Monitoring Activity-Dependent Peptide Release from the CNS Using Single-Bead Solid-Phase Extraction and MALDI TOF MS Detection

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• 作者：Nathan G. Hatcher ; Timothy A. Richmond ; Stanislav S. Rubakhin ; and Jonathan V. Sweedler
• 刊名：Analytical Chemistry
• 出版年：2005
• 出版时间：March 15, 2005
• 年：2005
• 卷：77
• 期：6
• 页码：1580 - 1587
• 全文大小：291K
• 年卷期：v.77,no.6(March 15, 2005)
• ISSN：1520-6882

文摘

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE)beads were used to collect peptides from specific locationsof well-characterized neurosecretory structures and evenindividual neuronal processes for off-line MALDI MSanalyses. Peptide binding parameters of single SPEbeads, including limits of collection, detection, and saturation capacity, were tested with ¹⁴C-labeled cytochromec as well as with mixtures of multiple neuropeptides(bradykinin, Aplysia acidic peptide 1-20, and insulin).MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysiabag cell neurons. With cultured cells, precise placementof SPE beads allowed peptide collection from distinctneurites with spatial localization on the order of 200 ges/entities/mgr.gif">m,and SPE beads could be replaced within time frames thatallowed analyte collection before and after cell stimulationparadigms. Comparison between pre- and poststimulationpeptide profiles in both model systems allowed a directedstrategy to determine which compounds were releasedwith neuronal activity. Single SPE bead MALDI MS offersa novel approach to investigate peptide signaling thatallows the detection and discovery of unknown intercellular signals secreted from a large variety of biologicaltissues.