Nutrikinetic modeling reveals order of genistein phase II metabolites appearance in human plasma.

SCOPE: Genistein from foods or supplements is metabolized by the gut microbiota and the human body, thereby releasing many different metabolites into systemic circulation. The order of their appearance in plasma and the possible influence of food format are still unknown. This study compared the nutrikinetic profiles of genistein metabolites. METHODS AND RESULTS: In a randomized cross-over trial, 12 healthy young volunteers were administered a single dose of 30 mg genistein provided as a genistein tablet, a genistein tablet in low fat milk, and soy milk containing genistein glycosides. A high mass resolution LC-LTQ-Orbitrap FTMS platform detected and quantified in human plasma: free genistein, seven of its phase-II metabolites and 15 gut-derived metabolites. Interestingly, a novel metabolite, genistein-4'-glucuronide-7-sulfate (G-4'G-7S) was identified. Nutrikinetic analysis using population-based modeling revealed the order of appearance of five genistein phase II metabolites in plasma: (1) genistein-4',7-diglucuronide, (2) genistein-7-sulfate, (3) genistein-4'-sulfate-7-glucuronide, (4) genistein-4'-glucuronide, and (5) genistein-7-glucuronide, independent of the food matrix. CONCLUSION: The conjugated genistein metabolites appear in a distinct order in human plasma. The specific early appearance of G-4',7-diG suggests a multistep formation process for the mono and hetero genistein conjugates, involving one or two deglucuronidation steps.

Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.

In the discovery of new enzymes genomic and cDNA expression libraries containing thousands of differential clones are generated to obtain biodiversity. These libraries need to be screened for the activity of interest. Removing so-called empty and redundant clones significantly reduces the size of these expression libraries and therefore speeds up new enzyme discovery. Here, we present a sensitive, generic workflow for high throughput screening of successful microbial protein over-expression in microtiter plates containing a complex matrix based on mass spectrometry techniques. MALDI-LTQ-Orbitrap screening followed by principal component analysis and peptide mass fingerprinting was developed to obtain a throughput of ∼12,000 samples per week. Alternatively, a UHPLC-MS(2) approach including MS(2) protein identification was developed for microorganisms with a complex protein secretome with a throughput of ∼2000 samples per week. TCA-induced protein precipitation enhanced by addition of bovine serum albumin is used for protein purification prior to MS detection. We show that this generic workflow can effectively reduce large expression libraries from fungi and bacteria to their minimal size by detection of successful protein over-expression using MS.

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.