RESUMO
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 â¼12,000 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 â¼2000 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.
Assuntos
Proteínas de Bactérias/análise , Proteínas Fúngicas/análise , Ensaios de Triagem em Larga Escala/métodos , Mapeamento de Peptídeos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aspergillus niger/química , Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Bases de Dados de Proteínas , Fermentação , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteômica/métodos , Soroalbumina Bovina/análise , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem , Ácido Tricloroacético/química , Tripsina/análise , Tripsina/química , Tripsina/metabolismoRESUMO
In the present study, the changeover from the Pico.Tag HPLC method to the AccQ.Tag(ultra) UPLC method for the analysis of amino acids in casein and bovine serum albumine hydrolysates is described. The total chromatographic run time of the AccQ.Tag(ultra) UPLC method was only 40% of the time required for the Pico.Tag HPLC method. Quantitative results of both methods for casein and bovine serum albumine hydrolysates compared fairly well. The derivatisation protocol for the formation of AQC derivatives of amino acids was automated using a Gilson Model 215 liquid handler. Comparison of the manual derivatisation protocol with the automated protocol showed lower coefficients of variation for the latter. Combination of the AccQ.Tag(ultra) UPLC method and automated derivatisation resulted in improved throughput compared to the Pico.Tag HPLC method.