Online microdialysis-dynamic nanoelectrospray ionization-mass spectrometry for monitoring neuropeptide secretion.
Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.
Jakubowski
Jennifer A
JA
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801, USA.
Hatcher
Nathan G
NG
Sweedler
Jonathan V
JV
D000818:Animals; D001048:Aplysia; D004867:Equipment Design; D017551:Microdialysis / Q000379:methods*; D009474:Neurons / Q000378:metabolism; D009479:Neuropeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D009862:Online Systems; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*
D009479:Neuropeptides