TY - JOUR A1 - Müller, Larissa A1 - Jakubowski, Norbert A1 - Hayen, H. A1 - Roos, P.H. T1 - Iodination of proteins, proteomes and antibodies with potassium triodide for LA-ICP-MS based proteomic analyses N2 - 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. PY - 2011 DO - https://doi.org/10.1039/c1ja10090d SN - 0267-9477 SN - 1364-5544 N1 - Geburtsname von Müller, Larissa: Wäntig, L. - Birth name of Müller, Larissa: Wäntig, L. VL - 26 SP - 1610 EP - 1618 PB - Royal Society of Chemistry CY - London AN - OPUS4-24209 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Polatajko, A. A1 - Feldmann, I. A1 - Hayen, H. A1 - Jakubowski, Norbert T1 - Combined application of a laser ablation-ICP-MS assay for screening and ESI-FTICR-MS for identification of a Cd-binding protein in Spinacia oleracea L. after exposure to Cd N2 - We have studied the binding of the toxic element Cd to plant proteins and have used for this purpose spinach (Spinacia oleracea L.) plants treated with 50 µM Cd(II) as a model system. Laser ablation ICP-MS has been applied for the screening of Cd-binding proteins after separation by native anodal polyacrylamide gel electrophoresis (AN-PAGE) and electroblotting onto membranes. The main Cd-carrying protein band was isolated and investigated by nano-electrospray ionization–Fourier transform ion cyclotron resonance (FTICR) mass spectrometry after tryptic digestion. By this procedure, the main Cd-binding protein was identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). The latter enzyme has been discussed in the literature to be affected in its activity by oxidative stress induced by Cd. However, in this paper it is demonstrated for the first time that RuBisCO directly binds Cd and thus may be directly altered by this toxic element. A commercially available protein standard was used to verify direct binding of Cd(II) to the protein, even without metabolisation. The resulting metal–protein complex was shown to be stable enough to survive AN-PAGE separation and electroblotting. By the use of size exclusion chromatography coupled with ICP-MS it was demonstrated that the RuBisCO protein standard shows similar metal binding properties to Cd. Furthermore, essential elements such as Mn(II), Fe(II) and Cu(II), which are known to possibly replace the RuBisCO activator Mg(II), were investigated in addition to Zn(II). Again, similar binding properties in comparison to the plant protein were observed. PY - 2011 DO - https://doi.org/10.1039/c1mt00051a SN - 1756-5901 SN - 1756-591X VL - 3 IS - 10 SP - 1001 EP - 1008 PB - RSC Publ. CY - Cambridge AN - OPUS4-24963 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Moreno-Gordaliza, E. A1 - Esteban-Fernández, D. A1 - Giesen, Charlotte A1 - Lehmann, K. A1 - Lázaro, A. A1 - Tejedor, A. A1 - Scheler, C. A1 - Canas, B. A1 - Jakubowski, Norbert A1 - Linscheid, M.W. A1 - Gómez-Gómez, M.M. T1 - LA-ICP-MS and nHPLC-ESI-LTQ-FT-MS/MS for the analysis of cisplatin-protein complexes separated by two dimensional gle elctrophoresis in biological samples N2 - A method for the analysis of Pt–protein complexes in biological samples, previously subjected to cisplatin treatment, has been developed. Proteins were separated by gel electrophoresis, and those bound to Pt were detected with high sensitivity by LA-ICP-(SF)-MS. Pt-containing spots were in-gel digested with trypsin, and the peptides produced identified using nHPLC-ESI-LTQ-FT-MS/MS. The influence of protein separation conditions, staining and gel processing prior to laser ablation on Pt–protein bonds preservation have been evaluated using standard proteins incubated with cisplatin. 