Analysis of secondary plant metabolites by indirect desorption electrospray ionization imaging mass spectrometry

Secondary metabolites in plant material can be imaged in a simple and robust way by creating an imprint of the plant material on a porous Teflon surface. The Teflon surface serves to extract compounds from the plant material for enhanced desorption electrospray ionization imaging analysis, while maintaining the spatial information of the sample. The method, which remedies for limitations in mass spectrometry imaging of compounds embedded in plant material, was demonstrated on leaves and petals of Hypericum perforatum and leaves of Datura stramonium.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

Versatile platform employing desorption electrospray ionization mass spectrometry for high-throughput analysis

A simple and easy-to-build high-throughput analysis system was constructed. The system consisted of three major components: (1) a multichannel device with 16 parallel capillaries, (2) a desorption electrospray ionization (DESI) source, and (3) a linear ion trap mass spectrometer. When analyses were performed, the multichannel device was moved horizontally on a translation stage controlled by a step motor. Our design expands the functions of DESI, in which the liquid sample in capillary was driven out by the nebulizing gas, ionized, and then transferred to a mass spectrometer. To assess the high-throughput performance of the system, 5 mg/L 1,3-diethyl-1,3-diphenylurea (DDU) solution and 10 mg/L angiotensin I solution were alternatively loaded into the reservoirs and capillaries in the multichannel device. Results indicated that analyses of the all the samples in 16 capillaries were completed within 1.6 min, which means a throughput of 600 samples/h. Reactive DESI experiment was also successfully performed with this system to show the feasibility of online derivatization. The relative standard deviations for a single capillary and five identical capillaries were 7.6 (n = 16) and 12.3%, respectively. Linear relative abundance response was achieved for DDU (r = 0.9971).     

Droplet dynamics and ionization mechanisms in desorption electrospray ionization mass spectrometry

A droplet pickup and other mechanisms have been suggested for the ionization of biomolecules like peptides and proteins by desorption electrospray ionization. To verify this hypothesis phase Doppler particle analysis was used to study the sizes and velocities of droplets involved in DESI. It was found that impacting droplets typically have velocities of 120 m/s and average diameters of 2-4 microm. Small differences in sprayer construction influence the operating conditions at which droplets of these dimensions are produced. Under these conditions, the kinetic energy per impacting water molecule is less than 0.6 meV and sputtering through momentum transfer during collisions or ionization by other electronic processes is unlikely. Droplets arrive at the surface with velocities well below the speed of sound in common materials, thereby excluding the possibility of ionization by shockwave formation. Some droplets appear to roll along the surface, increasing contact time and presumably the amount of material that is taken up into droplets during conditions typical of the DESI experiment.     

Carbohydrate analysis by desorption electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

We report the use of desorption electrospray ionization hybrid Fourier transform ion cyclotron resonance mass spectrometry (DESI-FT-ICR-MS) for the analysis of carbohydrates. Spectra of neat carbohydrates are presented along with their mass measurement accuracies and limits of detection. Furthermore, a comparison is made between the analyses of O-linked glycans from mucin by DESI-FT-ICR-MS and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. Finally, glycans from mucin are identified by using the high mass measurement accuracy and tandem MS capabilities afforded by the hybrid FT-ICR-MS platform.     

Thin-layer chromatography and mass spectrometry coupled using desorption electrospray ionization

Desorption electrospray ionization (DESI) was demonstrated as a means to couple thin-layer chromatography (TLC) with mass spectrometry. The experimental setup and its optimization are described. Development lanes were scanned by moving the TLC plate under computer control while directing the stationary DESI emitter charged droplet plume at the TLC plate surface. Mass spectral data were recorded in either selected reaction monitoring mode or in full scan ion trap mode using a hybrid triple quadrupole linear ion trap mass spectrometer. Fundamentals and practical applications of the technique were demonstrated in positive ion mode using selected reaction monitoring detection of rhodamine dyes separated on hydrophobic reversed-phase C8 plates and reversed-phase C2 plates, in negative ion full scan mode using a selection of FD&C dyes separated on a wettable reversed-phase C18 plate, and in positive ion full scan mode using a mixture of aspirin, acetaminophen, and caffeine from an over-the-counter pain medication separated on a normal-phase silica gel plate.     

