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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 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 Ptprotein 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.
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.
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.
Recent recommendations by the Food and Drug Administration1 and the European Medicines Agency2 are to limit the clinical use of linear gadolinium-based contrast agents (GBCAs) due to convincing evidence of deposition in tissues. Macrocyclic GBCA continued to be considered safe, provided that patients have normal renal function. To date, given the low sensitivity of conventional MRI, there has been a debate about the signal increase following the injections of a macrocyclic GBCA.
Complementarity of molecular and elemental mass spectrometric imaging of Gadovist™ in mouse tissues
(2019)
Drug biodistribution analyses can be considered a key issue in pharmaceutical discovery and development. Here, mass spectrometric imaging can be employed as a powerful tool to investigate distributions of drug compounds in biologically and medically relevant tissue sections. Both matrix-assisted laser desorption ionization–mass spectrometric imaging as molecular method and laser ablation inductively coupled plasma–mass spectrometric imaging as elemental detection method were applied to determine drug distributions in tissue thin sections. Several mouse organs including the heart, kidney, liver, and brain were analyzed with regard to distribution of Gadovist™, a gadolinium-based contrast agent already approved for clinical investigation. This work demonstrated the successful detection and localization of Gadovist™ in several organs. Furthermore, the results gave evidence that gadolinium-based contrast agents in general can be well analyzed by mass spectrometric imaging methods. In conclusion, the combined application of molecular and elemental mass spectrometry could complement each other and thus confirm analytical results or provide additional information.