2-DE separation under non-reducing conditions followed by either Coomassie blue brilliant or silver staining is appropriate for Pt–protein complexes, achieving a good separating resolution of the proteins in biological samples. Direct LA-ICP-MS analysis of glycerol-treated dried gels for Pt–protein monitoring resulted in better sensitivity, more reliable relative Pt signals and a simpler and less time-consuming approach compared to the analysis of blotted membranes. Ablation of gels allowed tackling protein identification of Pt-spots in the remaining non-ablated material in the gel, making it unnecessary to run several gels in parallel for separate Pt detection and protein identification. By using this approach, Pt coordinated to proteins, such as α-2-macroglobulin, transferrin, albumin or hemoglobin, was detected in the serum from a rat treated in vivo with cisplatin after nrSDS-PAGE separation. Furthermore, the first complete LA-ICP-MS metalloprotein contour map in a 2-DE gel has been produced, in this case for the detection of Pt–protein complexes in renal proximal tubule epithelial cells (RPTECs) incubated with cisplatin. Several proteins were identified in those spots containing Pt, which may have a connection with the drug-induced nephrotoxicity mainly affecting this cell type in the kidney. PY - 2012 DO - https://doi.org/10.1039/c2ja30016h SN - 0267-9477 SN - 1364-5544 VL - 27 IS - 9 SP - 1474 EP - 1483 PB - Royal Society of Chemistry CY - London AN - OPUS4-26562 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Richter, Janine A1 - Fettig, Ina A1 - Piechotta, Christian A1 - Philipp, Rosemarie A1 - Jakubowski, Norbert T1 - Tributylzinn in Gesamtwasserproben - Entwicklung eines Referenzverfahrens für die EU-Wasserrahmenrichtlinie PY - 2013 SN - 0016-3538 IS - 9 SP - 2 EP - 4 PB - GIT-Verlag CY - Darmstadt AN - OPUS4-29040 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Müller, Larissa A1 - Techritz, Sandra A1 - Jakubowski, Norbert A1 - Roos, P.H. T1 - A multi-parametric microarray for protein profiling: simultaneous analysis of 8 different cytochromes via differentially element tagged antibodies and laser ablation ICP-MS N2 - 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. KW - Multi-parametric KW - Multiplexing KW - Microarray KW - Immunoassay KW - LA-ICP-MS KW - Cytochrome P450 PY - 2013 DO - https://doi.org/10.1039/c3an00468f SN - 0003-2654 SN - 1364-5528 VL - 138 IS - 21 SP - 6309 EP - 6315 PB - Royal Society of Chemistry CY - Cambridge AN - OPUS4-29275 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - García Fernández, J. A1 - Sánchez-González, C. A1 - Bettmer, J. A1 - Llopi, J. A1 - Jakubowski, Norbert A1 - Panne, Ulrich A1 - Montes-Bayón, M. T1 - Quantitative assessment of the metabolic products of iron oxide nanoparticles to be used as iron supplements in cell cultures N2 - Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding Fe-57 as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1mM citrate to elute from the column. Exposed cells up to 0.5mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments. KW - Fe nanoparticles metabolism KW - Cells KW - HPLC-ICP-MS KW - Species-unspecific on-line isotope dilution PY - 2018 DO - https://doi.org/10.1016/j.aca.2018.08.003 SN - 0003-2670 VL - 1039 SP - 24 EP - 30 PB - Elsevier CY - Amsterdam AN - OPUS4-46817 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Giesen, C. A1 - Müller, Larissa A1 - Panne, Ulrich A1 - Jakubowski, Norbert T1 - History of inductively coupled plasma mass spectrometry-based immunoassays N2 - 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. KW - ICP-MS KW - LA-ICP-MS KW - Immunoassay KW - Elemental tagging KW - Multiplexing PY - 2012 DO - https://doi.org/10.1016/j.sab.2012.06.009 SN - 0584-8547 SN - 0038-6987 N1 - Geburtsname von Müller, Larissa: Wäntig, L. - Birth name of Müller, Larissa: Wäntig, L. VL - 76 SP - 27 EP - 39 PB - Elsevier CY - Amsterdam AN - OPUS4-27686 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Müller, Larissa A1 - Jakubowski, Norbert A1 - Hösl, Simone A1 - Scheler, C. A1 - Roos, P.H. A1 - Linscheid, M.W. T1 - Comparison of different chelates for lanthanide labeling of antibodies and application in a Western blot immunoassay combined with detection by laser ablation (LA-)ICP-MS N2 - 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. PY - 2012 DO - https://doi.org/10.1039/c2ja30068k SN - 0267-9477 SN - 1364-5544 N1 - Geburtsname von Müller, Larissa: Wäntig, L. - Birth name of Müller, Larissa: Wäntig, L. N1 - Geburtsname von Hösl, Simone: Hardt, S. - Birth name of Hösl, Simone: Hardt, S. VL - 27 IS - 8 SP - 1311 EP - 1320 PB - Royal Society of Chemistry CY - London AN - OPUS4-26256 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Löhr, Konrad