Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling.

Label-free protein characterization at surfaces is commonly achieved using digestion and/or matrix application prior to mass spectrometry. We report the assignment of undigested proteins at surfaces in situ using secondary ion mass spectrometry (SIMS). Ballistic fragmentation of proteins induced by a gas cluster ion beam (GCIB) leads to peptide cleavage producing fragments for subsequent OrbitrapTM analysis. In this work we annotate 16 example proteins (up to 272 kDa) by de novo peptide sequencing and illustrate the advantages of this approach by characterizing a protein monolayer biochip and the depth distribution of proteins in human skin.

Secondary Ion Yield and Fragmentation of Biological Molecules by Employing 35Cl Primary Ions within the MeV Energy Domain.

MeV-SIMS is an emerging mass spectrometry imaging method that employs fast, heavy ions to desorb secondary molecules from the analyzed sample. High yields and low fragmentation rates of large molecules, associated with the dominating electronic sputtering process, make it particularly useful in biomedical research, where insight into the distribution of organic molecules is vital. Both yield and fragmentation of desorbed molecules in MeV-SIMS rely on characteristics of the primary ion but may also be impaired by poor instrumental settings. After utilizing secondary ion optics in the linear mass spectrometer at the micro-analytical center of the Jozef Stefan Institute, we demonstrate very efficient detection of secondary ions. As a result, the secondary ion yield, using such settings, solely depends on the species and the characteristics of the primary ion. In order to analyze the yield dependence on the primary ion energy, and the corresponding stopping power within the electronic excitation regime, we used a continuous electron multiplier detector to measure the primary ion current during each measurement of the mass spectra. Secondary ion yield as a function of the primary ion energy and charge is presented as well as fragmentation rates of organic molecules arginine and leu-enkephalin. Other influential instrumental drawbacks are also studied, and their effect on the results is discussed.

High-Resolution Secondary Ion Mass Spectrometry Analysis of Cell Membranes.

This Feature describes the use a Cameca NanoSIMS instrument for directly imaging specific lipid and protein species in the plasma membranes of mammalian cells with approximately 100 nm-lateral resolution and discusses how these analyses have already begun to transform fundamental concepts in the field of membrane biology. Secondary ion generation is discussed with emphasis on the constraints that affect the detection and identification of membrane components, and then the sample preparation methodologies and data analysis strategies that address these constraints are described.

MeV-SIMS TOF Imaging of Organic Tissue with Continuous Primary Beam.

MeV-SIMS is an emerging mass spectrometry imaging method, which utilizes fast, heavy ions to desorb secondary molecules. High yields and low fragmentation rates of large molecules, associated with the electronic sputtering process, make it particularly useful in biomedical research, where insight into distribution of organic molecules is needed. Since the implementation of MeV-SIMS in to the micro-beam line at the tandem accelerator of Jozef Stefan Institute, MeV-SIMS provided some valuable observations on the distribution of biomolecules in plant tissue, as discussed by Jencic et al. (Nucl. Inst. Methods Phys. Res. B. 371, 205-210, 2016; Nucl. Inst. Methods Phys. Res. B. 404, 140-145, 2017). However, limited focusing ability of the chlorine ion beam only allowed imaging at the tissue level. In order to surpass shortcomings of the existing method, we introduced a new approach, where we employ a continuous, low-current primary beam. In this mode, we bombard thin samples with a steady chlorine ion flux of approx. 5000 ions/s. After desorbing molecules, chlorine ions penetrate through the thinly cut sample and trigger the time-of-flight "start" signal on a continuous electron multiplier detector, positioned behind the sample. Such bombardment is more effective than previously used pulsing-beam mode, which demanded several orders of magnitude higher primary ion beam currents. Sub-micrometer focusing of low-current primary ion beam allows imaging of biological tissue on a subcellular scale. Simultaneously, new time-of-flight acquisition approach also improves mass resolution by a factor of 5. Within the article, we compare the performance of both methods and demonstrate the application of continuous mode on biological tissue. We also describe the thin sample preparation protocol, necessary for measurements with low primary ion currents.

Metabolic Imaging at the Single-Cell Scale: Recent Advances in Mass Spectrometry Imaging.

