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A fast and mild method for iodine labelling of proteins is presented which is specifically designed and optimized for laser ablation (LA-)ICP-MS based proteomics. Single proteins (lysozyme, bovine serum albumin, cytochrome c and β-casein), whole proteomes (microsomal proteome of rats) and antibodies (anti-bovine casein, anti-bovine serum albumin) can be efficiently iodinated by means of potassium triiodide with minimal losses of antigen properties and antibody binding to iodinated proteins. A comparison with iodination by use of IODO-Beads is presented and it is shown that triiodide labelling is a fast, cheap and less laborious alternative without compromising the analytical figures of merit.
Carbohydrate functionalization of silver nanoparticles modulates cytotoxicity and cellular uptake
(2014)
Background
Increasing use of silver nanoparticles (Ag-NPs) in various products is resulting in a greater likelihood of human exposure to these materials. Nevertheless, little is still known about the influence of carbohydrates on the toxicity and cellular uptake of nanoparticles.
Methods
Ag-NPs functionalized with three different monosaccharides and ethylene glycol were synthesized and characterised. Oxidative stress and toxicity was evaluated by protein carbonylation and MTT assay, respectively. Cellular uptake was evaluated by confocal microscopy and ICP-MS.
Results
Ag-NPs coated with galactose and mannose were considerably less toxic to neuronal-like cells and hepatocytes compared to particles functionalized by glucose, ethylene glycol or citrate. Toxicity correlated to oxidative stress but not to cellular uptake.
Conclusions
Carbohydrate coating on silver nanoparticles modulates both oxidative stress and cellular uptake, but mainly the first has an impact on toxicity. These findings provide new perspectives on modulating the bioactivity of Ag-NPs by using carbohydrates.
We have developed lanthanide labeling strategies for antibodies to adapt conventional biochemical workflows like Western blot immunoassays for detection by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) analysis with a special interest to apply the multi-element capabilities of ICP-MS for the design of multiplexed immunoassays. In this paper the lanthanide labeling of antibodies with MeCAT was investigated and the reaction conditions were optimized for application in a Western blot immunoassay analyzed by LA-ICP-MS. Furthermore, the MeCAT labeling strategy was compared with two other commercially available labeling reagents, MAXPAR and SCN-DOTA. As a proof-of-principle experiment chemically induced alterations of cytochrome P450 protein expression were investigated and the suitability of the differentially labeled antibodies for Western blot immunoassays of a complex liver microsomal protein fraction was tested. Limits of detection (LODs) in the lower fmol range were reached in the Western blot application using MeCAT and MAXPAR as element labeling reagents, whereas even sub-fmol LODs can be achieved in a dot blot experiment for the pure antibodies.
A detailed characterization of metal-tagged antibodies is the prerequisite for the implementation of quantitative concepts in inductively coupled plasma–mass spectrometry (ICP-MS)-based bioanalysis or future medical diagnosis. In this paper, the common modification with bifunctional ligands containing maleimide residues as a reactive group was investigated in detail via size exclusion chromatography (SEC)-ICP-MS and liquid chromatography–time-of-flight (LC-TOF)-MS to determine the preservation of the antibody structure after tagging. Mouse monoclonal IgG modified with metal-coded tags (MeCATs) was used as a model system. Several antibody fragments were identified carrying different numbers of metal tags. In a second step, a functionality test was performed with isolated fragments where the antigen specificity was tested in a dot blot immunoassay.
A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS
(2017)
High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(III) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.
Development of theranostic concepts that include inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS (LA-ICP-MS) imaging can be hindered by the lack of a direct comparison to more standardly used methods for in vitro and in vivo evaluation; e.g. fluorescence or nuclear medicine. In this study a bimodal (or rather, hybrid) tracer that contains both a fluorescent dye and a chelate was used to evaluate the existence of a direct link between mass spectrometry (MS) and in vitro and in vivo molecular imaging findings using fluorescence and radioisotopes. At the same time, the hybrid label was used to determine whether the use of a single isotope label would allow for MS-based diagnostics.
Actual research demonstrates that LA-ICP-MS is capable of being used as an imaging tool with cellular resolution. The aim of this investigation was the method development for LA-ICP-MS to extend the versatility to quantitative and multiplexing imaging of single eukaryotic cells. For visualization of individual cells selected, lanthanide-labeled antibodies were optimized for immuno-imaging of single cells with LA-ICP-MS. The molar content of the artificial introduced labels per cell was quantified using self-made nitrocellulose-coated slides for matrix-matched calibration and calculated amounts were in the range of 3.1 to 17.8 atmol per cell. Furthermore, the quantification strategy allows a conversion of 2D intensity profiles based on counts per second (cps) to quantitative 2D profiles representing the molar amount of the artificial introduced elemental probes per pixel for each individual cell.
