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Heat stabilisation of the proteome in tissue biopsies

Figure 1. Phosphatase activity measured using SensoLyte pNPP phosphatise kit (Anaspec). Mouse brain cortexes were snap frozen and samples were solubilised using sonification in 1X PBS, pH 7.4 with or without chemical inhibitors. Activity was reduced in samples in which inhibitors had been added but not eliminated. In stabilised samples, the activity is at background level. Svensson M et al. Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics. J Proteome Res 2009; 8: 974–981.
Figure 2. Immunhistochemistry image of the phosphorylated form of CREB in mouse cerebellum, magnified 100 x. The upper panel shows the result of standard execution. The positively coloured cell nuclei, seen as larger red-brown spots, are mainly located closest to the surface, i.e. to the right, where the formalin has fixed the tissue first. In stabilised tissue, the middle and lower panels, positively coloured cell nuclei can be seen throughout the section, incuding after the sample has been stored for 24 h in room temperature prior to formalin fixation. The sections are coloured with haematoxylin; cell nuclei without phosphorylated CREB are small and blue.

Understanding the significance in disease states of post-translational modifications (PTMs) such as phosphorylation and their role as drug targets and biomarkers has become increasingly important. It is crucial to accurately determine the state of phosphorylation of a protein to elucidate its eventual role in the development of a disease.
by Dr M. Svensson

Removing a sample from its surrounding tissue causes a release of degradation mediators, which alter the levels and composition of proteins and PTMs. Consequently analytical results of biomarker studies reflect a mixture of the in vivo proteome and ex vivo degradation products. A new system, the Stabilizor T1, has been developed that utilises rapid and uniform thermal inactivation to eliminate all enzymatic degradation. This allows all solid tissues, e.g. biopsies, to be processed without using chemical inhibitors.

To demonstrate the system’s efficiency, phosphatase activity was compared in stabilised and non-stabilised mouse brain tissue, extracted with and without phosphatase- and protease inhibitors. It can be seen from Figure 1 that in the stabilised samples, phosphatase activity was neglible, but even with added inhibitors it was quite high in the untreated samples.

The system is compatible with the immunohistochemical imaging methods that are commonly used for diagnosis of cancer. Figure 2 shows that samples that have been stabilised prior to standard formalin fixation maintain a uniform phosphorylation state of CREB protein throughout the tissue for up to 24 hours at room temperature. The standard formalin fixation procedure only preserves this modified protein at the surface.

The Stabilizor system thus significantly reduces post-mortem protein modifications and allows the study of the true in vivo distribution of proteins and their modifications, of crucial importance in cancer research.

The author
Dr Marcus Svensson
Senior Researcher
Denator AB
Sweden




















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