There is an increasing appreciation that every cell, even of the same type, is different. This complexity, when additionally combined with the variety of different cell types in tissue, is driving the need for spatially resolved omics at the single-cell scale. Rapid advances are being made in genomics and transcriptomics, but progress in metabolomics lags. This is partly because amplification and tagging strategies are not suited to dynamically created metabolite molecules. Mass spectrometry imaging has excellent potential for metabolic imaging. This review summarizes the recent advances in two of these techniques: matrix-assisted laser desorption ionization (MALDI) and secondary ion mass spectrometry (SIMS) and their convergence in subcellular spatial resolution and molecular information. The barriers that have held back progress such as lack of sensitivity and the breakthroughs that have been made including laser-postionization are highlighted as well as the future challenges and opportunities for metabolic imaging at the single-cell scale.

Integration of paper-based microarray and time-of-flight secondary ion mass spectrometry (ToF-SIMS) for parallel detection and quantification of molecules in multiple samples automatically.

With its low-cost fabrication and ease of modification, paper-based analytical devices have developed rapidly in recent years. Microarrays allow automatic analysis of multiple samples or multiple reactions with minimal sample consumption. While cellulose paper is generally used, its high backgrounds in spectrometry outside of the visible range has limited its application to be mostly colorimetric analysis. In this work, glass-microfiber paper is used as the substrate for a microarray. The glass-microfiber is essentially chemically inert SiOx, and the lower background from this inorganic microfiber can avoid interference from organic analytes in various spectrometers. However, generally used wax printing fails to wet glass microfibers to form hydrophobic barriers. Therefore, to prepare the hydrophobic-hydrophilic pattern, the glass-microfiber paper was first modified with an octadecyltrichlorosilane (OTS) self-assembled monolayer (SAM) to make the paper hydrophobic. A hydrophilic microarray was then prepared using a CO2 laser scriber that selectively removed the OTS layer with a designed pattern. One microliter of aqueous drops of peptides at various concentrations were then dispensed inside the round patterns where OTS SAM was removed while the surrounding area with OTS layer served as a barrier to separate each drop. The resulting specimen of multiple spots was automatically analyzed with a time-of-flight secondary ion mass spectrometer (ToF-SIMS), and all of the secondary ions were collected. Among the various cluster ions that have developed over the past decade, pulsed C60+ was selected as the primary ion because of its high secondary ion intensity in the high mass region, its minimal alteration of the surface when operating within the static-limit and spatial resolution at the ∼µm level. In the resulting spectra, parent ions of various peptides (in the forms [M+H]+ and [M+Na]+) were readily identified for parallel detection of molecules in a mixture. By normalizing the ion intensity of peptides with respect to the glass-microfiber matrix ([SiOH]+), a linear calibration curve for each peptide was generated to quantify these components in a mixture.

Sample Preparation of Corn Seed Tissue to Prevent Analyte Relocations for Mass Spectrometry Imaging.

Corn seed tissue sections were prepared by the tape support method using an adhesive tape, and mass spectrometry imaging (MSI) was performed. The effect of heat generated during sample preparation was investigated by time-of-flight secondary mass spectrometry (TOF-SIMS) imaging of corn seed tissue prepared by the tape support and the thaw-mounted methods. Unlike thaw-mounted sample preparation, the tape support method does not cause imaging distortion because of the absence of heat, which can cause migration of the analytes on the sample. By applying the tape-support method, the corn seed tissue was prepared without structural damage and MSI with accurate spatial information of analytes was successfully performed. Graphical Abstract ᅟ.

Secondary ion yields for vacuum-type electrospray droplet beams measured with a triple focus time-of-flight analyzer.

RATIONALE: We previously developed a massive cluster ion beam gun for secondary ion mass spectrometry (SIMS) in which the primary beam source is a vacuum electrospray. The secondary ion yields produced by this method had not yet been measured with a commercial time-of-flight (TOF) secondary ion mass spectrometer, and the ionization performance was unknown. METHODS: A vacuum-type electrospray droplet ion gun was connected to a triple-focus TOF analyzer. The flight time of the secondary ions was measured using a sample-bias pulsing method, because a short pulse of the electrospray droplet beam could not be obtained. The secondary ion yields of an amino acid sample produced by the electrospray droplet beams and atomic Ga ion beams were compared. RESULTS: TOF secondary ion spectra were measured for the amino acid and peptide samples with a mass resolution of ~500 using the sample-bias pulsing method. The secondary ion yield of the amino acid sample produced with the 10 kV vacuum-type electrospray droplet beams was much higher than that produced by 10 kV Ga ion beams. In addition, the secondary ion yields for the peptide sample and amino acid samples were almost similar. CONCLUSIONS: This is the first report on secondary ion yields produced with vacuum-type electrospray droplet ion beams and measured with a semi-commercial TOF analyzer. The enhancement of secondary ion yields, in particular for relatively high-mass molecules, would be very useful in the SIMS analysis of a wide variety of biological samples. Copyright © 2016 John Wiley & Sons, Ltd.