We developed new procedures for internal standardization and calibration to be used for laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for elemental micro mapping imaging of biological samples like Western blot membranes and tissue sections. These procedures are based on printing of metal spiked inks onto the top of thin layer samples for simultaneous internal standardization and calibration of LA-ICP-MS. In the case of internal standardization the ink is spiked with indium as an internal standard and homogenously printed over the entire membrane (size 56 cm2) prior to LA-ICP-MS detection, a standard deviation (RSD) value of 2% was achieved. In the second approach the metal content of lanthanide tagged proteins and antibodies after biological work flows was quantified by LA-ICP-MS on nitro-cellulose membranes. In this case the inks spiked with varying metals were printed with different densities on the same nitrocellulose membranes in well-defined squares to produce matrix-matched calibration standards. For validation and calibration the ink squares were excised and the specific metal content was measured by liquid ICP-MS after solubilization of the membrane slice. For the printed calibration standard limits of detection (LOD) of <4 fmol for different metals and relative process standard deviations of 12% only were determined via LA-ICP-MS.
Today, quantitative data play a pivotal role in the understanding of biological processes. This is particularly true for the proteome: protein quantification always follows protein identification. To obtain useful and reliable quantitative data, rather sophisticated strategies using electrospray and MALDI mass spectrometry have been developed, which allow relative and sometimes even absolute quantification. All of those strategies have merits and limitations. In order to overcome some of these limits, methods based on the reliable and sensitive detection and quantification of heavy metals present in proteins using inductively coupled plasma (ICP)-MS have been reported. With specific labels carrying heavy metals, the applicability of ICP-MS has been extended to almost every protein. One of such covalently bound metal tags, allowing the quantification of low abundant proteins, uses 1,4,7,10-tetraazacyclododecane N,N',N'',N''-tetraacetic acid (DOTA) chelate complexes carrying lanthanides as the metal core. In this review the scope and limitations of peptide and protein quantification will be addressed. The metal tags do not only provide low detection limits, but also due to the large number of different lanthanides and lanthanide isotopes, multiplexing capabilities and previously unknown accuracy based on inherently possible isotope dilution methods came into reach. The developed workflows, including electrophoretic and chromatographic separation and preconcentration techniques, will be addressed to allow a comparison with already established procedures.
The analysis of single cells is a growing research field in many disciplines such as toxicology, medical diagnosis, drug and cancer research or metallomics, and different methods based on microscopic, mass spectrometric, and spectroscopic techniques are under investigation. This review focuses on the most recent trends in which inductively coupled plasma mass spectrometry (ICP-MS) and ICP optical emission spectrometry (ICP-OES) are applied for single-cell analysis using metal atoms being intrinsically present in cells, taken up by cells (e.g., nanoparticles), or which are artificially bound to a cell. For the latter, especially element tagged antibodies are of high interest and are discussed in the review. The application of different sample introduction systems for liquid analysis (pneumatic nebulization, droplet generation) and elemental imaging by laser ablation ICP-MS (LA-ICP-MS) of single cells are highlighted. Because of the high complexity of biological systems and for a better understanding of processes and dynamics of biologically or medically relevant cells, the authors discuss the idea of 'multimodal spectroscopies.'
The development of iron oxide nanoparticles for biomedical applications requires accurate histological evaluation. Prussian blue iron staining is widely used but may be unspecific when tissues contain substantial endogenous iron. Here we tested whether microscopy by laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is sensitive enough to analyze accumulation of very small iron oxide particles (VSOP) doped with europium in tissue sections.
A new laser ablation (LA)-ICP-MS method for single cell and cell nucleus imaging was developed. Therein, iodine was employed as an elemental dye for fibroblast cells and for thin tissue sections. At an incubation time of 60 s, iodine is located mainly within the cell nuclei. This effect was illustrated in fibroblast cells, and iodine signal within the cell nucleus was as high as 5 × 104 cps at 4 µm laser spot size. The surrounding cytoplasm was iodinated as well, but to a lesser extent. The spatial resolution attained was sufficient to detect even smaller cell nuclei within a liver biopsy tissue. Furthermore, iodine was successfully employed for biomolecule labeling and we demonstrated that iodine signal increased with increasing thickness of a palatine tonsil tissue. Thus, the use of iodine as an internal standard to correct for tissue inhomogeneities in LA-ICP-MS was investigated for the simultaneous detection of two tumor markers (Her 2 and CK 7) in breast cancer tissue. Additionally, lanthanide background resulting from glass ablation can be corrected for by Eu standardization.
