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–MS2 approach including MS2 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.

► We developed an automated high throughput workflow to filter expression libraries.
► Complex matrix was removed by TCA precipitation, enhanced by BSA spiking, in 96 wells format.
► Both MALDI and UHPLC high throughput MS-based analysis methods were applied.
► Empty and redundant clones were successfully filtered from expression libraries, resulting in strongly reduced smart libraries.
► Workflows have been developed and directly applied in an industrial biotechnological environment.