Cluster secondary ion mass spectrometry imaging of interfacial reactions of TiO2 microspheres embedded in ionic liquids.

RATIONALE: Our goal is to develop protocols for the elucidation of the identity and structure of reaction products embedded in a reaction medium. Results should find significance in a variety of disciplines ranging from the study of biological cells and tissues, to the steps associated with the functionalization of nanoparticles. METHODS: We utilize cluster secondary ion mass spectrometry (cluster-SIMS) to acquire three-dimensional (3D) information about 5-30 µm TiO2 microspheres imbedded into an ionic liquid. The method allows molecular depth profiling with submicron spatial resolution and depth profiling with a resolution of several tens of nanometers. The ionic liquid matrix enshrouds the spheres, allowing them to be introduced into the vacuum environment of the mass spectrometer. RESULTS: The results provide 3D chemical information about these microspheres as they are synthesized by interfacial sol-gel reactions. We show that with 40 keV C60 (+) , it is possible to erode through the reaction medium and map the distribution of those embedded TiO2 microspheres. Moreover, we demonstrate that it is possible to monitor surface modification of the particles and, via ion beam drilling, elucidate their internal structure. CONCLUSIONS: Using cluster-SIMS imaging, we are able to elucidate the identity and structure of reaction products embedded in a reaction medium, a problem of long-standing interest for materials characterization. With this strategy, we have provided a new approach that may be especially useful for the characterization of biological tissue and cells within the vacuum confines of the mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.

Fast Single-Cell Patterning for Study of Drug-Induced Phenotypic Alterations of HeLa Cells Using Time-of-Flight Secondary Ion Mass Spectrometry.

A facile single-cell patterning (ScP) method was developed and integrated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) for the study of drug-induced cellular phenotypic alterations. Micropatterned poly(dimethylsiloxane) (PDMS) stencil film and centrifugation-assisted cell trapping were combined for the preparation of on-surface single-cell microarrays, which exhibited both high site occupancy (>90%) and single-cell resolution (>97%). TOF-SIMS is a surface-sensitive mass spectrometry and is increasingly utilized in biological studies. Here we demonstrated, for the first time, its successful application in high-throughput single-cell analysis. Drug-induced phenotypic alterations of HeLa cells in the early stage of apoptosis were investigated using TOF-SIMS. The major molecular sources of variations were analyzed by principle component analysis (PCA).

Time-of-flight secondary ion mass spectrometry imaging of biological samples with delayed extraction for high mass and high spatial resolutions.

RATIONALE: In Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS), pulsed and focused primary ion beams enable mass spectrometry imaging, a method which is particularly useful to map various small molecules such as lipids at the surface of biological samples. When using TOF-SIMS instruments, the focusing modes of the primary ion beam delivered by liquid metal ion guns can provide either a mass resolution of several thousand or a sub-µm lateral resolution, but the combination of both is generally not possible. METHODS: With a TOF-SIMS setup, a delayed extraction applied to secondary ions has been studied extensively on rat cerebellum sections in order to compensate for the effect of long primary ion bunches. RESULTS: The use of a delayed extraction has been proven to be an efficient solution leading to unique features, i.e. a mass resolution up to 10000 at m/z 385.4 combined with a lateral resolution of about 400 nm. Simulations of ion trajectories confirm the experimental determination of optimal delayed extraction and allow understanding of the behavior of ions as a function of their mass-to-charge ratio. CONCLUSIONS: Although the use of a delayed extraction has been well known for many years and is very popular in MALDI, it is much less used in TOF-SIMS. Its full characterization now enables secondary ion images to be recorded in a single run with a submicron spatial resolution and with a mass resolution of several thousand. This improvement is very useful when analyzing lipids on tissue sections, or rare, precious, or very small size samples.

Mass spectrometric imaging of brain tissue by time-of-flight secondary ion mass spectrometry--How do polyatomic primary beams C60⁺, Ar2000⁺, water-doped Ar2000⁺ and (H2O)6000⁺ compare?

