Quantitation Of Neuropeptides Research Articles

Perspectives on Gender Parity in Bioanalysis: An Interview with Stephanie Cape

Stephanie Cape, is the Director of Bioanalytical Science at Covance, Madison, WI, USA. She received her BS in Chemistry from University of IL- Urbana/Champaign in 2003 and her PhD in Analytical Chemistry from the University of Wisconsin - Madison in 2007. Dr Cape's graduate research was focused on developing high-resolution mass spectrometric methods to enable discovery, characterization, imaging and quantitation of neuropeptides. Stephanie joined the field of regulated bioanalysis as a method development/validation chemist initially at PPD and subsequently at Covance. Within the Covance Bioanalytical team, she has held a variety of leadership roles including oversight of bioanalytical study direction, validation and method development. Additionally, Dr Cape is an active member of the broader scientific community through involvement in multiple industry groups focused on global best practices and innovation in the field of bioanalysis.

On-Column Dimethylation with Capillary Liquid Chromatography-Tandem Mass Spectrometry for Online Determination of Neuropeptides in Rat Brain Microdialysate.

We have developed a method for online collection and quantitation of neuropeptides in rat brain microdialysates using on-column dimethylation with capillary liquid chromatography-tandem mass spectrometry (cLC-MS2). This method addresses a number of the challenges of quantifying neuropeptides with cLC-MS. It is also a completely automated and robust method for the preparation of stable isotope labeled-peptide internal standards to correct for matrix effects and thus ensure accurate quantitation. Originally developed for tissue-derived proteomics samples ( Raijmakers et al. Mol. Cell. Proteomics 2008 , 7 , 1755 - 1762 ), the efficacy of on-column dimethylation for native peptides in microdialysate has not been demonstrated until now. We have modified the process to make it more amenable to the time scale of microdialysis sampling and to reduce the accumulation of nonvolatile contaminants on the column and, thus, loss of sensitivity. By decreasing labeling time, we have a temporal resolution of 1 h from sample loading to elution and our peptide detection limits are in the low pM range for 5 μL injections of microdialysate. We have demonstrated the effectiveness of this method by quantifying basal and potassium stimulated concentrations of the neuropeptides leu-enkephalin and met-enkephalin in the rat hippocampus. To our knowledge, this is the first report of quantitation of these peptides in the hippocampus using MS.

Extraction and quantitation of neuropeptides in bone by radioimmunoassay

The feasibility of extracting and quantifying neuropeptides in bone by radioimmunoassay was investigated in a study including 60 diaphyseal rat femora. Substance P, calcitonin gene-related peptide, neuropeptide Y and vasoactive intestinal polypeptide, previously identified in bone by immunohistochemistry, were extracted from separate homogenates of bone, periosteum and bone marrow in a solution of 4% EDTA and 2 M acetic acid. Measurable amounts of all four neuropeptides in bone, periosteum and bone marrow were obtained by radioimmunoassay in a reproducible manner. The neuropeptide immunoreactivities were characterized by reversephase high performance liquid chromatography. Among the four neuropeptides analyzed, neuropeptide Y consistently exhibited the highest concentrations in the different tissues. Overall, cortical bone showed the lowest neuropeptide concentrations. The concentration of vasoactive intestinal polypeptide was higher in periosteum than in bone marrow, whereas that of calcitonin gene-related peptide was uniform in these tissues. The distributional differences observed in bone tissue may be explained by a variety of physiological roles attributed to neuropeptides such as regulation of nociception, vasoactivity, immune function and local bone metabolism. The described methodology offers a new means of investigating a neuropeptidergic involvement in various disorders of the skeleton.

Chemical consideration of solid phase matrix interaction leads to improved quantitation of neuropeptides

Chemical consideration of solid phase matrix interaction leads to improved quantitation of neuropeptides