A1 - Traub, Heike A1 - Wanka, Antje Jutta A1 - Panne, Ulrich A1 - Jakubowski, Norbert T1 - Quantification of metals in single cells by LA-ICP-MS: Comparison of single spot analysis and imaging N2 - LA-ICP-MS is increasingly used for single cell analysis in two different detection modes using either the imaging mode with subcellular resolution or alternatively single spot analysis of cells with a larger laser spot size. This study compares the analytical figures of merit of both detection modes (signal to noise, precision, accuracy, throughput), as well as ease of operation and data evaluation. Adherent 3T3 fibroblast cells were stained with two metal dyes (mDOTA-Ho, Ir-DNA-intercalator) and several dozen cells were measured using both modes. We found a ten times higher throughput for single spot analysis, which has as well a straightforward data analysis, shortening the total analysis time further. The signal to noise ratio for single spot analysis was found to be slightly better compared to the signal to noise of pixels in imaging. The mean metal intensity per single cell differed by only 10% between both modes and obtained distributions were found to show no statistically significant differences. Using matrix matched calibration based on standards spotted onto nitrocellulose membrane, we achieved detection limits (10s) of 12 fg for Ir and 30 fg for Ho and quantified 57 +/-35 fg Ir and 1192 +/- 707 fg Ho per single cell. Compared to a conventional ICP-MS measurement of a digest of about 60000 cells, 54% of Ir content and 358% Ho content was found using quantitative LA-ICP-MS. The difference might be a consequence of the two metal dyes binding to different structures of the cell and therefore might behave differently in sample preparation for conventional and LA-ICP-MS. KW - Cells KW - Laser ablation KW - ICP-MS KW - Metals KW - Quantification PY - 2018 DO - https://doi.org/10.1039/c8ja00191j SN - 0267-9477 VL - 33 IS - 9 SP - 1579 EP - 1587 PB - RSC Royal Society of Chemistry CY - London AN - OPUS4-46441 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Loehr, Konrad A1 - Jakubowski, Norbert A1 - Wanka, Antje Jutta A1 - Traub, Heike A1 - Panne, Ulrich T1 - Quantification of metals in single cells by LA-ICP-MS comparison of single spot analysis and imaging N2 - LA-ICP-MS is increasingly used for single cell analysis in two different detection modes using either the imaging mode with subcellular resolution or alternatively single spot analysis of cells with a larger laser spot size. This study compares the analytical figures of merit of both detection modes (signal to noise, precision, accuracy, throughput), as well as ease of operation and data evaluation. Adherent 3T3 fibroblast cells were stained with two metal dyes (mDOTA-Ho, Ir-DNA-intercalator) and several dozen cells were measured using both modes. We found a ten times higher throughput for single spot analysis, which has as well a straightforward data analysis, shortening the total analysis time further. The signal to noise ratio for single spot analysis was found to be slightly better compared to the signal to noise of pixels in imaging. The mean metal intensity per single cell differed by only 10% between both modes and obtained distributions were found to show no statistically significant differences. Using matrix matched calibration based on standards spotted onto nitrocellulose membrane, we achieved detection limits (10 σ) of 12 fg for Ir and 30 fg for Ho and quantified 57 ± 35 fg Ir and 1,192 ± 707 fg Ho per single cell. Compared to a conventional ICP-MS measurement of a digest of ~60,000 cells, 54 % of Ir content and 358 % Ho content was found using quantitative LA-ICP-MS. The difference might be a consequence of the two metal dyes binding to different structures of the cell and therefore might behave differently in sample preparation for conventional and LA-ICP-MS. KW - Single cell analysis KW - LA-ICP-MS PY - 2018 DO - https://doi.org/10.1039/c8ja00191j SN - 0267-9477 SN - 1364-5544 VL - 33 IS - 9 SP - 1579 EP - 1587 PB - Royal Society of Chemistry AN - OPUS4-45903 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -