Secondary ion mass spectrometry imaging and multivariate data analysis reveal co‐aggregation patterns of Populus trichocarpa leaf surface compounds on a micrometer scale

Spatially resolved analysis of a multitude of compound classes has become feasible with the rapid advancement in mass spectrometry imaging strategies. In this study, we present a protocol that combines high lateral resolution time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) imaging with a multivariate data analysis (MVA) approach to probe the complex leaf surface chemistry of Populus trichocarpa. Here, epicuticular waxes (EWs) found on the adaxial leaf surface of P. trichocarpa were blotted on silicon wafers and imaged using TOF‐SIMS at 10 μm and 1 μm lateral resolution. Intense M^+● and M^?● molecular ions were clearly visible, which made it possible to resolve the individual compound classes present in EWs. Series of long‐chain aliphatic saturated alcohols (C₂₁–C₃₀), hydrocarbons (C₂₅–C₃₃) and wax esters (WEs; C₄₄–C₄₈) were clearly observed. These data correlated with the ⁷Li‐chelation matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) analysis, which yielded mostly molecular adduct ions of the analyzed compounds. Subsequently, MVA was used to interrogate the TOF‐SIMS dataset for identifying hidden patterns on the leaf's surface based on its chemical profile. After the application of principal component analysis (PCA), a small number of principal components (PCs) were found to be sufficient to explain maximum variance in the data. To further confirm the contributions from pure components, a five‐factor multivariate curve resolution (MCR) model was applied. Two distinct patterns of small islets, here termed ‘crystals’, were apparent from the resulting score plots. Based on PCA and MCR results, the crystals were found to be formed by C₂₃ or C₂₉ alcohols. Other less obvious patterns observed in the PCs revealed that the adaxial leaf surface is coated with a relatively homogenous layer of alcohols, hydrocarbons and WEs. The ultra‐high‐resolution TOF‐SIMS imaging combined with the MVA approach helped to highlight the diverse patterns underlying the leaf's surface. Currently, the methods available to analyze the surface chemistry of waxes in conjunction with the spatial information related to the distribution of compounds are limited. This study uses tools that may provide important biological insights into the composition of the wax layer, how this layer is repaired after mechanical damage or insect feeding, and which transport mechanisms are involved in deploying wax constituents to specific regions on the leaf surface.     

Ultra-structural cell distribution of the melanoma marker iodobenzamide: improved potentiality of SIMS imaging in life sciences

Background  

Analytical imaging by secondary ion mass spectrometry (SIMS) provides images representative of the distribution of a specific ion within a sample surface. For the last fifteen years, concerted collaborative research to design a new ion microprobe with high technical standards in both mass and lateral resolution as well as in sensitivity has led to the CAMECA NanoSims 50, recently introduced onto the market. This instrument has decisive capabilities, which allow biological applications of SIMS microscopy at a level previously inaccessible. Its potential is illustrated here by the demonstration of the specific affinity of a melanoma marker for melanin. This finding is of great importance for the diagnosis and/or treatment of malignant melanoma, a tumour whose worldwide incidence is continuously growing.     

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy)

This paper reviews the most recent methodological advances in the field of biological imaging using dynamic secondary ion mass spectrometry (SIMS). After a short reminder of the basic principle of SIMS imaging, the latest high-resolution dynamic SIMS equipment is briefly described. This new ion nanoprobe (CAMECA NanoSIMS 50) has a lateral resolution of less than 50 nm with primary Cs+ ion, the ability to detect simultaneously 5 different ions from the same micro-volume and a very good transmission even at high mass resolution (60% at M/DeltaM=5000). Basic considerations related to sample preparation, mass resolution and primary ion implantation are given. The decisive capability of this new instrument, and more generally of high-resolution dynamic SIMS imaging in biology, are illustrated with the most recent examples of utilization.     

