Search

The colocation of elemental species with host biomolecules such as lipids and metabolites may shed new light on the dysregulation of metabolic pathways and how these affect disease pathogeneses. Alkali metals have been the subject of extensive research, are implicated in various neurodegenerative and infectious diseases and are known to disrupt lipid metabolism. Desorption electrospray ionisation (DESI) is a widely used approach for molecular imaging, but previous work has shown that DESI delocalises ions such as potassium (K) and chlorine (Cl), precluding the subsequent elemental analysis of the same section of tissue. The solvent typically used for the DESI electrospray is a combination of methanol and water. Here we show that a novel solvent system, (50:50 (% v / v ) MeOH:EtOH) does not delocalise elemental species and thus enables elemental mapping to be performed on the same tissue section post-DESI. Benchmarking the MeOH:EtOH electrospray solvent against the widely used MeOH:H 2 O electrospray solvent revealed that the MeOH:EtOH solvent yielded increased signal-to-noise ratios for selected lipids. The developed multimodal imaging workflow was applied to a lung tissue section containing a tuberculosis granuloma, showcasing its applicability to elementally rich samples displaying defined structural information.

Characterizing proton beam damage in biological materials is of interest to enable the integration of proton microprobe elemental mapping techniques with other imaging modalities. It is also of relevance to obtain a deeper understanding of mechanical damage to lipids in tissues during proton beam cancer therapy. We have developed a novel strategy to characterize proton beam damage to lipids in biological tissues based on mass spectrometry imaging. This methodology is applied to characterize changes to lipids in tissues ex vivo, irradiated under different conditions designed to mitigate beam damage. This work shows that performing proton beam irradiation at ambient pressure, as well as including the application of an organic matrix prior to irradiation, can reduce damage to lipids in tissues. We also discovered that, irrespective of proton beam irradiation, placing a sample in a vacuum prior to desorption electrospray ionization imaging can enhance lipid signals, a conclusion that may be of future benefit to the mass spectrometry imaging community.

Elemental and molecular imaging play a crucial role in understanding disease pathogenesis. To accurately correlate elemental and molecular markers, it is desirable to perform sequential elemental and molecular imaging on a single-tissue section. However, very little is known about the impact of performing these measurements in sequence. In this work, we highlight some of the challenges and successes associated with performing elemental mapping in sequence with mass spectrometry imaging. Specifically, the feasibility of molecular mapping using the mass spectrometry imaging (MSI) techniques matrix-assisted laser desorption ionization (MALDI) and desorption electrospray ionization (DESI) in sequence with the elemental mapping technique particle-induced X-ray emission (PIXE) is explored. Challenges for integration include substrate compatibility, as well as delocalization and spectral changes. We demonstrate that while sequential imaging comes with some compromises, sequential DESI-PIXE imaging is sufficient to correlate sulfur, iron, and lipid markers in a single tissue section at the 50 μm scale.

A new tissue sample embedding and processing method is presented that provides downstream compatibility with numerous different histological, molecular biology, and analytical techniques. The methodology is based on the low temperature embedding of fresh frozen specimens into a hydrogel matrix composed of hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) and sectioning using a cryomicrotome. The hydrogel was expected not to interfere with standard tissue characterization methods, histologically or analytically. We assessed the compatibility of this protocol with various mass spectrometric imaging methods including matrix-assisted laser desorption ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). We also demonstrated the suitability of the universal protocol for extraction based molecular biology techniques such as rt-PCR. The integration of multiple analytical modalities through this universal sample preparation protocol offers the ability to study tissues at a systems biology level and directly linking results to tissue morphology and cellular phenotype.

The aim of this study was the characterization of the human Gbeta4 subunit of heterotrimeric G proteins. Human Gbeta4 is widely expressed. Its gene is located on chromosome 3 with a genomic structure indistinguishable from that of the genes of Gbeta1 to Gbeta3, but entirely different from Gbeta5. In vitro translation co-precipitation analyses revealed that Gbeta4 can form stable dimers with Ggamma1, Ggamma2, Ggamma3, Ggamma4, Ggamma5, Ggamma7, Ggamma10, Ggamma11, Ggamma12, and Ggamma13, dimers which were also able to stimulate phospholipase beta2.

The T-allele of a polymorphism (C825T) in the gene for the G-protein beta 3 subunit (GNB3) is associated with cardiovascular and metabolic disorders, distinct cellular features and altered drug responses. The molecular mechanisms that give rise to this complex phenotype have been linked to the occurrence of G beta 3s, a splice variant of GNB3. G beta 3s is predominantly expressed in cells with the 825T-allele. In the present study we describe the identification and characterization of an additional G beta 3 splice variant referred to as G beta 3s2. Its mRNA is expressed in heart, blood cells and tumour tissue, and its expression is also tightly associated with the GNB3 825T-allele. G beta 3s2 is generated by alternative splicing using non-canonical splice sites. G beta subunits belong to the family of propeller proteins and consist of seven regular propeller blades. Transcripts for G beta 3s2 are lacking 129 bp of the coding sequence of the wild-type G beta 3 protein. Thus the predicted structure consists of only six propeller blades, which resembles the structure of G beta 3s. Co-immunoprecipitation analyses indicated that G beta 3s2 dimerizes with different G gamma subunits, e.g. G gamma 5, G gamma 8(C) and G gamma 12. In Sf9 insect cells, expression of G beta 3s2 together with G gamma 12 enhances receptor-stimulated activation of G alpha(i2). Expression of G beta 3s2 in mammalian cells activated the mitogen-activated protein kinase cascade. Together, these results suggest that G beta 3s2 is a biologically active G beta variant which may play a role in the manifestation of the complex phenotype associated with the 825T-allele.