We optimized multiplexed immunohistochemistry (IHC) on breast cancer tissue. Up to 20 tumor markers are routinely evaluated for one patient, and thus, a common analysis results in a series of time consuming staining procedures. As an alternative, we used lanthanides for labeling of primary antibodies, which are applied in IHC. Laser ablation (LA) ICPMS was elaborated as a detection tool for multiplexed IHC of tissue sections. In this study, we optimized sample preparation steps and LA ICPMS parameters to achieve a sufficient signal-to-background ratio. The results prove the high selectivity of applied antibodies, which was sustained after labeling. Up to three tumor markers (Her 2, CK 7, and MUC 1) were detected simultaneously in a single multiplex analysis of a 5 µm thin breast cancer tissue at a laser spot size of 200 µm. Furthermore, the LA ICPMS results indicate a significantly higher expression level of MUC 1 compared to Her 2 and CK 7, which was not obvious from the conventionally stained tissue sections.
The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA-ICP-MS.
The paper presents a new multi-parametric protein microarray embracing the multi-analyte capabilities of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The combination of high throughput reverse phase protein microarrays with element tagged antibodies and LA-ICP-MS makes it possible to detect and quantify many proteins or biomarkers in multiple samples simultaneously. A proof of concept experiment is performed for the analysis of cytochromes particularly of cytochrome P450 enzymes, which play an important role in the metabolism of xenobiotics such as toxicants and drugs. With the aid of the LA-ICP-MS based multi-parametric reverse phase protein microarray it was possible to analyse 8 cytochromes in 14 different proteomes in one run. The methodology shows excellent detection limits in the lower amol range and a very good linearity of R² ≥ 0.9996 which is a prerequisite for the development of further quantification strategies.
Quantitative analysis of cytochrome P450 (CYP) patterns of cells and tissues is an important aspect in toxicological and pharmacological research as this group of enzymes is largely involved in the metabolism of toxic compounds and drugs. Here we present a method for the multi-parametric and simultaneous quantitative determination of several cytochromes P450 in liver microsomes of untreated and inducer treated rats. The method is based on the binding of specifically lanthanide labelled antibodies to electrophoretically separated and blotted CYP proteins and their subsequent identification and quantification by LA-ICP-MS. CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A1 were simultaneously quantified and the patterns between microsomal samples were compared. Microsomes of rats treated with 3-methylcholanthrene, phenobarbital and dexamethasone showed increased levels of CYP1A1, CYP2B1 and CYP3A1, respectively. These results coincide with data obtained by independent methods for CYP quantification, i.e. ethoxyresorufin O-deethylase activity for CYP1A1 and pentoxyresorufin O-depentylase for CYP2B1. The presented method is useful for multi-parametric CYP profiling and has further large potential with respect to the number of analysed parameters/proteins and sensitivity.
Formalin-fixed paraffin-embedded (FFPE) specimen from biopsy materials are a widespread sample format for pathologists and medical researchers. Pathologists are archiving vast numbers of FFPE samples which can be stored for decades. Conventional immunohistochemical staining (IHC) of biomarkers on FFPE tissue sections is one of the most important analytical techniques for cancer diagnosis and pathology in general. However standardization for IHC samples and quality management is tedious and differs significantly from clinic to clinic. Combining established IHC staining strategies with modern mass spectrometry mediated methods would increase it`s potential and enable access of large FFPE archives for multiplexed quantitation purposes. In this work element mass spectrometry and a new ink-jet printed internal standardization approach was successfully combined with IHC staining to facilitate quantitative multiplex assays for archived FFPE samples. The printing strategy improves elemental image resolution and reproducibility of paraffin embedded breast cancer tissue sections in laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using conventional IHC staining as a model system to investigate the new capabilities of this technique. For the internal standardization we applied a conventional CD-ink-jet printer to print a metal spiked ink onto the top of thin layer tissue sections with constant density. Printing was carried out in a direct comparison to an iodination of the tissue section as previously described as an alternative standardization method. The use of the printed internal standard allowed correction of the fluctuation during the laser ablation process and compensated instrumental drift effects. Mediated by the ink correction approach we achieved better signal-to-background-ratios (SBR) of 74 and better spatial resolution of 30 µm compared to iodination (SBR=23). This improved performance was demonstrated on tumorous areas in FFPE breast cancer tissue sections and allowing detection of Her-2 in tumorous areas of this tissue with significantly improved contrast.
Superficial white matter (SWM) contains the most cortico-cortical white matter connections in the human brain encompassing the short U-shaped association fibers. Despite its importance for brain connectivity, very little is known about SWM in humans, mainly due to the lack of noninvasive imaging methods. Here, we lay the groundwork for systematic in vivo SWM mapping using ultrahigh resolution 7 T magnetic resonance imaging. Using biophysical modeling informed by quantitative ion beam microscopy on postmortem brain tissue, we demonstrate that MR contrast in SWM is driven by iron and can be linked to the microscopic iron distribution. Higher SWM iron concentrations were observed in U-fiber–rich frontal, temporal, and parietal areas, potentially reflecting high fiber density or late myelination in these areas. Our SWM mapping approach provides the foundation for systematic studies of interindividual differences, plasticity, and pathologies of this crucial structure for cortico-cortical connectivity in humans.
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.