Biomarkers at the microscopic range: ToF-SIMS molecular imaging of Archaea-derived lipids in a microbial mat

Time‐of‐Flight Secondary Ion Mass Spectrometry (ToF‐SIMS) with a bismuth cluster primary ion source was used for analysing microbial lipid biomarkers in 10‐µm‐thick microscopic cryosections of methanotrophic microbial mats from the Black Sea. Without further sample preparation, archaeal isopranyl glycerol di‐ and tetraether core lipids, together with their intact diglycoside (gentiobiosyl‐) derivatives, were simultaneously identified by exact mass determination. Utilizing the imaging capability of ToF‐SIMS, the spatial distributions of these biomarkers were mapped at a lateral resolution of < 5 µm in 500 × 500 µm² areas on the mat sections. Using cluster projectiles in the burst alignment mode, it was possible to reach a lateral resolution of 1 µm on an area of 233 × 233 µm, thus approaching the typical size of microbial cells. The mappings showed different ‘provenances’ within the sections that are distinguished by individual lipid fingerprints, namely (A) the diethers archaeol and hydroxyarchaeol co‐occurring with glycerol dialkyl glycerol tetraethers (GDGT), (B) hydroxyarchaeol and dihydroxyarchaeol, and (C) GDGT and gentiobiosyl‐GDGT. Because ToF‐SIMS is a virtually nondestructive technique affecting only the outermost layers of the sample surface (typically 10–100 nm), it was possible to further examine the studied areas using conventional microscopy, and associate the individual lipid patterns with specific morphological traits. This showed that provenance (B) was frequently associated with irregular, methane‐derived CaCO₃ crystallites, whereas provenance (C) revealed a population of fluorescent, filamentous microorganisms showing the morphology of known methanotrophic ANME‐1 archaea. The direct coupling of imaging mass spectrometry with microscopic techniques reveals interesting perspectives for the in‐situ study of lipids in geobiology, microbial ecology, and organic geochemistry. After further developing protocols for handling different kinds of environmental samples, ToF‐SIMS could be used as a tool to attack many challenging problems in these fields, such as the attribution of biological source(s) to particular biomarkers in question, or the high‐resolution tracking of biogeochemical processes in modern and ancient natural environments.     

Imaging lipids with secondary ion mass spectrometry

This review discusses the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and magnetic sector SIMS with high lateral resolution performed on a Cameca NanoSIMS 50(L) to imaging lipids. The similarities between the two SIMS approaches and the differences that impart them with complementary strengths are described, and various strategies for sample preparation and to optimize the quality of the SIMS data are presented. Recent reports that demonstrate the new insight into lipid biochemistry that can be acquired with SIMS are also highlighted. This article is part of a Special Issue entitled Tools to study lipid functions.     

14N and 15N imaging by SIMS microscopy in soybean leaves

The distribution of ¹⁵N and ¹⁴N compounds in cryofixed and resin embedded sections of soybean (Glycine max L) leaves was studied by SIMS microscopy. The results indicate that, with a mass resolution M/ΔM higher than 6000, images of the nitrogen distribution can be obtained from the mapping of the two secondary cluster ions ¹²C¹⁴N^? and ¹²C¹⁵N^?, in samples of both control and ¹⁵N-labeled leaves. The ionic images were clearly related to the histological structure of the organ, and allow the detection of ¹⁴N and ¹⁵N at the subcellular level. Furthermore, relative measurements of the ¹²C¹⁴N^? and ¹²C¹⁵N^? beams made possible the quantification of the ¹⁵N atom% in the various tissues of the leaf.     

