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Neuropeptidomics study profiles hypothalamic “nucleus”, individual cells
The team also examined the peptide component of individual magnocellular neurons from within the SON. Although they detected far fewer molecules per Talk about fragmented thinking. The sample, the researchers did find a few brain is loaded with small peptide relatively abundant peptides. They could fragmentssprotein pieces carved from even detect compositional differences prohormones that change the behavior between nominally identical cells. “There of cells both near and far. is a trade-off,” says Sweedler. “If you Many such peptides have been charhave 100,000 cells, you get better depth acterized, yet according to of coverage. If you have a single Jonathan Sweedler of the Univercell, you only see those peptides at sity of Illinois Urbana-Champaign the highest level, but you can see (UIUC), many others remain to be cell-to-cell differences.” found. “Brain peptides are present Notably absent in the team’s at low levels, and they are difficult analyses was oxytocin, a SON-deto predict from genetic informarived neuropeptide important in tion alone,” Sweedler says, “so birth and in lactation. “Oxytocin they have to be measured, and really has to be there, there’s just they are still being discovered.” too much information about it,” In JPR (DOI 10.1021/pr800394e), says Sweedler, who says its abSweedler, Martha Gillette, Neil sence could be the result of anyKelleher, and colleagues at UIUC thing from poor extraction or iontake a stab at the problem for one ization to its rapid degradation. “I small piece of the brain, the sudo not claim that our approach is praoptic nucleus (SON) of the hycomprehensive and detects all the pothalamus, which releases peppeptides,” he stresses. tides that control osmotic balance, Pieces of mind. A fluorescent image (vertical) from a brain The present study is not the first lactation, and childbirth. slice through the SON of a 3-month-old rat and a labeled to investigate the peptidomics of According to Sweedler, the brain section showing vasopressin-containing magnocellular the brain, but it may be the first to neurons (horizontal) linked by mass spectra (blue and white) study had two primary goals: to examine so small a structure. of peptide profiles from the SON. compile a list of the neuropeptides Whereas other studies have tar(the “neuropeptidome”) found in geted such intact brain regions as the SON and to develop protocols the hypothalamus or hippocamfor detecting peptides from such pus, this study focused on an indicolleagues extracted the peptides by ussmall brain regions. vidual “nucleus” of cells within the hying a three-step sequential extraction. Conventional wisdom holds that these pothalamus; the SON contains only Finally, they pooled the extracts and peptides begin degrading within seconds ∼50,000 cells. subjected them to a battery of MS of the death of an animal. To stabilize The technique can easily be applied techniques. neuropeptides long enough to analyze to other brain regions, Sweedler notes. A total of 85 peptides were detected them, researchers typically microwave or In a recent report (Proc. Natl. Acad. from whole SON samples, including 20 heat the brain samples immediately beSci. U.S.A. 2008, 105, 12,527-12,532), that were novel. Most of the peptides were fore or after decapitation of the animals Sweedler’s team profiled the peptides derived from known prohormones, and being studied, thereby inactivating proreleased from the suprachiasmatic had termini that correlated to known proteolytic enzymes. Yet this technique, says nucleus, the portion of the hypothalatease cleavage sites. However, some pepSweedler, so disrupts anatomic strucmus that governs circadian rhythms, as tides arose instead from proteins such as tures that precise sample excision bea function of stimulation protocol or myelin-basic protein and ubiquitin, which comes difficult. time of day. are not prohormones, or they bore unParadoxically, physiologists studying Next up, Sweedler says, are studies usual cleavage sites. These novel peptides phenomena such as circadian rhythm to determine which SON neuropepmay or may not be functional, Sweedler and learning have shown that brain tides are released under specific condinotes, because the researchers didn’t test slices can be cultured ex vivo for several tions. “Presumably, only a subset of bioactivity. Some of these peptides may be days, and to all appearances, the slices the peptides we detect would change artifacts of the sample preparation procebehave as they do in vivo, neuropeptides with a particular stress,” he says. dures rather than products of natural celand all. Apparently, given proper condi—Jeffrey M. Perkel lular processes. tions, the peptide pools rapidly regenerLARRY MILLET, UIUC
ate after their initial depletions. Sweedler’s team set out to determine whether brain slice technology could be applied to the neuropeptidome. “This will allow us to link physiological studies with peptidomics,” he explains. The researchers prepared rat brain slices containing the hypothalamus and harvested supraoptic nuclei by taking tissue punches. Then, Sweedler and
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Journal of Proteome Research • Vol. 7, No. 11, 2008
10.1021/pr800672a
© 2008 American Chemical Society