Mapping lipid alterations in traumatically injured rat spinal cord by desorption electrospray ionization imaging mass spectrometry

Desorption electrospray ionization (DESI) mass spectrometry (MS) is used in an imaging mode to interrogate the lipid profiles of 15 μm thin tissue cross sections of injured rat spinal cord and normal healthy tissue. Increased relative intensities of fatty acids, diacylglycerols, and lysolipids (between +120% and +240%) as well as a small decrease in intensities of lipids (-30%) were visualized in the lesion epicenter and adjacent areas after spinal cord injury. This indicates the hydrolysis of lipids during the demyelination process due to activation of phospholipase A(2) enzyme. In addition, signals corresponding to oxidative degradation products, such as prostaglandin and hydroxyeicosatetraenoic acid, exhibited increased signal intensity by a factor of 2 in the negative ion mode in lesions relative to the normal healthy tissue. Analysis of malondialdehyde, a product of lipid peroxidation and marker of oxidative stress, was accomplished in the ambient environment using reactive DESI mass spectrometry imaging. This was achieved by electrospraying reagent solution containing dinitrophenylhydrazine as high-velocity charged droplets onto the tissue section. The hydrazine reacts selectively and rapidly with the carbonyl groups of malondialdehyde, and signal intensity of twice the intensity was detected in the lesions compared to healthy spinal cord. With a small amount of tissue sample, DESI-MS imaging provides information on the composition and distribution of specific compounds (limited by the occurrence of isomeric lipids with very similar fragmentation patterns) in lesions after spinal cord injury in comparison with normal healthy tissue allowing identification of the extent of the lesion and its repair.     

Direct ionization of large proteins and protein complexes by desorption electrospray ionization-mass spectrometry

Desorption electrospray ionization-mass spectrometry (DESI-MS) has advantages for rapid sample analysis with little or no sample pretreatment, but performance for large biomolecules has not been demonstrated. In this study, liquid sample DESI, an extended version of DESI used for analysis of liquid samples, was shown to have capabilities for direct ionization of large noncovalent protein complexes (>45 kDa) and proteins (up to 150 kDa). Protein complex ions (e.g., superoxide dismutase, enolase, and hemoglobin) desorbed from solution by liquid sample DESI were measured intact, indicating the capability of DESI for preserving weak noncovalent interactions. Doping the DESI spray solvent with supercharging reagents resulted in protein complex ions having increased multiple charging without complex dissociation. Ion mobility measurements of model protein cytochrome c showed that the supercharging reagent favored the more compact conformation for the lower charged protein ions. Liquid sample DESI of hydrophobic peptide gramicidin D suggests that the ionization mechanism involves a droplet pick-up mixing process. Measurement of liquid samples significantly extends the mass range of DESI-MS, allowing the analysis of high-mass proteins such as 150 kDa immunoglobulin G (IgG) and thus represents the largest protein successfully ionized by DESI to date.     

Localization and quantification of drugs in animal tissues by use of desorption electrospray ionization mass spectrometry imaging

Mass spectrometric imaging (MSI) has emerged as a powerful technique to obtain spatial arrangement of individual molecular ions in animal tissues. Ambient desorption electrospray ionization (DESI) technique is uniquely suited for such imaging experiments, as it can be performed on animal tissues in their native environment without prior treatments. Although MSI has become a rapid growing technique for localization of proteins, lipids, drugs, and endogenous compounds in different tissues, quantification of imaged targets has not been explored extensively. Here we present a novel MSI approach for localization and quantification of drugs in animal thin tissue sections. DESI-MSI using an Orbitrap mass analyzer in full scan mode was performed on 6 μm coronal brain sections from rats that were administered 2.5 mg/kg clozapine. Clozapine was localized and quantified in individual brain sections 45 min postdose. External calibration curves were prepared by micropipetting standards with internal standard (IS) on top of the tissues, and average response factors were calculated for the scans in which both clozapine and IS were detected. All response factors were normalized to area units. Quantifications from DESI-MSI revealed 0.2-1.2 ng of clozapine in individual brain sections, results that were further confirmed by extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis.     

Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization

Reaction kinetics studied by mass spectrometry (MS) has previously been limited to millisecond time resolution. This paper presents the development of a submillisecond time-resolved mass spectrometric method for fast reaction kinetic study, based on the capability of desorption electrospray ionization (DESI) for direct and fast ionization of a high-speed liquid jet stream. The principle underlying this methodology is that two reactant solutions undergo rapid mixing to produce a free liquid jet which is ionized by DESI at different positions corresponding to different reaction times. Due to the high velocity of the liquid jet, high time resolution can be achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol (DCIP) and L-ascorbic acid (L-AA) was chosen as an example to demonstrate this concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 μs. The good agreement of the measured value of (116 ± 3) s(-1) with that measured by the stopped-flow optical method (105 ± 2) s(-1) validates the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.     