RATIONALE: To discover the degree to which water-containing cluster beams increase secondary ion yield and reduce the matrix effect in time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging of biological tissue. METHODS: The positive SIMS ion yields from model compounds, mouse brain lipid extract and mouse brain tissue together with mouse brain images were compared using 20 keV C60(+), Ar2000(+), water-doped Ar2000(+) and pure (H2O)6000(+) primary beams. RESULTS: Water-containing cluster beams where the beam energy per nucleon (E/nucleon) ≈ 0.2 eV are optimum for enhancing ion yields dependent on protonation. Ion yield enhancements over those observed using Ar2000(+) lie in the range 10 to >100 using the (H2 O)6000 (+) beam, while with water-doped (H2O)Ar2000(+) they lie in the 4 to 10 range. The two water-containing beams appear to be optimum for tissue imaging and show strong evidence of increasing yields from molecules that experience matrix suppression under other primary beams. CONCLUSIONS: The application of water-containing primary beams is suggested for biological SIMS imaging applications, particularly if the beam energy can be raised to 40 keV or higher to further increase ion yield and enhance spatial resolution to ≤1 µm.

Development of an organic lateral resolution test device for imaging mass spectrometry.

An organic lateral resolution test device has been developed to measure the performance of imaging mass spectrometry (IMS) systems. The device contains periodic gratings of polyethylene glycol (PEG) and lipid bars covering a wide range of spatial frequencies. Microfabrication technologies were employed to produce well-defined chemical interfaces, which allow lateral resolution to be assessed using the edge-spread function (ESF). In addition, the design of the device allows for the direct measurement of the modulation transfer function (MTF) to assess image quality. Scanning electron microscopy (SEM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) were used to characterize the device. TOF-SIMS imaging was used to measure the chemical displacement of biomolecules in matrix-assisted laser desorption/ionization (MALDI) matrix crystals. In a proof-of-concept experiment, the platform was also used to evaluate MALDI matrix application methods, specifically aerosol spray and sublimation methods.

Development and validation of TOF-SIMS and CLSM imaging method for cytotoxicity study of ZnO nanoparticles in HaCaT cells.

Zinc oxide nanoparticles (ZnO NPs) exhibit novel physiochemical properties and have found increasing use in sunscreen products and cosmetics. The potential toxicity is of increasing concern due to their close association with human skin. A time-of-flight secondary ion mass spectrometry (TOF-SIMS) and confocal laser scanning microscopy (CLSM) imaging method was developed and validated for rapid and sensitive cytotoxicity study of ZnO NPs using human skin equivalent HaCaT cells as a model system. Assorted material, chemical, and toxicological analysis methods were used to confirm their shape, size, crystalline structure, and aggregation properties as well as dissolution behavior and effect on HaCaT cell viability in the presence of various concentrations of ZnO NPs in aqueous media. Comparative and correlative analyses of aforementioned results with TOF-SIMS and CLSM imaging results exhibit reasonable and acceptable outcome. A marked drop in survival rate was observed with 50µg/ml ZnO NPs. The CLSM images reveal the absorption and localization of ZnO NPs in cytoplasm and nuclei. The TOF-SIMS images demonstrate elevated levels of intracellular ZnO concentration and associated Zn concentration-dependent (40)Ca/(39)K ratio, presumably caused by the dissolution behavior of ZnO NPs. Additional validation by using stable isotope-labeled (68)ZnO NPs as tracers under the same experimental conditions yields similar cytotoxicity effect. The imaging results demonstrate spatially-resolved cytotoxicity relationship between intracellular ZnO NPs, (40)Ca/(39)K ratio, phosphocholine fragments, and glutathione fragments. The trend of change in TOF-SIMS spectra and images of ZnO NPs treated HaCaT cells demonstrate the possible mode of actions by ZnO NP involves cell membrane disruption, cytotoxic response, and ROS mediated apoptosis.

Quantitative surface analysis of a binary drug mixture--suppression effects in the detection of sputtered ions and post-ionized neutrals.

A systematic mass spectrometric study of two of the most common analgesic drugs, paracetamol and ibuprofen, is reported. The drugs were studied by means of secondary ion mass spectrometry (SIMS) and secondary neutral mass spectrometry (SNMS) using laser post-ionization (LPI) both in pure samples and in a two-component mixture. Ion suppression within the two-component system observed in SIMS mode is ameliorated using LPI under room temperature analysis. However, suppression effects are apparent in LPI mode on performing the analysis at cryogenic temperatures, which we attribute to changes in the desorption characteristics of sputtered molecules, which influences the subsequent post-ionization efficiency. This suggests different mechanisms of ion suppression in SIMS and LPI modes.