Ultrasensitive radioisotope,stable-isotope,and trace-element analysis in the biological sciences using tandem accelerator mass spectrometry

The new technique of tandem accelerator mass spectrometry (TAMS) has improved the sensitivity for measurement of several long-lived radioisotopes and certain stable isotopes by many orders of magnitude. Nuclear physics tandems and new small dedicated accelerators are now able to measure¹⁴C,¹⁰Be,²⁶Al,³²Si,³⁶Cl,⁴¹Ca, and¹²⁹I in natural materials. Sensitivities down to 10⁵ atoms per sample can be achieved in favorable cases. By accelerating ions to MeV energies, one can eliminate molecules and uniquely identify the atomic numbers below 20. Although most applications to date have been in the earth sciences, the opportunity now exists for important new applications in biology and toxicology. Trace elements can be measured at the parts per billion (10⁹) level using a secondary ion mass spectrometry (SIMS) ion source. Radioactive tracer measurements can be made for elements, such as aluminum, for which there are no isotopes with suitable half-lives for conventional decay counting methods. For¹⁴C, counting times become much shorter and dose levels can be reduced.     

High resolution imaging by organic secondary ion mass spectrometry

Secondary-ion mass spectrometry (SIMS) is based on the acceleration of high-energy primary ions onto a target. Secondary electrons, neutrals and ions are emitted from the target, reflecting its chemical composition. This enables simultaneous analysis and localization of target molecules, giving valuable information that is difficult or impossible to obtain with other analytical methods. The secondary ions can be extracted and detected by any type of mass analyzer. SIMS is unique in its ability to detect several target molecules simultaneously in small samples and to image their localization at subcellular resolution. The recent development of bioimaging SIMS opens up new possibilities in biotechnology and biological research with applications in biomedicine and pathology. The current development of this technique has the potential to become as important for biotechnology as the advent of the electron microscope, confocal microscope or in situ hybridization.     

纳米二次离子质谱技术(NanoSIMS)在微生物生态学研究中的应用

超高分辨率显微镜成像技术与同位素示踪技术相结合的纳米二次离子质谱技术(NanoSIMS)具有较高的灵敏度和离子传输效率、极高的质量分辨率和空间分辨率(＜ 50 nm),代表着当今离子探针成像技术的最高水平.利用稳定性或者放射性同位素在原位或者微宇宙条件下示踪目标微生物,然后将样品进行固定、脱水、树脂包埋或者导电镀膜处理,制备成可供二次离子质谱分析的薄片,进一步通过NanoSIMS成像分析,不仅能够在单细胞水平上提供微生物的生理生态特征信息,而且能够准确识别复杂环境样品中的代谢活跃的微生物细胞及其系统分类信息,对于认识微生物介导的元素生物地球化学循环机制具有重要意义.介绍了纳米二次离子质谱技术的工作原理和技术路线,及其与同位素示踪技术、透射电子显微镜(TEM)、扫描电子显微镜(SEM)、荧光原位杂交技术(FISH)、催化报告沉积荧光原位杂交技术(CARD-FISH)、卤素原位杂交技术(Halogen In Situ Hybridization,HISH)等联合使用在微生物生态学研究方面的应用.     

Delineating Anopheles gambiae coactivator associated arginine methyltransferase 1 automethylation using top–down high resolution tandem mass spectrometry

Coactivator‐associated arginine methyltransferase 1 (CARM1), originally defined as a coactivator for steroid receptors, is a member of the protein arginine methyltransferases. Here, we report the discovery and characterization of an automethylation event by AgCARM1, a CARM1 homologue in the mosquito Anopheles gambiae, using top–down high resolution tandem mass spectrometry, which allows fine mapping of modifications in the intact protein accurately and quantitatively without priori knowledge. Unexpectedly, we found that AgCARM1 has already been predominantly dimethylated during its expression in Escherichia coli. A single arginine methylation site, R485, was identified which is conserved among CARM1 in insects. No methylation was observed in the intact AgCARM1^R485K mutant where R485 is mutated to lysine, which confirms that R485 is the only detectable methylation site. Using AgCARM1 methyltransferase defective mutants, we confirmed that this is an automethylation event and show the automethylation of AgCARM1 occurs intermolecularly. This study represents the first comprehensive characterization of an automethylation event by top–down mass spectrometry. The unexpected high percentage of automethylated recombinant AgCARM1 expressed in E. coli may shed light on other bacterially expressed post‐translational modifying enzymes, which could be modified but overlooked in biochemical and structural studies. Top–down high resolution tandem mass spectrometry thus provides unique opportunities for revealing unexpected protein modification, localizing specific modification to one amino acid, and delineating molecular mechanism of an enzyme.     

