A Multifaceted Mass Spectrometric Method to Probe Feeding Related Neuropeptide Changes in Callinectes sapidus and Carcinus maenas
Food intake is regulated by various neuromodulators, including numerous neuropeptides. However, it remains elusive at the molecular and cellular level as to how these important chemicals regulate internal processes and which regions of the neuronal organs are responsible for regulating the behavior. Here we report a comparative neuropeptidomic analysis of the brain and pericardial organ (PO) in response to feeding in two well-studied crustacean physiology model organisms, Callinectes sapidus and Carcinus maenas, using mass spectrometry (MS) techniques. A multifaceted MS-based approach has been developed to obtain complementary information on the expression changes of a large array of neuropeptides in the brain and PO. The method employs stable isotope labeling of brain and PO extracts for relative MS quantitation, capillary electrophoresis (CE)-MS for fractionation and high-specificity analysis, and mass spectrometric imaging (MSI) for in-situ molecular mapping of peptides. A number of neuropeptides, including RFamides, B-type allatostatins (AST-B), RYamides, and orcokinins exhibit significant changes in abundance after feeding in this investigation. Peptides from the AST-B family found in PO tissue were shown to have both altered expression and localization changes after feeding, indicating that they may be a class of vital neuropeptide regulators involved in feeding behavior.
KeywordsPeptidomics Neuropeptides Isotopic labeling Mass spectrometric imaging Capillary electrophoresis
The authors thank the University of Wisconsin-Biotechnology Center Mass Spectrometry Facility, Dr. Amy Harms, and Dr. Mike Sussman for access to the MALDI-TOF/TOF instrument. This work was supported by the National Institutes of Health through grants 1R01DK071801 and R01NS029436. K.D acknowledges a predoctoral fellowship supported by the National Institutes of Health, under Ruth L. Kirschstein National Research Service Award T32 HL 007936 from the National Heart Lung and Blood Institute to the University of Wisconsin-Madison Cardiovascular Research Center.
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