High-Throughput Platform Quantifies Metabolic Pathway Proteins in Bacteria

The Science

A multi-institutional team analyzed hundreds of peptides in the bacterium Pseudomonas putida KT2440. These protein fragments came from proteins in various metabolic pathways. The scientists used two different microflow liquid chromatography–-selected reaction monitoring (LC-SRM) platforms for this analysis, a nanoflow platform and a microflow platform. The results from the two platforms were highly correlated. This demonstrated that the microflow platform works well to quantify metabolic pathway enzymes without sacrificing sensitivity for speed.

The Impact

LC-SRM is a popular technique to quantify targeted proteins on a large scale. Scientists use it to get detailed insights into the intrinsic metabolism of a biological system. They need these details to be able to use synthetic biology and engineer organisms with new functions. P. putida, the bacterium used in this study, has considerable potential for a wide range of biotechnology applications, such as bioproduct production through bioengineering.

Summary

In this study, a multi-institutional team of scientists monitored 339 peptides representing 132 enzymes in P. putida KT2440 grown on various carbon sources ranging from sugars to lignin-derived aromatic compounds. To do this, they used microflow and nanoflow liquid chromatography, as well as the triple quadrupole mass spectrometer at the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility at Pacific Northwest National Laboratory (PNNL). This work is part of EMSL’s Biomolecular Pathways Integrated Research Platform.

The scientists compared the performance of the micro- and nano-flow LC-SRM platforms. They found that microflow LC-SRM had comparable sensitivity for the majority of detected peptides. It also had better mass spectrometry signal and chromatography stability than nanoflow LC- SRM.

The scientists also quantified key enzymes in common metabolic pathways, such as central carbon metabolism and β-ketoadipate pathways. This analysis revealed how P. putida enzyme expression levels changed in response to various carbon sources and media composition. The increased throughput and measurement reliability of this microflow LC-SRM platform make it an exceptional test tool for synthetic biology–guided engineering. Those features reduce the time of Design-Build-Test-Learn cycles and thereby accelerate the path to microbial production and commercialization of natural products.

Principal Investigator(s)

Kristin Burnum-Johnson
Pacific Northwest National Laboratory
[email protected]

Yuqian Gao
Pacific Northwest National Laboratory
[email protected]

Funding

Funding was provided by the Bioenergy Technologies Office, within the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy, for the Agile BioFoundry. A portion of this research was performed at Pacific Northwest Natonal Laboratory using the Environmental Molecular Sciences Laboratory (EMSL), a DOE Office of Science user facility sponsored by the Office of Biological and Environmental Research, within the DOE Office of Science.

References

Gao, Y. et al. “High-throughput large-scale targeted proteomics assays for quantifying pathway proteins in Pseudomonas putida KT2440.” Frontiers in Bioengineering and Biotechnology 8, 603488 (2020). [DOI:10.3389/fbioe.2020.603488]. OSTI 1728466.