Determination of naphthodianthrones in plant extracts from Hypericum perforatum L. by liquid chromatography–electrospray mass spectrometry

The determination of the naphthodianthrone constituents in extracts of dried blossoms of Hypericum perforatum L. by combined HPLC–electrospray mass spectrometry is described. Hypericin (1), pseudohypericin (2) and their precursor compounds produce intensive negative quasi-molecular ions by deprotonation provided a non-acidic eluent system is used in the HPLC separation. From the [M–H]⁻ ions formed in the electrospray ionization process characteristic daughter ion spectra can be obtained by collisional activation which have been studied by tandem mass spectrometry.     

A triazole‐based fluorescence probe for detecting Hg2+ ions and its biological application

A new compound, ethyl 5‐phenyl‐2‐(p‐tolyl)‐2H‐1,2,3‐triazole‐4‐carboxylate was successfully introduced and synthesized as a novel rhodamine B derivative named REPPC, and characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and high resolution mass spectrometry (HRMS). It showed an obvious fluorescence and UV–visible light absorption enhancement towards Hg²⁺ ion without interference from common metal ions in N,N‐dimethylformamide–H₂O (pH 7.4). The spirolactam ring moiety of rhodamine in REPPC was converted to the open‐ring form generating a 1:1 complex with the intervention of a mercury ion, verified by electrospray ionization‐mass spectroscopy testing and density functional theory calculation. REPPC was used to visualize the level of mercury ions in living HeLa cells with encouraging results.     

A laser ablation technique maps differences in elemental composition in roots of two barley cultivars subjected to salinity stress

In saline soils, high levels of sodium (Na⁺) and chloride (Cl^?) ions reduce root growth by inhibiting cell division and elongation, thereby impacting on crop yield. Soil salinity can lead to Na⁺ toxicity of plant cells, influencing the uptake and retention of other important ions [i.e. potassium (K⁺)] required for growth. However, measuring and quantifying soluble ions in their native, cellular environment is inherently difficult. Technologies that allow in situ profiling of plant tissues are fundamental for our understanding of abiotic stress responses and the development of tolerant crops. Here, we employ laser ablation‐inductively coupled plasma‐mass spectrometry (LA‐ICP‐MS) to quantify Na, K and other elements [calcium (Ca), magnesium (Mg), sulphur (S), phosphorus (P), iron (Fe)] at high spatial resolution in the root growth zone of two genotypes of barley (Hordeum vulgare) that differ in salt‐tolerance, cv. Clipper (tolerant) and Sahara (sensitive). The data show that Na⁺ was excluded from the meristem and cell division zone, indicating that Na⁺ toxicity is not directly reducing cell division in the salt‐sensitive genotype, Sahara. Interestingly, in both genotypes, K⁺ was strongly correlated with Na⁺ concentration, in response to salt stress. In addition, we also show important genetic differences and salt‐specific changes in elemental composition in the root growth zone. These results show that LA‐ICP‐MS can be used for fine mapping of soluble ions (i.e. Na⁺ and K⁺) in plant tissues, providing insight into the link between Na⁺ toxicity and root growth responses to salt stress.     

Use of secondary ion mass spectrometry to image44calcium uptake in the cell walls of apple fruit

Summary Calcium, an important agent in regulating cell wall autolysis during fruit ripening, interacts with pectic acid polymers to form cross-bridges that influence cell separation. In the present study, secondary ion mass spectrometry (SIMS) was used to determine whether the cell walls of apple fruit were able to take up exogenously applied⁴⁴Ca, which was infiltrated into mature fruit. SIMS, which has the ability to discriminate between isotopes, allowed localization of the exogenously applied⁴⁴Ca and the native⁴⁰Ca. The results indicated that the total amount of calcium present in the cell walls was enriched with⁴⁴Ca and that heterogeneity of⁴⁴Ca distribution occurred in the pericarp. Isotope ratio images showed microdomains in the cell wall, particularly in the middle lamella intersects that oppose the intercellular spaces. These domains may be the key areas that control cell separation. These data suggest that exogenously applied calcium may influence cell wall autolysis.Abbreviations SIMS secondary ion mass spectrometry     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

