title: Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.
abstract: 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.
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613AX Delft, The Netherlands. Michiel.Akeroyd@DSM.com
authors.forename: Michiel
authors.initials: M
authors.lastname: Akeroyd
authors.forename: Maurien
authors.initials: M
authors.lastname: Olsthoorn
authors.forename: Jort
authors.initials: J
authors.lastname: Gerritsma
authors.forename: Diana
authors.initials: D
authors.lastname: Gutker-Vermaas
authors.forename: Laurens
authors.initials: L
authors.lastname: Ekkelkamp
authors.forename: Tjeerd
authors.initials: T
authors.lastname: van Rij
authors.forename: Paul
authors.initials: P
authors.lastname: Klaassen
authors.forename: Wim
authors.initials: W
authors.lastname: Plugge
authors.forename: Ed
authors.initials: E
authors.lastname: Smit
authors.forename: Kerstin
authors.initials: K
authors.lastname: Strupat
authors.forename: Thibaut
authors.initials: T
authors.lastname: Wenzel
authors.forename: Marcel
authors.initials: M
authors.lastname: van Tilborg
authors.forename: Rob
authors.initials: R
authors.lastname: van der Hoeven
mesh_terms: D001234:Aspergillus niger / Q000737:chemistry; D001412:Bacillus subtilis / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D030562:Databases, Protein; D005285:Fermentation; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014238:Trichloroacetic Acid / Q000737:chemistry; D014357:Trypsin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D012710:Serum Albumin, Bovine; D014238:Trichloroacetic Acid; D014357:Trypsin