Chemical imaging of trichome specialized metabolites using contact printing and laser desorption/ionization mass spectrometry

Cell transfer by contact printing coupled with carbon-substrate-assisted laser desorption/ionization was used to directly profile and image secondary metabolites in trichomes on leaves of the wild tomato Solanum habrochaites. Major specialized metabolites, including acyl sugars, alkaloids, flavonoids, and terpenoid acids, were successfully detected in positive ion mode or negative ion mode, and in some cases in both modes. This simple solvent-free and matrix-free sample preparation for mass spectrometry imaging avoids tedious sample preparation steps, and high-spatial-resolution images were obtained. Metabolite profiles were generated for individual glandular trichomes from a single Solanum habrochaites leaf at a spatial resolution of around 50 μm. Relative quantitative data from imaging experiments were validated by independent liquid chromatography–mass spectrometry analysis of subsamples from fresh plant material. The spatially resolved metabolite profiles of individual glands provided new information about the complexity of biosynthesis of specialized metabolites at the cellular-resolution scale. In addition, this technique offers a scheme capable of high-throughput profiling of metabolites in trichomes and irregularly shaped tissues and spatially discontinuous cells of a given cell type.

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Fe, Cu and Zn in cryostat sections of human eye lenses and for depth profiling analysis in bovine lenses. To ensure a tight temperature control throughout the experiments, a new Peltier-cooled laser ablation cell was employed. For quantification purposes, matrix-matched laboratory standards were prepared from a pool of human lenses from eye donors and spiked with standard solutions containing different concentrations of natural abundance Fe, Cu and Zn. A normalisation strategy was also carried out to correct matrix effects, lack of tissue homogeneity and/or instrumental drifts using a thin gold film deposited on the sample surface. Quantitative images of cryo-sections of human eye lenses analysed by LA-ICP-MS revealed a homogeneous distribution of Fe, Cu and Zn in the nuclear region and a slight increase in Fe concentration in the outer cell layer (i.e. lens epithelium) at the anterior pole. These results were assessed also by isotope dilution mass spectrometry, and Fe, Cu and Zn concentrations determined by ID-ICP-MS in digested samples of lenses and lens capsules.

IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging of biological tissue sections using a layer of deposited ice as an energy-absorbing matrix was investigated. Dynamics of plume ablation were first explored using a nanosecond exposure shadowgraphy system designed to simultaneously collect pictures of the plume with a camera and collect the Fourier transform ion cyclotron resonance FT-ICR mass spectrum corresponding to that same ablation event. Ablation of fresh tissue analyzed with and without using ice as a matrix were compared using this technique. Effect of spot-to-spot distance, number of laser shots per pixel, and tissue condition (matrix) on ion abundance were also investigated for 50 μm-thick tissue sections. Finally, the statistical method called design of experiments was used to compare source parameters and determine the optimal conditions for IR-MALDESI of tissue sections using deposited ice as a matrix. With a better understanding of the fundamentals of ablation dynamics and a systematic approach to explore the experimental space, it was possible to improve ion abundance by nearly one order of magnitude.

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

High-Mass MALDI-MS Using Ion Conversion Dynode Detectors: Influence of the Conversion Voltage on Sensitivity and Spectral Quality

With the development of special ion conversion dynode (ICD) detectors for high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the mass-to-charge ratio is no longer a limiting factor. Although these detectors have been successfully used in the past, there is lack of understanding of the basic processes in the detector. We present a systematic study to investigate the performance of such an ICD detector and separate the contributions of the MALDI process from the ones of the ion-to-secondary ion and the secondary ion-to-electron conversions. The performance was evaluated as a function of the voltages applied to the conversion dynodes and the sample amount utilized, and we found that the detector reflects the MALDI process correctly: limitations such as sensitivity or deviations from the expected signal intensity ratios originate from the MALDI process itself and not from the detector.

Recent methodological advances in MALDI mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is widely used for characterization of large, thermally labile biomolecules. Advantages of this analytical technique are high sensitivity, robustness, high-throughput capacity, and applicability to a wide range of compound classes. For some years, MALDI-MS has also been increasingly used for mass spectrometric imaging as well as in other areas of clinical research. Recently, several new concepts have been presented that have the potential to further advance the performance characteristics of MALDI. Among these innovations are novel matrices with low proton affinities for particularly efficient protonation of analyte molecules, use of wavelength-tunable lasers to achieve optimum excitation conditions, and use of liquid matrices for improved quantification. Instrumental modifications have also made possible MALDI-MS imaging with cellular resolution as well as an efficient generation of multiply charged MALDI ions by use of heated vacuum interfaces. This article reviews these recent innovations and gives the author’s personal outlook of possible future developments.