Quantitative analysis with desorption/ionization on silicon mass spectrometry using electrospray deposition

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is demonstrated as a quantitative analytical tool when coupled to electrospray deposition (ESD). In this study, we illustrate the utility of DIOS-MS in the quantitative analysis of a peptide and two amino acids with deuterated and structural analogues used as internal standards. An important feature of this approach is the incorporation of ESD to improve sample homogeneity across the porous silicon surface. ESD allowed for a marked improvement in quantitative analysis due to its applicability to LC-DIOS, and because of the absence of matrix, sample can be deposited at very low flow rates (150 nL/min). Experiments comparing the traditional dried droplet and ESD methods show that ESD samples exhibit significantly improved quantitation and much higher sample-to-sample reproducibility.     

Interfacing capillary-based separations to mass spectrometry using desorption electrospray ionization

The powerful hybrid analysis method of capillary-based separations followed by mass spectrometric analysis gives substantial chemical identity and structural information. It is usually carried out using electrospray ionization. However, the salts and detergents used in the mobile phase for electrokinetic separations suppress ionization efficiencies and contaminate the inlet of the mass spectrometer. This report describes a new method that uses desorption electrospray ionization (DESI) to overcome these limitations. Effluent from capillary columns is deposited on a rotating Teflon disk that is covered with paper. As the surface rotates, the temporal separation of the eluting analytes (i.e., the electropherogram) is spatially encoded on the surface. Then, using DESI, surface-deposited analytes are preferentially ionized, reducing the effects of ion suppression and inlet contamination on signal. With the use of this novel approach, two capillary-based separations were performed: a mixture of the rhodamine dyes at milligram/milliliter levels in a 10 mM sodium borate solution was separated by capillary electrophoresis, and a mixture of three cardiac drugs at milligram/milliliter levels in a 12.5 mM sodium borate and 12.5 mM sodium dodecyl sulfate solution was separated by micellar electrokinetic chromatography. In both experiments, the negative effects of detergents and salts on the MS analyses were minimized.     

Chemical analysis of complex organic mixtures using reactive nanospray desorption electrospray ionization mass spectrometry

Reactive nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry was utilized for the analysis of secondary organic aerosol produced through ozonolysis of limonene (LSOA). Previous studies have shown that LSOA constituents are multifunctional compounds containing at least one aldehyde or ketone groups. In this study, we used the selectivity of the Girard's reagent T (GT) toward carbonyl compounds to examine the utility of reactive nano-DESI for the analysis of complex organic mixtures. In these experiments, 1-100 μM GT solutions were used as the working solvents for reactive nano-DESI analysis. Abundant products from the single addition of GT to LSOA constituents were observed at GT concentrations in excess of 10 μM. We found that LSOA dimeric and trimeric compounds react with GT through a simple addition reaction resulting in formation of the carbinolamine derivative. In contrast, reactions of GT with monomeric species result in the formation of both the carbinolamine and the hydrazone derivatives. In addition, several monomers did not react with GT on the time scale of our experiment. These molecules were characterized by relatively high values of the double bond equivalent and low oxygen content. Furthermore, because addition of a charged GT tag to a neutral molecule eliminates the discrimination against the low proton affinity compounds in the ionization process, reactive nano-DESI analysis enables quantification of individual compounds in the complex mixture. For example, we were able to estimate for the first time the amounts of dimers and trimers in the LSOA mixture. Specifically, we found that the most abundant LSOA dimer was detected at the ～0.5 pg level and the total amount of dimers and trimers in the analyzed sample was ～11 pg. Our results indicate that reactive nano-DESI is a valuable approach for examining the presence of specific functional groups and for the quantification of compounds possessing these groups in complex mixtures.     

Study of electrochemical reactions using nanospray desorption electrospray ionization mass spectrometry

The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool for studying mechanisms of redox reactions, identification of products and intermediates, and online derivatization/recognition of analytes. This work reports a new coupling interface for EC/MS by employing nanospray desorption electrospray ionization, a recently developed ambient ionization method. We demonstrate online coupling of nanospray desorption electrospray ionization MS with a traditional electrochemical flow cell, in which the electrolyzed solution emanating from the cell is ionized by nanospray desorption electrospray ionization for MS analysis. Furthermore, we show first coupling of nanospray desorption electrospray ionization MS with an interdigitated array (IDA) electrode enabling chemical analysis of electrolyzed samples directly from electrode surfaces. Because of its inherent sensitivity, nanospray desorption electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 μL of 1 μM solution of dopamine on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization as a novel interface for electrochemical mass spectrometry research.     

Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry

A method for the determination of underivatized amino acids based on capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) is described. To analyze free amino acids simultaneously a low acidic pH condition was used to confer positive charge on whole amino acids. The choice of the electrolyte and its concentration influenced resolution and peak shape of the amino acids, and 1 M formic acid was selected as the optimal electrolyte. Meanwhile, the sheath liquid composition had a significant effect on sensitivity and the highest sensitivity was obtained when 5 mM ammonium acetate in 50% (v/v) methanol-water was used. Protonated amino acids were roughly separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow electrospray ionization interface. Under the optimized conditions, 19 free amino acids normally found in proteins and several physiological amino acids were well determined in less than 17 min. The detection limits for basic amino acids were between 0.3 and 1.1 mumol/L and for acidic and low molecular weight amino acids were less than 6.0 mumol/L with pressure injection of 50 mbar for 3 s (3 nL) at a signal-to-noise ratio of 3. This method is simple, rapid, and selective compared with conventional techniques and could be readily applied to the analysis of free amino acids in soy sauce.     

Automated sampling and imaging of analytes separated on thin-layer chromatography plates using desorption electrospray ionization mass spectrometry

Modest modifications to the atmospheric sampling capillary of a commercial electrospray mass spectrometer and upgrades to an in-house-developed surface positioning control software package (HandsFree TLC/MS) were used to enable the automated sampling and imaging of analytes on and within large area surface substrates using desorption electrospray ionization mass spectrometry. Sampling and imaging of rhodamine dyes separated on TLC plates were used to illustrate some of the practical applications of this system. Examples are shown for user-defined spot sampling from separated bands on a TLC plate (one or multiple spots), scanning of a complete development lane (one or multiple lanes), or imaging of analyte bands in a development lane (i.e., multiple lane scans with close spacing). The post data acquisition processing and data display aspects of the software system are also discussed.     

Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry

Mass spectrometry in conjunction with atmospheric pressure ionization methods enables the in vivo investigation of biochemical changes with high specificity and sensitivity. Laser ablation electrospray ionization (LAESI) is a recently introduced ambient ionization method suited for the analysis of biological samples with sufficient water content. With LAESI mass spectrometric analysis of chimeric Aphelandra squarrosa leaf tissue, we identify the metabolites characteristic for the green and yellow sectors of variegation. Significant parts of the related biosynthetic pathways (e.g., kaempferol biosynthesis) are ascertained from the detected metabolites and metabolomic databases. Scanning electron microscopy of the ablated areas indicates the feasibility of both two-dimensional imaging and depth profiling with a approximately 350 microm lateral and approximately 50 microm depth resolution. Molecular distributions of some endogenous metabolites show chemical contrast between the sectors of variegation and quantitative changes as the ablation reaches the epidermal and mesophyll layers. Our results demonstrate that LAESI mass spectrometry opens a new way for ambient molecular imaging and depth profiling of metabolites in biological tissues and live organisms.     

Direct analysis of lipids in single zooplankter individuals by matrix-assisted laser desorption/ionization mass spectrometry

A highly sensitive method to analyze the intact lipids in a single zooplankter individual at the level of a few tenths of a microgram was developed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with a direct sampling technique. The sampling procedure involved (1) putting a zooplankter individual sample onto the MALDI sample plate, (2) cutting the sample into a few pieces by means of tweezers, (3) depositing aliquots of matrix and cationization reagent solutions on the zooplankter sample, and (4) irradiating with a N2 laser to cause MALDI. By using this technique, the mass spectra of the single zooplankter samples showed a series of ions generated from phospholipids with 34 or 36 carbons in the acyl groups and neutral lipids such as triglycerides and diacylglyceryl ethers with 50-54 carbons in their acyl and alkenyl groups. Accordingly, this method enabled us to estimate the relative quantity between "structured lipids" (phospholipids) and "storage lipids" (neutral lipids) in an individual zooplankter, which should give us a good clue to elucidate the roles of each class of lipids in its growth.     

Direct monitoring of heat-stressed biopolymers with temperature-controlled electrospray ionization mass spectrometry

The ability to monitor protein aggregation at the molecular level is critical for progress in many areas of life sciences ranging from understanding mechanisms of amyloidosis and etiology of conformational diseases to development of safe and efficient biopharmaceutical products. Despite the spectacular progress in understanding the mechanisms of protein aggregation in recent years, many aspects of the aggregating proteins behavior remain unclear because of the extreme difficulty in tracking evolution of these notoriously complex and heterogeneous systems. Here, we introduce a mass spectrometry-based methodology that allows the early stages of heat-induced aggregation to be studied by monitoring both conformational changes and formation of oligomers as a function of temperature. The new approach allows biopolymer behavior (both reversible and irreversible processes) to be monitored in a wide temperature range. Validation of the methodology is carried out by comparing temperature profiles of model proteins and nucleic acids deduced from mass spectrometry measurements and differential scanning calorimetry. Application of the methodology to study heat-induced aggregation of human glucocerebrosidase unequivocally links loss of conformational fidelity to formation of soluble oligomers, which serve as precursors to aggregation.