Meeting the Challenge of Biological Changes Postsampling

Understanding the role of proteins, peptides, and their post-translational modifications, e.g., phosphorylations, in normal and diseased biological samples is crucial to elucidate in vivo biological mechanisms or identifying and defining these biological molecules’ potential use as drugs, drug targets, or disease biomarkers.

Yet biological change begins from the moment a tissue sample is removed from its native environment. This disruption of normal control mechanisms causes drastic alterations at the molecular level. This is manifested by alterations in phosphorylation states, the breakdown of proteins due to uncontrolled proteolytic activity, and changes to metabolite levels where molecules such as adenosine triphosphate (ATP) are metabolized in order to release energy.

In short, sample quality deteriorates rapidly, and vital information about the in vivo components may be lost or distorted. Needless to say, this is a major analytical challenge because the reduction in sample quality increases the risks of incorrect data interpretation and drawing misleading conclusions. In order to obtain meaningful scientific results, it is crucial to efficiently control sample components directly after sampling.

Preventing biological changes

An additive-free sample preservation technology that preserves the quality of biological samples throughout the entire workflow has been developed. The Stabilizor system (Denator, Gothenburg, Sweden; www.denator.com) utilizes rapid conductive heating to generate fast, homogeneous, and irreversible thermal denaturation of proteins. This results in complete and permanent elimination of enzymatic activity and a stable sample that will not degrade or change.

The technology builds on the principle of thermal denaturation: In its active, native form, proteins and their activity are dependent on the protein’s tertiary structure (3-D fold). During heat stabilization, the proteins unfold into inactive, random configurations and, upon cooling, refold into a new denatured and inactivated structure, keeping the sample from any further enzymatic changes.

A permanent, inhibitor-free alternative

Conventional approaches of reducing and inhibiting these biological changes after sampling include snap-freezing, the addition of inhibitor cocktails, changes to pH, or cross-linking. These established measures may halt, suppress, and even reduce enzymatic activity, but they are not permanent solutions to inactivation since enzymes’ native structure can resume during further sample handling if the sample environment changes. Residual enzymatic activity can also be present due to incomplete inhibition due to the composition of the cocktail used. In addition, inhibitors may also interfere with downstream analytical techniques.

It has been demonstrated that kinase and phosphatase activities, critical in the control of phosphorylation states, are eliminated in heat-stabilized tissue. In a study from Harvard Medical School, it was shown that heat stabilization abolishes 99.6% of the kinase activity that otherwise would be present.1 In addition, in a comparison with snap-frozen samples, the phosphatase activity in heat-stabilized samples was shown to be equivalent to background assay levels. Even in the presence of inhibitors, snap-frozen samples contained significantly higher enzyme activity.2 This increases the accuracy and quality of the obtained analytical results. After heat stabilization of either fresh or frozen tissue samples, in a Stabilizor system, biological components can be extracted and analyzed using common buffers and techniques.

Case study: Preserving phosphorylation states

The ability to determine phosphorylation states that reflect the in vivo status as closely as possible is crucial to understanding the role of phosphoproteins. This is of particular interest for diseases such as cancer and neurodegeneration, and also in disease prognosis and in determining the action of a potential therapeutic.

Prof. Katheleen Gardiner is heading a group of scientists in the division of Genetics in the Department of Pediatrics at the University of Colorado (Boulder). They are focusing their efforts on understanding the mechanisms of Down syndrome on a molecular level by analyzing protein expression and pathways in mouse model systems. A major challenge when studying proteins and their pathways in the mouse brain is to obtain reproducible and accurate measurement of molecular events. The protein profiles are highly affected by the changes occurring in the tissue sample after sampling.

Figure 1 ‒ Phosphorylation states remain constant in heat-stabilized samples. Statistical significance determined by unpaired Student’s t-test.

