Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of Escherichia coli Colonies

Abstract

High-throughput molecular screening of microbial colonies and DNA libraries are critical procedures that enable applications such as directed evolution, functional genomics, microbial identification, and creation of engineered microbial strains to produce high-value molecules. A promising chemical screening approach is the measurement of products directly from microbial colonies via optically guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Measuring the compounds from microbial colonies bypasses liquid culture with a screen that takes approximately 5 s per sample. We describe a protocol combining a dedicated informatics pipeline and sample preparation method that can prepare up to 3,000 colonies in under 3 h. The screening protocol starts from colonies grown on Petri dishes and then transferred onto MALDI plates via imprinting. The target plate with the colonies is imaged by a flatbed scanner and the colonies are located via custom software. The target plate is coated with MALDI matrix, MALDI-MS analyzes the colony locations, and data analysis enables the determination of colonies with the desired biochemical properties. This workflow screens thousands of colonies per day without requiring additional automation. The wide chemical coverage and the high sensitivity of MALDI-MS enable diverse screening projects such as modifying enzymes and functional genomics surveys of gene activation/inhibition libraries.Key features• Mass spectrometry analyzes a range of compounds from E. coli colonies as a proxy for liquid culture testing enzyme mutant libraries.• Colonies are transferred to a MALDI target plate by a simple imprinting method.• The screen compares the ratio among several products or searches for the qualitative presence of specific compounds.• The protocol requires a MALDI mass spectrometer.Graphical overviewOverview of the MALDI-MS analysis of microbial colonies for screening mutant libraries. Microbial cells containing a mutant library for enzymes/metabolic pathways are first grown in agar. The colonies are then imprinted onto a MALDI target plate using a filter paper intermediate. An optical image of the MALDI target plate is analyzed by custom software to find the locations of individual colonies and direct subsequent MALDI-MS analyses to the selected colonies. After applying MALDI matrix onto the target plate, MALDI-MS analysis of the colonies is performed. Colonies showing the desired product profiles are found by data analysis via the software, and the colonies are picked for downstream analysis.