Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation.Leibniz AssociationEU Horizon 2020Indiana University Graduate Training Program in Quantitative and Chemical Biolog

Spatially resolved quantification of the 5-HT2A receptor in brain tissues using praseodymium labelled antibodies in combination with LA-ICP-MS detection

Depression is a commonly occurring mental disease. Its therapy is carried out with medication as well as with psychotherapy. The family of 5-HT receptors has an important part for that because the effect of antidepressants can be enhanced by modulation of certain serotonin receptors. For example, a range of antidepressants and antipsychotics bind to the subcategory of 5-HT2A receptors. They are mainly located in the whole central nervous system. In the last years the interest in representing the distribution of elements is growing rapidly. It helps to define different processes more closely and helps to gain fundamental information. Therefore, it is relevant to know the distribution of these serotonin receptors. One opportunity to gain this information is found in immunohistochemistry. It is a simple and powerful technique. To represent the distribution, an antibody reacts specifically with the analyzed receptor. For visualization the primary antibody is detected directly or indirectly. For the direct method the primary antibody is tagged, for the indirect method a secondary antibody binds to the primary. The tag is either an enzyme or a fluorophore. Notwithstanding, this part is a limiting step because it is not always possible to find a suitable opportunity of visualization. In this work the 5-HT2A receptor is used as an example. An immunohistochemical assay is used with an indirect visualization using a secondary antibody that is tagged with a fluorophore. Following, an image of the distribution is created with a fluorescence microscopy. Since there is a limitation using immunohistochemistry, an alternative method is needed: With laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) it is possible to extend the range of representable receptors. For the purpose of unambiguous association of the selected receptor, it has to bind specifically to a labelled antibody. It is labelled to an element that is not naturally occurring in the tissue. For this reason, the rare earth element praseodymium is selected. Now it is possible to gain information about the distribution of this special serotonin receptor by representing the distribution of praseodymium with LA-ICP-MS. Nevertheless, there are some problems for this analytical method. During the measurement there is not only a change in the composition of the sample matrix but also variations in the experimental conditions. For this reason an internal standard is used and it has to grant a variety of requirements. It does not solve these problems it offers a possibility to take them into account. However, it has to meet some requirements. The elements gold and indium are candidates, whereas gold is applied as an additional layer on the sample and indium is present as a thin layer on the sample slides. Finally, indium turns out to be the better choice. To quantify the results from LA-ICP-MS, all kinds of approaches exist. In many cases either certified reference material or matrix matched standards are used. Nevertheless, these two opportunities are either barely available or it is a time-consuming procedure and not easy in handling. To avoid these problems, standards are used that simulate the analyzed tissue. Since the analyzed tissue is brain, gelatin is used because it imitates animal material. For the gelatin standards, the rare earth elements lanthanum, cerium, praseodymium and europium are used. They make it possible to quantify the distribution image of the 5-HT2A receptor.6