In their evaluation of the heat-stabilization technology compared to conventional snap-freezing of hippocampal lysates, it was confirmed that phosphorylation states were preserved by heat stabilization. Phosphorylation states continued to change in snap-frozen samples left for 30 min at room temperature, but remained constant in heat-stabilized samples under the same conditions3 (Figure 1).

Use of the Stabilizor system has enabled the research group to obtain reproducible and consistent protein profile studies, which is advantageous in their pursuit of drugs with the potential for significant benefits to individuals with Down syndrome.

Case study: Detecting more, novel endogenous peptides

Many endogenous peptides exist at very low concentrations compared to other larger proteins that are present at much higher concentrations. To detect and determine the role of these endogenous peptides is particularly challenging when proteolytic activity after sampling produces protein fragments that mask many these endogenous peptides.

Dr. Michelle Colgrave, a senior scientist at CSIRO Animal, Food and Health Sciences in Brisbane, Australia, is working to characterize the sequence, structure, and function of proteins associated with commercially important livestock production traits and diseases. As part of that work, she identifies novel proteins and characterizes their function as well as their post-translational modifications.

Figure 2 ‒ Neuropeptide profiling of the bovine hypothalamus. LC-MS profile: neurosecretory peptides VGF [489–506], CCK [21–44], GnRH, AL11, CLIP, HCNP, and BAM-1745 detected in heat-stabilized samples are diminished in snap-frozen samples. Degradation products of stathmin precursor (stathmin [2–20] and stathmin [2–22]) seen only in snap-frozen samples.

In a comparison with conventional snap-frozen samples, Dr. Colgrave and her team were able to reveal a greater number of neuropeptides in bovine hypothalamus tissue by using heat stabilization. Snap-frozen samples revealed fewer numbers of neuropeptides and instead showed large numbers of protein degradation products. As an example, the degradation products of protein stathmin (stathmin [2–20] and stathmin [2–22] peptide fragments) were prominent. The group was able to identify 140 candidate neuropeptides in heat-stabilized samples. Their results, using mass spectrometry, clearly demonstrate how heat stabilization enables the detection of more, potentially novel, endogenous peptides by preventing proteolytic degradation after sampling4 (Figure 2).

Figure 3 ‒ Heat stabilization can be used for almost any kind of tissue sample, and has been verified to be compatible with many downstream analytical techniques.

Conclusion

By using heat-stabilization, scientists have been able to drastically improve the consistency of sample quality and discover new, biologically relevant information without needing to use additives or modify buffer composition. This is of particular significance to research areas such as neuroscience, oncology, and proteomic research, where short-lived molecules and potential biomarkers need to be confidently identified.

Compatibility with a number of downstream analytical techniques has been verified, including LC-MS, phospho-shotgun-MS, matrix-assisted laser desorption ionization (MALDI) imaging-MS, Western blot with phospho-specific antibodies, 1-D and 2-D gel electrophoresis (GE) with phospho-specific stains, radioimmunoassay (RIA), and reversed-phase protein arrays (RPPAs) (Figure  3).

References

  1. Smejkal, G.B.; Rivas-Morello, C. et al. Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis. Electrophoresis  2011 Aug, 32(16), 2206‒15.
  2. Svensson, M.; Boren, M. et al. Heat stabilization of the tissue proteome: a new technology for improved proteomics. J. Proteome Res.  2009 Feb, 8(2), 974‒81. 
  3. Ahmed, M.M.; Gardiner, K.G. et al. Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization. J. Neurosci. Methods  2011 Mar 15, 196(1), 99‒106.
  4. Colgrave, M.L.; Xi, L. et al. Neuropeptide profiling of the bovine hypothalamus: thermal stabilization is an effective tool in inhibiting post-mortem degradation. Proteomics 2011 Apr, 11(7), 1264‒76.

Marcus Söderquist, Ph.D. (marcus@denator.com), is Senior Scientist at Denator, Gothenburg, Sweden; www.denator.com.

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