Pursuing Experimental Reproducibility: An Efficient Protocol for the Preparation of Cerebrospinal Fluid Samples for NMR-based Metabolomics and Analysis of Sample Degradation.

NMR-based metabolomics investigations of human biofluids offer great potential to uncover new biomarkers. In contrast to protocols for sample collection and biobanking, procedures for sample preparation prior to NMR measurements are still heterogeneous, thus compromising the comparability of the resulting data. Herein, we present results of an investigation of the handling of cerebrospinal fluid (CSF) samples for NMR metabolomics research. Origins of commonly observed problems when conducting NMR experiments on this type of sample are addressed, and suitable experimental conditions in terms of sample preparation and pH control are discussed. Sample stability was assessed by monitoring the degradation of CSF samples by NMR, hereby identifying metabolite candidates, which are potentially affected by sample storage. A protocol was devised yielding consistent spectroscopic data as well as achieving overall sample stability for robust analysis. We present easy to adopt standard operating procedures with the aim to establish a shared sample handling strategy that facilitates and promotes inter-laboratory comparison, and the analysis of sample degradation provides new insights into sample stability.

Interaction between ghrelin and the ghrelin receptor (GHS-R1a), a NMR study using living cells.

The study of the interaction of ghrelin (1), the endogenous ligand for the GH secretagogues receptor (GHS-R1a), and des-acyl ghrelin (2) with the GHS-R1a by NMR using living cells is presented, using GHS-R1a stably transfected cell lines (CHO and HEK 293) and wild type cells. Therefore, the interaction of 1 and 2 with the GHS-R1a receptor has been performed using quasi-physiological conditions. Ghrelin (1), showed a higher number of residues affected by chemical shift perturbation (CSP) or chemical shift exchange (CSE) effects: Ser3, Phe4, Leu5, Val12, Gln13/Gln14, Lys16/Lys19, Glu17 and Lys24 were much more affected in 1 than in des-acyl ghrelin (2). The chemical shift index CSI values indicated the presence of a possible alpha-helical region between Glu8 and Lys20 for ghrelin (1). After analysing the NMR data, two possible structures have arisen, which present different proline rotamers: the EEZE and the EZEZ conformers, at positions Pro7, Pro21, Pro22 and Pro27, respectively, keeping a left-handed alpha-helix from Glu8 to Lys20. These experimental evidences might imply that the GHS-R1a receptor is acting as a prolyl-cis/trans isomerase.