Mass Spectrometric Detection of Neuropeptides Using Affinity-Enhanced Microdialysis with Antibody-Coated Magnetic Nanoparticles

Abstract

Microdialysis (MD) is a useful sampling tool for many applications due to its ability to permit sampling from an animal concurrent with normal activity. MD is of particular importance in the field of neuroscience, in which it is used to sample neurotransmitters (NTs) while the animal is behaving in order to correlate dynamic changes in NTs with behavior. One important class of signaling molecules, the neuropeptides (NPs), however, presented significant challenges when studied with MD, due to the low relative recovery (RR) of NPs by this technique. Affinity-enhanced microdialysis (AE-MD) has previously been used to improve recovery of NPs and similar molecules. For AE-MD, an affinity agent (AA), such as an antibody-coated particle or free antibody, is added to the liquid perfusing the MD probe. This AA provides an additional mass transport driving force for analyte to pass through the dialysis membrane, and thus increases the RR. In this work, a variety of AAs have been investigated for AE-MD of NPs in vitro and in vivo, including particles with C18 surface functionality and antibody-coated particles. Antibody-coated magnetic nanoparticles (AbMnP) provided the best RR enhancement in vitro, with statistically significant (p<0.05) enhancements for 4 out of 6 NP standards tested, and RR increases up to 41-fold. These particles were then used for in vivo MD in the Jonah crab, Cancer borealis, during a feeding study, with mass spectrometric (MS) detection. 31 NPs were detected in a 30 min collection sample, compared to 17 when no AA was used. The use of AbMnP also increased the temporal resolution from 4–18 hrs in previous studies to just 30 min in this study. The levels of NPs detected were also sufficient for reliable quantitation with the MS system in use, permitting quantitative analysis of the concentration changes for 7 identified NPs on a 30 min time course during feeding.

An ultrasensitive nano UHPLC–ESI–MS/MS method for the quantification of three neuromedin-like peptides in microdialysates

“…Indeed, the recovery for peptides is smaller compared with the classical neurotransmitters because of their higher molecular weight and their tendency to stick to the membrane material and other parts of the probe. Other possibilities to improve the recovery of neuropeptides are the use of affinityenhanced microdialysis [46] or alternative sampling devices [47]. In addition, we observed that our peptides of interest considerably stick to the wall of the outlet tubing, thereby drastically reducing the peptide amount which is collected for analysis (unpublished data).…”

Sources and range of long-term variability of rhythmic motor patternsin vivo.

“…We found that in unfed animals, the phase relationships of the pyloric neurons rarely deviated from the canonical patterns, indicating that the pyloric rhythm's structure remains stable. This was despite several studies showing that, in vivo, the STG is continuously exposed to both hormonal and neural modulation (Diehl et al, 2013;Hedrich et al, 2011;Behrens et al, 2008;Schmerberg and Li, 2013;Christie et al, 1995), which have the potential to modify the phase relationships of the involved motor neurons (Marder and Weimann, 1992).…”

Mass spectrometric measurement of neuropeptide secretion in the crab, Cancer borealis, by in vivo microdialysis

“…The crabs were allowed to adjust to the tanks for at least one week after shipment before performing hemolymph extraction or microdialysis. Details of animal housing procedures were described elsewhere 19 . Animals were housed, treated and sacrificed following the animal care protocol in accordance with the University of Wisconsin-Madison’s animal care guidelines.…”

“…The procedure for in vivo microdialysis surgery on Jonah crabs was adapted from previous publications 18, 19 . After the probe was surgically implanted in the crab, the animal was allowed to recover for at least 24 hr before dialysate was collected for MS analysis.…”

“…Diffusion can occur between the perfusion fluid and the extracellular space as the perfusion fluid passes through the probe tip, and molecules such as NPs will diffuse into the perfusion fluid, driven down their concentration gradient. Microdialysis has been widely used to monitor a wide range of molecules including electrolytes 15 , amines and amino acids 16, 17 , NPs 18, 19 and proteins 17, 20 .…”