Implementation of a Gaussian Beam Laser and Aspheric Optics for High Spatial Resolution MALDI Imaging MS

We have investigated the use of a Gaussian beam laser for MALDI Imaging Mass Spectrometry to provide a precisely defined laser spot of 5 μm diameter on target using a commercial MALDI TOF instrument originally designed to produce a 20 μm diameter laser beam spot at its smallest setting. A Gaussian beam laser was installed in the instrument in combination with an aspheric focusing lens. This ion source produced sharp ion images at 5 μm spatial resolution with signals of high intensity as shown for images from thin tissue sections of mouse brain.

Microchip-based strategy for enrichment of acetylated proteins

We have developed a simple microchip-based method for the separation and enrichment of acetylated proteins and peptides using a microchip technique. Poly (dimethylsiloxane) (PDMS) microfluidic channels were modified by passing an acidic solution of hydrogen peroxide through them. This resulted in hydrophilic silanol-covered surfaces onto which poly (diallyldimethylammonium chloride) (PDDA) can be coated. Protein A/G beads were then captured by the PDDA layer and antibodies can then be immobilized via the protein A/G. This technique enables efficient capture of antigens due to the optimal spacing and orientation of surface molecules. Two solutions, one containing 72.5 fmol?μL^?1 of acetylated bovine serum albumin (BSA-Ac), the other 72.5 fmol?μL^?1 of tryptic BSA-Ac digest were then enriched. High selectivities were obtained, and a 82.4 % recovery of the acetylated proteins was attained. This on-chip platform was then coupled to MALDI-MS to provide information on the acetylation sites of proteins and peptides. Additional peaks were observed in the mass spectra after enrichment and were assigned to acetylated peptides. This is significant with respect to understanding the mechanism and function of acetylation. In our opinion, this microchip-based technique has a large potential for detecting acetylated proteins and peptides in complex biological mixtures, and in acetylomics in general.

Cryo ultra-low-angle microtomy for XPS-depth profiling of organic coatings

In X-ray photoelectron spectroscopy (XPS) Ar⁺ ion sputtering is usually used for depth profiling. However, for such samples as organic coatings, this is not feasible because of degradation. Also, measurement of a depth profile on a conventionally prepared cross-section is not possible if, for example, sample thickness is below the smallest available measurement spot size of the XPS system. In our approach we used a rotary microtome to cut samples under a shallow tilting angle of 0.5° to obtain an extended cross-section suitable for XPS investigations. We also used liquid nitrogen cooling to ensure an exposed area of higher quality: topography measurements with a novel optical 3D microscope and by atomic force microscopy revealed the linearity of the inclined sections. With our cryo ultra-low-angle microtomy (cryo-ULAM) preparation technique we were able to determine, by XPS, elemental and chemical gradients within a 25 μm thick polyester-based organic coating deposited on steel. The gradients were related to, for example, depletion of the crosslinking agent in the sub-surface region. Complementary reflection electron energy-loss spectroscopy measurements performed on the cryo-ULAM sections also support the findings obtained from the XPS depth profiles.

Laser Beam Filtration for High Spatial Resolution MALDI Imaging Mass Spectrometry

We describe an easy and inexpensive way to provide a highly defined Gaussian shaped laser spot on target of 5 μm diameter for imaging mass spectrometry using a commercial MALDI TOF instrument that is designed to produce a 20 μm diameter laser beam on target at its lowest setting. A 25 μm pinhole filter on a swivel arm was installed in the laser beam optics outside the vacuum ion source chamber so it is easily flipped into or out of the beam as desired by the operator. The resulting ion images at 5 μm spatial resolution are sharp since the satellite secondary laser beam maxima have been removed by the filter. Ion images are shown to demonstrate the performance and are compared with the method of oversampling to achieve higher spatial resolution when only a larger laser beam spot on target is available.

Recent developments in nanoparticle-based MALDI mass spectrometric analysis of phosphoproteomes

Mass spectrometry-based strategies are widely used for mapping of post-translational modifications of phosphoproteins. However, the presence of large amounts of non-phosphopeptides seriously interferes by suppressing the intensities of signals for phosphopeptides in direct MALDI-MS techniques due to the low stoichiometry of protein phosphorylation. Several MALDI-MS approaches are known which use either nanoparticles (NPs) as affinity probes, or NPs as microwave heat absorbers. They assist in the enrichment of trace levels of phosphopeptides from complex protein digests and require minimal sample pretreatment, digestion times, and sample volume. This leads to enhance sensitivity and selectivity in the analysis of the phosphoproteomes. This review (with 89 refs.) summarizes and discusses recent developments in the field, with a particular focus on the potential use of nanomaterials such as metal oxides, metal NPs, NPs-coated target plates, and as core-shell nanocomposites acting as affinity probes and as heat absorbers in MALDI-MS analysis of phosphoproteomes.

