteroatom content, higher average carbon number, and lower aromaticity for early stage deposits and the produced bitumen compared to later stage samples that show increased water solubility. Little was previously known about those species other than the presence of organic acid/acid salt functionality. Moreover, we report the first in vacuo isolation and tandem mass spectrometry of individual components of petroleum mixtures. Tandem mass spectrometry by infrared multiphoton dissociation of trapped ions indicates dicarboxylic acid structures for predominant CcHhO4 and dicarboxylic acid structures with thiophenic sulfur for predominant CcHhSO4 species. In this manner, we provide a highly detailed landscape for the organic acid content of inverted SAGD heat-exchanger deposits, to further illuminate the nature of fouling in those systems.

Mass spectral analysis and imaging of tissue by ToF-SIMS—The role of buckminsterfullerene, C 60+, primary ions

Recent developments in desorption/ionisation mass spectrometry techniques have made their application to biological analysis a realistic and successful proposition. Developments in primary ion source technology, mainly through the advent of polyatomic ion beams, have meant that the technique of secondary ion mass spectrometry (SIMS) can now access the depths of information required to allow biological imaging to be a viable option. Here the role of the primary ion C 60 + is assessed with regard to molecular imaging of lipids and pharmaceuticals within tissue sections. High secondary ion yields and low surface damage accumulation are demonstrated on both model and real biological samples, indicating the high secondary ion efficiency afforded to the analyst by this primary ion when compared to other cluster ion beams used in imaging. The newly developed 40 keV C 60 + ion source allows the beam to be focused such that high resolution imaging is demonstrated on a tissue sample, and the greater yields allow the molecular signal from the drug raclopride to be imaged within tissue section following in vivo dosing. The localisation shown for this drug alludes to issues regarding the chemical environment affecting the ionisation probability of the molecule; the importance of this effect is demonstrated with model systems and the possibility of using laser post-ionisation as a method for reducing this consequence of bio-sample complexity is demonstrated and discussed.

Molecule specific imaging in biology: What are the challenges and the important applications?

It is becoming clear that mass spectrometry is a powerful tool for the analysis and imaging of complex organic and biological systems. The discussion session “Meeting the Challenge of Molecule Specific Analysis and Imaging in Organic and Bio-Systems” will attempt to illustrate the challenges to be met in mass spectral imaging and analysis of biological samples. Some of these challenges will be discussed here including the issues of sensitivity needed to obtain high resolution images at the cellular and sub-cellular level, information that can be obtained with alternative techniques and where will they be better versus when SIMS will be better suited to the need, and finally the need for depth profiling and mechanisms to achieve this goal for cellular samples.

Immuohistochemical and MALDI Imaging Reveal Changes in Expression and Phosphorylation of Annexin I and II in Chemical‐Induced Renal Tumors

2,3,5-Tris-(glutathion-S-yl)hydroquinone (TGHQ) is a nephrocarcinogen in the Eker rat, predisposed to renal tumors by a mutation in one allele (Tsc-2EK/+) of the tuberous sclerosis tumor suppressor, Tsc-2, gene. In the present study, both Tsc-2+/+ (wild-type) and Tsc-2EK/+ (mutant) rats were treated with TGHQ (i.p.) for 1–8 months. TGHQ induced cell proliferation in the kidney in the outer stripe of the outer medulla region in both Tsc-2+/+ and Tsc-2EK/+ rats, but only induced tumors in Tsc-2EK/+ rats. TGHQ increased transcription (cDNA microarray) and expression (immunohistochemistry, IHC) of annexin I and II (AI & AII) in injured cells (Tsc-2+/+ and Tsc-2EK/+) and preneoplastic lesions and renal tumors (Tsc-2EK/+). AII appeared in the cytoplasm in injured cells and the membrane in pre- and neoplastic cells, suggesting movement from the cytoplasm to membranes during compensatory proliferation following cell injury. MALDI-TOF mass spectral imaging of rat kidney slices from a TGHQ treated (8 mo) Tsc-2EK/+ and an untreated Tsc-2+/+ rat revealed the same distribution of AI and II as seen with IHC. Moreover an increase in mono- and bisphosphorylated AI and AII were detected in TGHQ treated Tsc-2EK/+ rats by MS-based tissue imaging technique. The role of native and post-translationally modified AI and AII in TGHQ-induced cell injury, cell proliferation and tumor formation is under investigation (GM39338, P30ES06694).

198Infrared & mass spectral imaging of prostate cancer cells

198Infrared & mass spectral imaging of prostate cancer cells

Interpretation of static time-of-flight ion mass spectral images of adsorbed protein films on topographically complex surfaces

In an earlier study, we demonstrated the use of statistical modeling using mixture models and principal components analysis (PCA) in characterizing samples having a relatively simple chemistry and coupled with topographical features [Proc. Am. Vac. Soc. (in press)]. This study involves the extension to analysis of samples with more complex chemistry, such as with proteins. In this study, polystyrene microspheres were adsorbed with different proteins and statistical models such as PCA and mixture models were used to analyze the ToF-SIMS images. Discrimination of protein spectra from the images was then performed using the afore-mentioned techniques, without a priori information about the type of protein adsorbed onto the sphere surfaces. In one set of experiments, each sample was prepared by coating the microspheres with a certain type of protein. Using PCA, we could differentiate between these samples based on their protein spectra. In another experiment, the sample consisted of a set of microspheres, each coated with a different kind of protein. Mixture models were used to contrast between these microspheres on ToF-SIMS images.

Detection of xenophobic response in the periostracum of the bivalve,Corbicula fluminea, through laser-induced mass spectrometry

Aqueous-phased xenobiotic contaminant exposure can biochemically modify newly generated periostracum of the Asian freshwater bivalve,Corbicula fluminea. Laser-induced desorption of partially polymerized periostracum produces spectra distinguishable from mass spectral images generated from uncontaminated periostracum. Organic xenobiotic contamination putatively impedes full polymerization of the periostracin protein. The detection of the effects of pollution on periostracum by the laser microprobe mass analyzer constitutes a novel bioprobe for the definitive but qualified detection of xenobiotic contamination.