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RESEARCH PROFILE
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MS/MS spectra of samples collected from a rat brain under (a) basal conditions and + (b) depolarization conditions induced with a highly concentrated K solution.
of several hundred picomolar. Although the low concentrations of peptides were detectable by LC/MS/MS, Kennedy says his group is interested in further lowering the detection limit by improving sample-preparation and sampling techniques. Kennedy explains that increasing the sensitivity of the method is important because, “Potassium stimulation is a really massive, sledgehammer approach to getting a signal. Even with that, we’re not identifying a huge number of peptides, so we have to [improve] sensitivity to look at more subtle changes that might happen in different behavioral states or [under] drug interventions.” It is unclear whether some of the detected peptides were produced as a direct consequence of K+-induced depolarization or whether they were a result of cellular damage to the brain upon probe insertion. In fact, Kennedy and colleagues reported finding peptides that were blood or intracellular proteins. But Kennedy points out that brain damage by probe insertion is in itself interesting, as it provides a case study for studying brain trauma. “There is a lot of interest in finding markers for brain trauma. This [technique] may help you identify factors that are released in response to trauma,” he says. Once the investigators identified peptides in the extracellular fluid that
were novel, they determined whether the peptides were capable of stimulating neuronal activity by re-introducing the individual peptides into a specific region of the rat brain. As the dialysis probe introduced the peptide, the investigators simultaneously measured the concentrations of eight amino acid neurotransmitters using CE laser-induced fluorescence detection (CE-LIF). The researchers found that three of the six peptides elicited a response from at least one of the eight amino acid neurotransmitters. Thus, they confirmed that some of the novel peptides identified by LC/MS/MS were capable of altering neurochemical signaling, just as expected for a neurotransmitter. Kennedy says his group continues to analyze extracellular fluid from rat brains to discover new types of neuropeptides. The investigators would also like to try alternative screening methods for neurochemical activity, such as receptor screens, in which peptides are exposed to an array of receptors to find out which receptor each of the peptides activates. Kennedy adds, “We’re [also interested] in other brain regions and picking up other peptides. We find we get a lot more coverage of the enkephalins and some other potential hormone-like signaling peptides.” a —Rajendrani Mukhopadhyay
O C T O B E R 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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COURTESY OF ROBERT KENNEDY, UNIVERSITY OF MICHIGAN
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Neuropeptides transmit information between neurons in the brain and are known to play critical roles in functions such as learning, appetite regulation, and sleep. In the September 15 issue of Analytical Chemistry (pp 5523–5533), Robert Kennedy and colleagues at the University of Michigan, Ann Arbor, and the University of Florida describe a method to analyze the neuropeptides present in the extracellular fluid of live rat brains. Kennedy and colleagues collected extracellular fluid samples from 13 anesthetized male rats by inserting a microdialysis probe into their brains. Once samples were collected from the rat brains under basal or resting conditions, the investigators induced localized depolarization. Depolarization occurs when there is a shift in the neuronal cell membrane potential to a less negative value. The investigators introduced a highly concentrated K+ solution into the rat brains and collected samples under depolarization conditions. Kennedy says, “We thought it would be really neat if you could measure what’s actually being released under different conditions in the brain.” Samples collected under both basal and depolarization conditions were analyzed by capillary LC/MS/MS. The spectra obtained under the two different conditions were subtracted from each other, and the resulting spectra were scanned using Sequest and Mascot database searches. Spectra identified by both database searches were then submitted to Lutefisk for partial de novo sequencing as a final evaluation step. The investigators identified 29 peptides, 25 of which were novel. The peptides included neuropeptide precursors, blood proteins, and transporters. In particular, the investigators found a novel group of non-opioid peptides derived from proenkephalin A. Enkephalins are neurotransmitter peptides that are thought to play significant roles in addiction and pain perception. The identified peptides were present in the extracellular fluid at concentrations
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Peptides on the brain
