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Multifunctional composite nanoprobes consisting of iron oxide nanoparticles linked to silver and gold nanoparticles, Ag–Magnetite and Au–Magnetite, respectively, were introduced by endocytic uptake into cultured fibroblast cells. The cells containing the non-toxic nanoprobes were shown to be displaceable in an external magnetic field and can be manipulated in microfluidic channels. The distribution of the composite nanostructures that are contained in the endosomal system is discussed on the basis of surfaceenhanced Raman scattering (SERS) mapping, quantitative laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping, and cryo soft X-ray tomography (cryo soft-XRT). Cryo soft-XRT of intact, vitrified cells reveals that the composite nanoprobes form intra-endosomal aggregates. The nanoprobes provide SERS signals from the biomolecular composition of their surface in the endosomal environment. The SERS data indicate the high stability of the nanoprobes and of their plasmonic properties in the harsh environment of endosomes and lysosomes. The spectra point at the molecular composition at the surface of the Ag–Magnetite and Au–Magnetite nanostructures that is very similar to that of other Composite structures, but different from the composition of pure silver and gold SERS nanoprobes used for intracellular investigations. As shown by the LA-ICP-MS data, the uptake efficiency of the magnetite composites is approximately two to three times higher than that of the pure gold and silver nanoparticles.
The study of the distribution of the cytostatic drugs cisplatin, carboplatin, and oxaliplatin along the kidney may help to understand their different nephrotoxic behavior. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) allows the acquisition of trace element images in biological tissues. However, results obtained are affected by several variations concerning the sample matrix and instrumental drifts. In this work, an internal standardization method based on printing an Ir-spiked ink onto the surface of the sample has been developed to evaluate the different distributions and accumulation levels of the aforementioned drugs along the kidney of a rat model. A conventional ink-jet printer was used to print fresh sagittal kidney tissue slices of 4 μm. A reproducible and homogenous deposition of the ink along the tissue was observed. The ink was partially absorbed on top of the tissue. Thus, this approach provides a pseudo-internal standardization, due to the fact that the ablation sample and internal standard take place subsequently and not simultaneously. A satisfactory normalization of LA-ICP-MS bioimages and therefore a reliable comparison of the kidney treated with different Pt-based drugs were achieved even for tissues analyzed on different days. Due to the complete ablation of the sample, the transport of the ablated internal standard and tissue to the inductively coupled plasma-mass spectrometry (ICP-MS) is practically taking place at the same time. Pt accumulation in the kidney was observed in accordance to the dosages administered for each drug. Although the accumulation rate of cisplatin and oxaliplatin is high in both cases, their Pt distributions differ. The strong nephrotoxicity observed for cisplatin and the absence of such side effect in the case of oxaliplatin could explain these distribution differences. The homogeneous distribution of oxaliplatin in the cortical and medullar areas could be related with its higher affinity for cellular transporters such as MATE2-k.
In a proof of concept study, metal-coded affinity tags based on click chemistry (MeCAT-Click) were used to analyze the proteome of Escherichia coli (E. coli) in response to heat stress. This allows high labeling efficiency, high detection sensitivity, and multiplex capabilities, which are pivotal for its application to protein quantification. Two approaches are presented for relative quantification of differentially lanthanide-labeled proteins. The first approach uses isotope-labeling, where ESI-MS was utilized to quantify the differentially labeled proteins from different states of E. coli. With this approach, 14 proteins were found with changed abundance, among them five proteins upregulated.
In the second approach, differentially labeled samples were separated by two dimensional gel electrophoresis (2 DE) and scanned by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Comparison of the signal intensities of the different lanthanides was used to quantify different sample states. Based on this information, ESI-MS was used to identify the proteins with different abundance. The sensitivity of LA-ICP-MS allowed us to find one upregulated protein that was nearly invisible by silver staining ("Probable replication endonuclease from retron EC67"). The advantage of this approach is to locate low abundant proteins with differential expression using LA-ICP-MS, which may be overlooked otherwise.
Biological significance: This paper demonstrates the successful application of a novel metal labeling strategy to quantify the proteins from complex biological samples. In comparison with former metal labeling strategies, it reduces the steric hindrance and improves the labeling efficiency during the labeling process, which ensure its successful application. This methodology is compatible with both molecular and elemental mass spectrometry. ESI-MS/MS in combination with software-based search allows the identification and relative quantification of labeled proteins. In addition, LA-ICP-MS helps to locate the labeled proteins in 2-DE gels with superior detection capability, thus, target proteins with low abundance can be precisely followed. Its excellent sensitivity allows one to track the proteins of interest that are barely visible by silver staining.
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.
Trace element concentrations in gold grains from various geological units in South Africa were measured in situ by field emission-electron probe microanalysis (FE-EPMA), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and synchrotron micro X-ray fluorescence spectroscopy (SR-µ-XRF). This study assesses the accuracy, precision and detection limits of these mostly non-destructive analytical methods using certified reference materials and discusses their application in natural sample measurement. FE-EPMA point analyses yielded reproducible and discernible concentrations for Au and trace concentrations of S, Cu, Ti, Hg, Fe and Ni, with detection limits well below the actual concentrations in the gold. LA-ICP-MS analyses required larger gold particles (> 60 µm) to avoid contamination during measurement. Elements that measured above detection limits included Ag, Cu, Ti, Fe, Pt, Pd, Mn, Cr, Ni, Sn, Hg, Pb, As and Te, which can be used for geochemical characterisation and gold fingerprinting. Although LA-ICP-MS measurements had lower detection limits, precision was lower than FE-EPMA and SR-µ-XRF. The higher variability in absolute values measured by LA-ICP-MS, possibly due to micro-inclusions, had to be critically assessed. Non-destructive point analyses of gold alloys by SR-µ-XRF revealed Ag, Fe, Cu, Ni, Pb, Ti, Sb, U, Cr, Co, As, Y and Zr in the various gold samples. Detection limits were mostly lower than those for elements measured by FE-EPMA, but higher than those for elements measured by LA-ICP-MS.