CO2 laser ionization of acoustically levitated droplets

For many analytical purposes, direct laser ionization of liquids is desirable. Several studies on supported droplets, free liquid jets, and ballistically dispensed microdroplets have been conducted, yet detailed knowledge of the underlying mechanistics in ion formation is still missing. This contribution introduces a simple combination of IR-MALDI mass spectrometry and an acoustical levitation device for contactless confinement of the liquid sample. The homebuilt ultrasonic levitator supports droplets of several millimeters in diameter. These droplets are vaporized by a carbon dioxide laser in the vicinity of the atmospheric pressure interface of a time of flight mass spectrometer. The evaporation process is studied by high repetition rate shadowgraphy experiments elucidating the ballistic evaporation of the sample and revealing strong confinement of the vapor by the ultrasonic field of the trap. Finally, typical mass spectra for pure glycerol/water matrix and lysine as an analyte are presented with and without the addition of trifluoracetic acid, and the ionization mechanism is briefly discussed. The technique is a promising candidate for a reproducible mass spectrometric detection scheme for the field of microfluidics.

Rapid enrichment of phosphopeptides by BaTiO3 nanoparticles after microwave-assisted tryptic digest of phosphoproteins, and their identification by MALDI-MS

We show that BaTiO₃ nanoparticles (NPs) can be used as a novel substrate for the rapid enrichment of phosphopeptides from microwave tryptic digests of α-casein and non-fat milk prior to their identification by MALDI-MS. Protein digestion is achieved by microwave tryptic digest for 50?s, and the resulting phosphopeptides can be effectively adsorbed on the surfaces of the NPs. The phosphopeptides were selectively detected via MALDI-MS. Digestion, enrichment and detection are accomplished within ～60?min. The method was applied to the indentification of 24 phosphopeptides from α-casein and of 21 phosphopeptides (of the α-casein type) from nonfat milk.

Laser-Induced Azomethine Ylide Formation and Its Covalent Entrapment by Fulleropyrrolidine Derivatives During MALDI Analysis

Two novel monofunctionalized fulleropyrrolidine derivatives (Prato adducts) were prepared and characterized by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MALDI experiments conducted in the positive-ion mode on pure and mixed samples of both monofunctionalized fullerene derivatives revealed the efficient formation of bisadducts (in the case of the pure samples) and mixed bisadducts (in the case of a mixed sample). Bisadducts were not observed in the ESI experiments and thus not present in the sample. A mechanism for the MALDI formation of these bisadduct ions is proposed in which an azomethine ylide fragment is formed in situ from the monofunctionalized fulleropyrrolidine species upon laser irradiation. This fragment, which can survive as an intact moiety in the gas phase in the special environment provided by the MALDI experiment, is then able to attach to a fulleropyrrolidine monoadduct which acts as a dipolarophile, thus leading to the formation of a bisadduct fullerene derivative. The unprecedented re-attachment of the azomethine ylide implies that the establishment of the ligand attainment of Prato adducts based on MALDI analysis alone can lead to wrong assignments.

Overcoming Selectivity and Sensitivity Issues of Direct Inject Electrospray Mass Spectrometry via DAPNe-NSI-MS

Direct inject electrospray mass spectrometry offers minimal sample preparation and a “shotgun” approach to analyzing samples. However, complex matrix effects often make direct inject an undesirable sample introduction technique, particularly for trace level analytes. Highlighted here is our solution to the pitfalls of direct inject mass spectrometry and other ambient ionization methods with a focus on trace explosives. Direct analyte-probed nanoextraction coupled to nanospray ionization mass spectrometry solves selectivity issues and reduces matrix effects while maintaining minimal sample preparation requirements. With appropriate solvent conditions, most explosive residues can be analyzed with this technique regardless of the nature of the substance (i.e., nitroaromatic, oxidizing salt, or peroxide).

Identification of selenium in the leaf protein of sunflowers by a combination of 2D-PAGE and laser ablation ICP-MS

We report on a method for the identification of selenium-containing proteins in an extract of sunflower leafs. It is based on the separation of the proteins by 2-dimensional gel electrophoresis, followed by detection of selenium via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The laser system was operated in a raster mode at 100?μm?s^-1 and proved to be an efficient alternative in the search for selenoproteins in the spots of the gels. The instrumental parameters were optimized in terms of plasma energy and application of optimal reaction cell conditions, and the detection of the mass ⁸⁰Se¹⁶O⁺ which enabled the elimination of interfering species. Selenium was identified in 9.6% of the analyzed spots, indicating its random incorporation into the primary structure of the proteins.