In situ molecular imaging of a hydrated biofilm in a microfluidic reactor by ToF-SIMS.

A novel microfluidic reactor for biofilm growth and in situ characterization using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was constructed to enable two-dimensional chemical imaging of hydrated biofilms. We demonstrate the detection of characteristic fatty acid fragments from microfluidic reactor-grown biofilms and illustrate advantages of hydrated-state ToF-SIMS imaging.

Secondary ion mass spectrometry imaging of biological cells and tissues.

Secondary ion mass spectrometry (SIMS) is capable of providing detailed atomic and molecular characterization of the surface chemistry of (bio)molecular samples. It is one of a range of mass spectrometry imaging techniques that combine the high sensitivity and specificity of mass spectrometry with the capability to view the distribution of analytes within solid samples. The technique is particularly suited to the detection and imaging of small molecules such as lipids and other metabolites. A limit of detection in the ppm range and spatial resolution <1 µm can be obtained. Recent progress in instrumental developments, including new cluster ion beams, the implementation of tandem mass spectrometry (MS/MS), and the application of multivariate data analysis protocols promise further advances. This chapter presents a brief overview of the technique and methodology of SIMS using exemplar studies of biological cells and tissue.

Peptide dissociation patterns in secondary ion mass spectrometry under large argon cluster ion bombardment.

RATIONALE: The analysis of organic and biological substances by secondary ion mass spectrometry (SIMS) has greatly benefited from the use of cluster ions as primary bombarding species. Thereby, depth profiling and three-dimensional (3D) imaging of such systems became feasible. Large Ar(n)(+) cluster ions may constitute a further improvement in this direction. METHODS: To explore this option, large Ar(n)(+) cluster ions (with n ~1500 Ar atoms per cluster) were used to investigate the emission of positive secondary ions from two peptide specimens (angiotensin I and bradykinin) by orthogonal time-of-flight SIMS using bombarding energies 6, 10 and 14 keV. RESULTS: For both peptides, the protonated molecular ion is observed in the mass spectra. In addition, distinct fragmentation patterns were observed; these indicate that fragment ions under Ar cluster irradiation form primarily via cleavage of bonds along the peptide backbone whereas the rapture of side chains occurs much less frequently. These features appear to be similar to low-energy collision-induced dissociation pathways. CONCLUSIONS: Tentatively, these findings can then be ascribed to the concerted action of the large number of Ar atoms in the impact zone of cluster at the surface: these low-energy Ar species (with an average energy of few eV) may effect the cleavage of the peptide bonds and lead, eventually, to the emission of the fragment ions.

High mass accuracy and high mass resolving power FT-ICR secondary ion mass spectrometry for biological tissue imaging.

Biological tissue imaging by secondary ion mass spectrometry has seen rapid development with the commercial availability of polyatomic primary ion sources. Endogenous lipids and other small bio-molecules can now be routinely mapped on the sub-micrometer scale. Such experiments are typically performed on time-of-flight mass spectrometers for high sensitivity and high repetition rate imaging. However, such mass analyzers lack the mass resolving power to ensure separation of isobaric ions and the mass accuracy for elemental formula assignment based on exact mass measurement. We have recently reported a secondary ion mass spectrometer with the combination of a C60 primary ion gun with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) for high mass resolving power, high mass measurement accuracy, and tandem mass spectrometry capabilities. In this work, high specificity and high sensitivity secondary ion FT-ICR MS was applied to chemical imaging of biological tissue. An entire rat brain tissue was measured with 150 µm spatial resolution (75 µm primary ion spot size) with mass resolving power (m/Δm(50%)) of 67,500 (at m/z 750) and root-mean-square measurement accuracy less than two parts-per-million for intact phospholipids, small molecules and fragments. For the first time, ultra-high mass resolving power SIMS has been demonstrated, with m/Δm(50%) > 3,000,000. Higher spatial resolution capabilities of the platform were tested at a spatial resolution of 20 µm. The results represent order of magnitude improvements in mass resolving power and mass measurement accuracy for SIMS imaging and the promise of the platform for ultra-high mass resolving power and high spatial resolution imaging.