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

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Genomic and cDNA expression libraries are crucial for discovering new enzymes, but screening thousands of clones is time-consuming.
• Identifying and removing empty or redundant clones is essential to streamline the process of new enzyme discovery.
• High-throughput screening methods are needed to efficiently analyze these large expression libraries.

Purpose of the Study:

• To develop a sensitive and generic workflow for high-throughput screening of microbial protein over-expression.
• To enable rapid identification of successful protein production in large expression libraries.
• To reduce the size of expression libraries, thereby accelerating new enzyme discovery.

Main Methods:

• Utilized mass spectrometry techniques, specifically MALDI-LTQ-Orbitrap screening with principal component analysis and peptide mass fingerprinting.
• Developed an alternative UHPLC-MS(2) approach for microorganisms with complex protein secretomes.
• Employed TCA-induced protein precipitation with bovine serum albumin for protein purification before MS detection.

Main Results:

• Achieved a throughput of approximately 12,000 samples per week using the MALDI-LTQ-Orbitrap method.
• Reached a throughput of approximately 2000 samples per week with the UHPLC-MS(2) approach.
• Demonstrated the workflow's effectiveness in reducing large fungal and bacterial expression libraries by detecting successful protein over-expression via MS.

Conclusions:

• The presented mass spectrometry-based workflow offers a sensitive and generic solution for high-throughput screening of protein over-expression.
• This approach significantly accelerates new enzyme discovery by efficiently reducing the size of microbial expression libraries.
• The workflow is applicable to diverse microbial sources, including fungi and bacteria, facilitating biodiversity exploration.