Online ISSN: 2515-8260

Keywords : Overall

SRM-based quantification of malignant biliary stenosis biomarkers in human bile

Annarita Farina; Annie Adrait; Jean-Marc Dumonceau; Myriam Delhaye; Jean-Louis Frossard; Yohann Coute

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 2, Pages -

The differential diagnosis of biliary stenosis is a critical problem for gastroenterologists. An early identification of malignant lesions would enable the rapid resort to surgical resection which currently represents the only potentially curative option. Unfortunately, the diagnostic value of all available methods (e.g. imaging technics, standard serum biomarkers) is limited by relatively poor accuracy and negative predictive value. Recently, our group and others highlighted new potential cancer biomarkers in bile by using comparative proteomic analysis. Nevertheless, to date, only a few candidates have been verified for their diagnostic performances in discriminating between malignant and non-malignant stenoses. In addition, no data have yet been collected on the simultaneous measurement of these proteins with the intent of evaluating the diagnostic interest of a panel of biomarkers. To overcome the limitation of classical verification tools and give a new impetus to the translation of bile biomarkers into clinical diagnostics, mass spectrometry-based quantification could represent a rapid and cost-effective opportunity thanks to its capacity for multiplexed, high-throughput analysis, combined with its analytical specificity and reliable quantification. Here we developed the first Selected Reaction Monitoring (SRM) assay for the multiplexed measurement of cancer biomarkers in human bile. For this purpose, 8 potential biomarker candidates previously highlighted by proteomic analysis were selected. Equal volumes of bile collected from patients presenting with malignant and non-malignant biliary stenosis were stacked on the top of a SDS-PAGE gel. Proteins were then digested in-gel with trypsin and proteotypic peptides of each candidate biomarker were quantified by nanoLC-SRM on a 5500-QTrap mass spectrometer (ABSciex) using heavy synthetic peptides as standards (PEPotecTM, Thermofisher). SRM data were finally analysed using Skyline software and manual validation. The developed assay proved to be valuable and reliable to quantify all the selected candidates. Moreover, the results confirmed the simultaneous overexpression of some of the proteins in bile samples from malignant stenoses.

Metabolic phenotyping in mouse and man: Mind the differences!

Denise Sonntag; Guido Krebiehl; Torben Friedrich

European Journal of Molecular & Clinical Medicine, 2015, Volume 2, Issue 2, Pages -

Metabolic phenotyping comprises the quantification of endogenous metabolites in biofluids, cells, and tissues. It provides insights into normal as well as aberrant metabolic pathways and biological processes, which is important for the understanding of disease phenotypes. It also allows the identification of biological markers, which can serve as early disease indicators and therapeutic markers for the evaluation of treatment effects. As metabolic markers are not species-restricted, the concept of metabolic phenotyping is highly applicable for translational research. Species independence allows the use of established animal and cell culture models for various diseases within a preclinical context. However, differences in the metabolic set-up of study organisms compared to humans needs to be taken into consideration to prevent misleading conclusions from otherwise valid experimental designs. To determine species-related metabolic differences, a targeted metabolomics approach was applied using a mass spectrometry platform for the quantification of a predefined set of endogenous metabolites, i.e. amino acids, biogenic amines, phosphatidylcholines, sphingomyelins, hexoses, steroid hormones and others. Results from this species comparison on the metabolic level will be presented.