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Quantification of bioactive peptides by mass spectrometry - Applicability in drug discovery

Final Activity Report Summary - PEPTIDE QUANT (Quantification of bioactive peptides by mass spectrometry - Applicability in drug discovery)

The main objective of this project is to improve the state-of-the-art in the field of absolute quantification of bioactive peptides and proteins in biological fluids by means of liquid chromatography - tandem mass spectrometry analysis (LC-MS/MS). Melanotan-II (MT-II) and the glucose-dependent insulinotropic polypeptide (GIP1-42) were chosen for this purpose. They were chosen primarily as representatives of low and high molecular weight peptides, because of their known biological importance, and because methods for their detection and quantification could benefit from significant improvement.

MT-II (molecular weight of approximately 1 kDa) is a synthetic analogue of the natural melanocortin peptide, a-melanocyte-stimulating hormone (a-MSH). MT-II is well known for the anorexic effects it elicits in rodents. During this project, we improved assay limits of MT-II in mouse brain and in plasma and demonstrated the peptide's low permeability to mouse brain. This is significant because it demonstrates that a super potent melanocortin receptor agonist, often used as a pharmacological tool for efficacy studies, does not penetrate into the brain in efficacious doses. GIP1-42, is a gastric peptide incretin hormone (molecular weight of ~5 kDa). It is released postprandially, but is rapidly degraded by Dipeptidyl Peptidase IV (DP-IV) which cleaves the first two aminoacids to form the inactive GIP3-42. GIP is a biomarker for diabetes patients treated with DP-IV inhibitors. We established a novel method for the simultaneous determination of the pair of GIP peptides via proteolysis and analysis of the product peptides by LC-MS/MS.

Current methodologies towards peptide and protein quantification include immunoassays and mass spectrometric techniques. Immunoassays, although very sensitive, lack the necessary selectivity for distinction between peptide and its metabolites, or various recombinant forms of a protein, leading to cross-reactivity. In addition LC-MS method development is rapid, and it is not matrix specific, which potentially makes one assay suitable for both preclinical and clinical studies, where different species are used. However, although it is generally accepted that MS is ideal for the quantification of trace levels of analytes with a good range of calibration curve, there are particular challenges associated with the analysis of especially large peptides. Tryptic cleavage may be necessary to yield smaller and 'handy' fragments. These surrogate shorter peptides can be detected with higher sensitivity compared with the precursor protein.

The analysis of bioactive peptides and proteins in plasma and other biological samples is critical for the understanding of disease mechanisms and the development of novel therapies. Contributions to the current state of the art of these analytical approaches, through the methodologies developed in the current programme, will have a significant impact on the wider biomedical research. Absolute peptide and protein quantification by means of LC-MS/MS plays an important role in the pipeline for diagnostics (biomarker validation) and drug discovery.

Currently, the rate of attrition of medicines in clinical development, due to lack of clinical efficacy or toxicity remains high. Potentially the discovery and use of good biomarkers (often peptides or proteins) will help in reducing attrition rates, thus facilitating new drug development. In addition, high throughput proteomic technologies lack the ability of absolute quantification of large, biologically significant peptides and proteins, producing results that are typically either qualitative or semi-quantitative in nature. It is vital that results from proteomic studies (putative biomarkers for example) are validated by absolute quantification studies.

We believe that the methodologies developed in this project, are bridging this gap and we intend to use them in our on-going studies in the field of cancer biomarker validation. Such projects were initiated in the last two years, largely due to the expertise developed during the current programme.