Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%–35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10−6% of total proteins, by weight. Signal peptide recovery ranged between 5%–100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction

Effects of controlled ingestion of kaolinite (5%) on food intake, gut morphology and in vitro motility in rats.

International audienceGeophagia is found in various animal species and in humans. We have previously shown that spontaneously ingested kaolinite interacts with the intestinal mucosa modifies nutrient absorption and slows down gastric emptying and intestinal transit in rats in vivo. However, the precise mechanisms involved are not elucidated. The aim of this work was to investigate the effects of controlled kaolinite ingestion on food intake, gut morphology and in vitro motility in rats. Male Wistar rats were fed with 5% kaolinite in standard food pellets during 7, 14 and 28 days. Body mass and food consumption were measured daily. Intestinal morphological and proteomic analyses were conducted. The length of mucosal lacteals was evaluated. Plasmatic levels of leptin and adiponectin were determined. Finally, organ bath studies were conducted to evaluate smooth muscle contractility. Food consumption was significantly increased during the first two weeks of kaolinite ingestion without any mass gain compared to controls. Kaolinite induced weak variations in proteins that are involved in various biological processes. Compared to control animals, the length of intestinal lacteals was significantly reduced in kaolinite group whatever the duration of the experiment. Leptin and adiponectin plasmatic levels were significantly increased after 14 days of kaolinite consumption. Changes in spontaneous motility and responses to electrical nerve stimulation of the jejunum and proximal colon were observed at day 14. Altogether, the present data give evidence for a modulation by kaolinite-controlled ingestion on satiety and anorexigenic signals as well as on intestinal and colonic motility