Within the last two decades small peptides, such as neurotransmitters (e.g. endorphins), growth factors (e.g. bombesin and gastrin-releasing peptide), antibacterial (e.g. magainins) and regulators of body functions (e.g. angiotensin), have been identified to have important roles within biological systems. These discoveries have also led to increased interest in small peptides as future therapeutic agents or as targets for therapy. The endogenous opioid peptides have been extensively studied as potential selective analgetics and for their potential to treat drug addiction. The analytical methodology needed to investigate the pharmacokinetics of such important peptides in vivo has, however, neither kept abreast with nor contributed sufficiently to the rapid advances in the knowledge of their biological function. This deficiency has been partly due to the high potency of these small peptides, their poor spectral properties, rapid metabolism, polarity and considerable mass. As a result, most investigators have adopted immunoassays is complex as well as expensive, requiring a lengthy development procedure for each peptide. Here we proposed to develop an alternative method for the analysis of small peptides based upon partial sequence homology. In this approach, the well established quantitative technique of gas chromatography/mass spectrometry (GC/MS) with negative ion chemical ionization (NICI) will be coupled with improvements in capillary GC and selective enzymatic cleavage of peptides using immobilized enzyme systems. Briefly, peptides will be isolated from the biological matrix by solid phase extraction, the peptide of interest purified by HPLC, then subjected to selective enzymic digestion using immobilized trypsin or chymotrypsin to yield specific di-or tripeptide which are unique to that peptide. These di- nd tripeptide when derivatized by acylation with fluorinated anhydrides and alkylation with pentafluorobenzyl bromide will be subjected to GC/NICI MS. Using a deuterium is sufficient for quantification of potent peptides in biological fluids. This method, implemented with less expensive and widely available GC/MS instruments, will first be evaluated by characterizing each steps of endogenous peptide, dynophin A (Fragment 1-13). The method will have utility for the analysis of a broad range of peptides and should be easily extended for the analysis of proteins. The quantitative analytical method will allow future studies of the pharmacokinetics and pharmacodynamics of opiate peptides by a wide range of investigators, thus stimulating advances in this area and others.
| Marquez, C D; Lee, M L; Weintraub, S T et al. (1997) Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization. J Chromatogr B Biomed Sci Appl 700:9-21 |
| Marquez, C D; Weintraub, S T; Smith, P C (1997) Quantitative analysis of two opioid peptides in plasma by liquid chromatography-electrospray ionization tandem mass spectrometry. J Chromatogr B Biomed Sci Appl 694:21-30 |