Emerging Lyngbya wollei toxins: A new high resolution mass spectrometry method to elucidate a potential environmental threat

Mass spectrometric methods for the quantitative and qualitative analyses of algal biotoxins are often complicated by co-eluting compounds that present analytically as interferences. This issue is particularly critical for organic polyamines, where co-eluting materials can suppress the formation of cations during electrospray ionization. Here we present an extraction procedure designed specifically to overcome matrix-derived ion suppression of algal toxins in samples of Lyngbya wollei, a filamentous benthic algae known to produce several saxitoxin analogues. Lyngbya wollei samples were collected from a large, persistent harmful algal bloom in Lake Wateree, SC. Six known Lyngbya wollei-specific toxins (LWT1–6) were successfully resolved and quantified against saxitoxin using hydrophilic interaction liquid chromatography coupled with triple quadrupole and quadrupole time-of-flight mass spectrometry. The parent ions [M²⁺ – H⁺]⁺ were observed for LWTs 1–6 and the [M]²⁺ ion was observed for LWT5. High resolution mass spectra and unique fragmentation ions were obtained for LWTs 1–6. A dilution factor of 50 resulted in a linear calibration of saxitoxin in the algae matrix. Ion suppression was resolved by sample dilution, which led to linear, positive correlations between peak area and mass of the extracted sample (R² > 0.96). Optimized sample extraction method and instrument parameters are presented.     

Comparability analysis of protein therapeutics by bottom-up LC-MS with stable isotope-tagged reference standards

Comparability studies lie at the heart of assessments that evaluate differences amongst manufacturing processes and stability studies of protein therapeutics. Low resolution chromatographic and electrophoretic methods facilitate quantitation, but do not always yield detailed insight into the effect of the manufacturing change or environmental stress. Conversely, mass spectrometry (MS) can provide high resolution information on the molecule, but conventional methods are not very quantitative. This gap can be reconciled by use of a stable isotope-tagged reference standard (SITRS), a version of the analyte protein that is uniformly labeled with ¹³C₆-arginine and ¹³C₆-lysine. The SITRS serves as an internal control that is trypsin-digested and analyzed by liquid chromatography (LC)-MS with the analyte sample. The ratio of the ion intensities of each unlabeled and labeled peptide pair is then compared to that of other sample(s). A comparison of these ratios provides a readily accessible way to spot even minute differences among samples. In a study of a monoclonal antibody (mAb) spiked with varying amounts of the same antibody bearing point mutations, peptides containing the mutations were readily identified and quantified at concentrations as low as 2% relative to unmodified peptides. The method was robust, reproducible and produced a linear response for every peptide that was monitored. The method was also successfully used to distinguish between two batches of a mAb that were produced in two different cell lines while two batches produced from the same cell line were found to be highly comparable. Finally, the use of the SITRS method in the comparison of two stressed mAb samples enabled the identification of sites susceptible to deamidation and oxidation, as well as their quantitation. The experimental results indicate that use of a SITRS in a peptide mapping experiment with MS detection enables sensitive and quantitative comparability studies of proteins at high resolution.Key words: protein therapeutics, monoclonal antibodies, comparability, peptide mapping, mass spectrometry, LC-MS, stable isotope-tagged reference standard, SITRS     

Design of a stable isotope dilution gas chromatography/mass spectrometric assay for cAMP: Comparison with standard protein-binding and radioimmunoassay methods

