Quantitative Neuropeptidomics of Microwave-irradiated Mouse Brain and Pituitary

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 ␤ 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. Peptides perform many important functions throughout the body as hormones and neurotransmitters. Neuropeptides are involved in a wide variety of systems, including reward mechanisms, pain, memory, food intake and body weight regulation, circadian rhythms, and many others (1, 2). A large number of studies have examined the changes in levels of various peptides upon different treatments or in different physiological states. These studies typically measured peptide levels using radioimmunoassays (RIAs).1 Although this approach is sensitive, most antisera are not specific for a single peptide and are able to cross-react with N-and/or C-terminally extended peptides and with peptides modified by post-translational modifications such as acetylation, phosphorylation, or sulfation (3). Also RIAs ...

Neuropeptidomics: MS Applied to the Discovery of Novel Peptides from the Brain

“…Recently, other researchers have confirmed several of the novel endogenous peptides that we have published (30,33,45). Most of the peptides listed in Table 1 are cleaved enzymatically at characteristic processing sites for prohormone convertases.…”

“…Hence, neuropeptidomics is the technological approach for detailed analyses of endogenous peptides from the brain (5,23,(30)(31)(32)(33)(34). The levels of peptides in the brain reflect certain information about physiological status; this information can be revealed when MS is used to generate broad profiles of the dynamic neuropeptide pattern.…”

“…Another method to prevent postmortem degradation is to use a microwave to heat the brain instantly after it is removed (30). Although there is a delay of several seconds before the brain is heated to 80 °C, this does not seem to contribute to neuropeptide degradation.…”

“…Although there is a delay of several seconds before the brain is heated to 80 °C, this does not seem to contribute to neuropeptide degradation. Nevertheless, it should be noted that several hemoglobin degradation fragments were detected, although it is not clear whether these fragments were generated during the peptide-extraction procedure or the in vitro microwave heating or were, in fact, degradation products due to the postmortem delay before the sample was frozen (30).…”

Neuropeptide alterations in the tree shrew hypothalamus during volatile anesthesia

“…Enzyme inactivation should be performed immediately post-sampling, otherwise protein degradation products would contaminate the sample and the endogenous peptides would be difficult or impossible to analyze. Following decapitation, the head was immediately heated up to 80°C in 16 s using microwave irradiation as described in Che et al [16]. The brain was removed and stored at −80°C.…”