Online microdialysis‐dynamic nanoelectrospray ionization‐mass spectrometry for monitoring neuropeptide secretion

Abstract

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.

Methods for samples preparation in proteomic research

“…As a rule, the higher mass of the component, the lower recovery through microdialysis membrane. The advantage of the technique might be a possibility of its on-line coupling to LC [186][187][188].…”

Breast Tumor Microenvironment: Proteomics Highlights the Treatments Targeting Secretome

“…36 Jakubowski and co-workers developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly linked to a dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. 39 The device using online microdialysis-dynamic nanoelectrospray mass spectrometry was able to detect acidic peptide, acidic peptide 1-24, and delta-bag cell peptide that were secreted from the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Although the microdialysis method has been extensively applied for in vivo sampling of proteins, there are still several concerns when it was employed for sampling.…”

Selective Removal of Alkali Metal Cations from Multiply-Charged Ions via Gas-Phase Ion/Ion Reactions Using Weakly Coordinating Anions

“…The calculations also suggest that proton transfer is inherently a faster process because it is often observed with other reagent anions for which metal ion transfer is preferred thermodynamically, although not as much as the carborane anion. Therefore, these results demonstrate that the CHB 11 Cl 11 − is the most reliable reagent for the removal of metal cations from peptide ions with mixtures of excess protons and alkali metal cations.…”

“…The size of the ESI capillary can be decreased to effect nano-ESI (nESI), which has been shown to improve salt tolerance by approximately one order of magnitude [6]. Techniques implemented prior to ESI-MS, such as liquid chromatography [7,8], ion exchange [9], solid phase extraction [10], and dialysis [11,12] have been shown to aid in salt removal; however, these methods add both time and complexity to the experiment. Vapor exposure [13] and solution additives [3,14] have also been demonstrated to minimize metal cation adduction.…”