Identification, characterization and quantification of specific neuropeptides in rat spinal cord by liquid chromatography electrospray quadrupole ion trap mass spectrometry.

Abstract

Substance P and CGRP play a central role in neuropathic pain development and maintenance. Additionally, dynorphin A is an endogenous ligand of opioid receptors implicated in the modulation of neurotransmitters including neuropeptides, such as substance P and CGRP. This manuscript proposes a method to characterize, identify and quantify substance P, CGRP and dynorphin A in rat spinal cord by HPLC-ESI/MS/MS. Rat spinal cords were collected and homogenized into a TFA solution. Samples were chromatographed using a microbore C(8) 100 x 1 mm column and a 19 min linear gradient (0:100 --> 40:60; ACN:0.2% formic acid in water) at a flow rate of 75 microL/min for a total run time of 32 min. The peptides were identified in rat spinal cord based on full-scan MS/MS spectra. Substance P, CGRP and dynorphin A were predominantly identified by the presence of specific b CID fragments. Extracted ion chromatogram (XIC) suggested selected mass transitions of 674 --> [600 + 254], 952 --> [1215 + 963] and 717 --> [944 + 630] for substance P, CGRP and dynorphin A can be used for isolation and quantitative analysis. A linear regression (weighted 1/x) was used and coefficients of correlations (r) ranging from 0.990 to 0.999 were observed. The precision (%CV) and accuracy (%NOM) observed were 10.9-14.4% and 8.9-14.2%, 8.8-13.0% and 91.0-110.2% and 97.2-107.3% and 91.8-97.3% for substance P, CGRP and dynorphin A respectively. Following the analysis of rat spinal cords, the mean endogenous concentrations were 110.7, 2541 and 779.4 pmol/g for substance P, CGRP and dynorphin A respectively. The results obtained show that the method provides adequate figures of merit to support targeted peptidomic studies aimed to determine neuropeptide regulation in animal neuropathic and chronic pain models.

• Authors: Francis Beaudry, Catherine E Ferland, Pascal Vachon
• Journal: Biomedical chromatography : BMC (Biomed Chromatogr) Vol. 23 Issue 9 Pg. 940-50 (Sep 2009) ISSN: 1099-0801 [Electronic] England
• PMID: 19358311 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright (c) 2009 John Wiley & Sons, Ltd.

Chemical References

• Neuropeptides
• Substance P
• Dynorphins
• Calcitonin Gene-Related Peptide

Topics

• Animals
• Calcitonin Gene-Related Peptide (analysis, chemistry)
• Chromatography, High Pressure Liquid (methods)
• Dynorphins (analysis, chemistry)
• Linear Models
• Male
• Neuropeptides (analysis, chemistry)
• Rats
• Rats, Sprague-Dawley
• Reproducibility of Results
• Sensitivity and Specificity
• Spectrometry, Mass, Electrospray Ionization (methods)
• Spinal Cord (chemistry)
• Substance P (analysis, chemistry)