A stable isotope dilution assay is presented in which picomole quantities of cAMP can be determined with high precision and selectivity using gas chromatography and electron impact mass spectrometry with multiple ion detection techniques. Using synthetic [2,8-²H₂,6-¹⁵N]-cAMP as the internal standard, suitable specificity was obtained by monitoring the (MCH₃)⁺ fragment ions of the trimethylsilyl derivatives of cAMP and the internal standard at $\text{m}\text{z}$ 530 and $\text{m}\text{z}$ 533, respectively. The sensitivity of the assay as judged from the lower limit of detection of the mass spectrometer was 3.0 pmol. Rat liver and human urine cAMP levels were assayed using gas chromatography/mass spectrometry and were compared with levels determined by protein-binding assays and radioimmunoassays for the same samples. The intraassay coefficients of variation of the gas chromatography/mass spectrometry assay were 5.3% for the rat liver sample (cAMP level 832 pmol/g) and 6.0% for the urine sample (cAMP level 2.50 μmol/liter). Comparison of the levels of cAMP determined by the three assay methods showed correlation to within 10% variation.     

The imaging and quantification of aluminium in the human brain using dynamic secondary ion mass spectrometry (SIMS).

Dynamic secondary ion mass spectrometry (SIMS) has been utilised to study the post-mortem distribution of aluminium in air-dried frozen sections from unfixed, unstained human brain in order to minimise contamination of the tissue and avoid redistribution and extraction of endogenous tissue aluminium. Substrates, sputter-coated with silver, were found to be free of focal aluminum surface contamination and thus minimised substrate induced artefacts in the tissue aluminium ion image. SIMS imaging of aluminium secondary ions at a mass resolution that eliminated the major molecular interferences, combined with a photomontage technique provided a unique strategy for studying aluminium distribution in tissue unrivalled by other spatially resolved microanalytical techniques such as laser microprobe mass spectrometry or X-ray microanalysis. Using this strategy, high densities of focal aluminium accumulations have been demonstrated in the cerebral cortex of the majority of chronic renal dialysis patients studied. In contrast, such aluminium accumulations were absent in control patients. SIMS imaging of aluminium appeared to provide much better discrimination between the dialysis patient group and the control group than one of the most widely used techniques for measuring aluminium in bulk samples, graphite furnace atomic absorption spectrometry. Preliminary studies have shown the feasibility of quantifying focal aluminium SIMS images obtained from brain tissue using aluminium-loaded brain homogenates as reference standards.     

Ancient microbial activity recorded in fracture fillings from granitic rocks (Äspö Hard Rock Laboratory, Sweden)

Fracture minerals within the 1.8‐Ga‐old Äspö Diorite (Sweden) were investigated for fossil traces of subterranean microbial activity. To track the potential organic and inorganic biosignatures, an approach combining complementary analytical techniques of high lateral resolution was applied to drill core material obtained at ?450 m depth in the Äspö Hard Rock Laboratory. This approach included polarization microscopy, time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS), confocal Raman microscopy, electron microprobe (EMP) and laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS). The fracture mineral succession, consisting of fluorite and low‐temperature calcite, showed a thin (20–100 μm), dark amorphous layer lining the boundary between the two phases. Microscopic investigations of the amorphous layer revealed corrosion marks and, in places, branched tubular structures within the fluorite. Geochemical analysis showed significant accumulations of Si, Al, Mg, Fe and the light rare earth elements (REE) in the amorphous layer. In the same area, ToF‐SIMS imaging revealed abundant, partly functionalized organic moieties, for example, C_xH_y⁺, C_xH_yN⁺, C_xH_yO⁺. The presence of such functionalized organic compounds was corroborated by Raman imaging showing bands characteristic of C‐C, C‐N and C‐O bonds. According to its organic nature and the abundance of relatively unstable N‐ and O‐ heterocompounds, the organic‐rich amorphous layer is interpreted to represent the remains of a microbial biofilm that established much later than the initial cooling of the Precambrian host rock. Indeed, δ¹³C, δ¹⁸O and ⁸⁷Sr/⁸⁶Sr isotope data of the fracture minerals and the host rock point to an association with a fracture reactivation event in the most recent geological past.