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Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Published on: November 14, 2013
Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary
Fa-Yun Che1, Jihyeon Lim, Hui Pan
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Abstract:
In neuropeptidomics, the degradation of a small fraction of abundant proteins overwhelms the low signals from neuropeptides, and many neuropeptides cannot be detected by mass spectrometry without extensive purification. Protein degradation was prevented when mice were sacrificed with focused microwave irradiation, permitting the detection of hypothalamic neuropeptides by mass spectrometry. Here we report an alternative and very simple method utilizing an ordinary microwave oven to inhibit enzymatic degradation. We used this technique to identify brain and pituitary neuropeptides. Quantitative analysis using mass spectrometry in combination with stable isotopic labeling was performed to determine the effect of microwave irradiation on relative levels of neuropeptides and protein degradation fragments. Microwave irradiation greatly reduced the levels of degradation fragments of proteins. In contrast, neuropeptide levels were increased about 2-3 times in hypothalamus by the microwave irradiation but not increased in pituitary. In a second experiment, three brain regions (hypothalamus, hippocampus, and striatum) from microwave-irradiated mice were analyzed. Altogether 41 neuropeptides or fragments of secretory pathway proteins were identified after microwave treatment; some of these are novel. These peptides were derived from 15 proteins: proopiomelanocortin, proSAAS, proenkephalin, preprotachykinins A and B, provasopressin, prooxytocin, melanin-concentrating hormone, proneurotensin, chromogranins A and B, secretogranin II, prohormone convertases 1 and 2, and peptidyl amidating monooxygenase. Although some protein degradation fragments were still found after microwave irradiation, these appear to result from protein breakdown during the extraction and not to an enzymatic reaction during the postmortem period. Two of the protein fragments corresponded to novel protein forms: VAP-33 with a 7-residue N-terminal extension and beta tubulin with a glutathione on the Cys near the N terminus. In conclusion, microwave irradiation with an ordinary microwave oven effectively inhibits enzymatic postmortem protein degradation, increases the recovery of neuropeptides, and makes it possible to conduct neuropeptidomic studies with mouse brain tissues.
Insights
Using an ordinary microwave oven to sacrifice mice effectively inhibits protein degradation, significantly increasing neuropeptide detection in brain tissues for neuropeptidomics research.
Area of Science:
- Neuroscience
- Biochemistry
- Analytical Chemistry
Background:
- Neuropeptide detection is hindered by abundant protein degradation products.
- Extensive purification is often required for mass spectrometry-based neuropeptide identification.
Purpose of the Study:
- To present a simple method using an ordinary microwave oven to inhibit postmortem enzymatic protein degradation.
- To enhance neuropeptide detection and identification in brain tissues.
Main Methods:
- Mice were sacrificed using an ordinary microwave oven to prevent enzymatic degradation.
- Mass spectrometry with stable isotopic labeling was used for quantitative analysis.
- Neuropeptides and protein fragments were identified in hypothalamus, pituitary, hippocampus, and striatum.
Main Results:
- Microwave irradiation significantly reduced protein degradation fragments.
- Neuropeptide levels increased 2-3 fold in the hypothalamus but not the pituitary.
- 41 neuropeptides or protein fragments were identified, including novel forms.
Conclusions:
- Microwave irradiation is an effective and simple method to inhibit postmortem protein degradation.
- This technique enhances neuropeptide recovery and facilitates neuropeptidomic studies.
- Novel neuropeptides and protein forms were identified using this approach.
