Filtern
Dokumenttyp
Sprache
- Englisch (5)
Referierte Publikation
- ja (5)
Schlagworte
- Cytochrome P450 (1)
- Immunoassay (1)
- LA-ICP-MS (1)
- Microarray (1)
- Multi-parametric (1)
- Multiplexing (1)
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 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.
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.
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.
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.