Protein Discoveries in Biofuels Plant Made Possible by Emerging Mass Spectrometry Methods

Mowei Zhou, a chemist with the Environmental Molecular Sciences Laboratory, will discuss his latest protein discoveries for a plant with the potential to transform biofuels production as part of the ACS Spring conference taking place March 20–24. The discoveries were made possible using two emerging forms of mass spectrometry. 

Zhou’s presentation begins at 8:15 a.m. on Tuesday, March 22. 

As part of a recent study, Zhou and his team used top-down and native mass spectrometry in combination with the traditional method of bottom-up proteomics with mass spectrometry to identify 14 proteins that are generally considered to be associated with either dirigent protein function or the cell wall of the Forsythia × intermedia plant. Dirigent proteins are members of a class of proteins that dictate the spatial arrangement of atoms in a class of compounds synthesized by plants. 

For decades, scientists have traditionally used bottom-up proteomics with mass spectrometry to identify new proteins and analyze their structure. This technique requires the slicing of proteins into small segments called peptides using an enzyme-based chemical process before analysis. When piecing these components back together, however, not all information needed to complete the full protein picture may be present. 

Unlike the bottom-up method, top-down mass spectrometry involves measuring the fully intact protein, or the “top” part of the top-down sequencing. And similarly with native mass spectrometry, researchers can study proteins in their intact form. But instead of analyzing individual proteins, it allows them to analyze their “native” and active form, typically involving complexes among several proteins. 

Zhou said by using the two emerging mass spectrometry methods in combination with the traditional bottom-up approach to paint a clearer picture of protein structure for Forsythia × intermedia and its protein interactions, they were able to draw further connections to the structure of a substance called lignin within the plant. Lignin comprises the woody structure in plants and is found in the plant’s cell walls. Through degradation, it can be used as a feedstock for biofuels production. 

“We were able to extract this enzyme from the stem of the plant, which grows in people’s gardens in this area,” Zhou said. “We found that this enzyme from the plant controls the stereochemistry of the metabolites in the plant. Those small molecules can serve as the building blocks of lignin.” 

Zhou said that before his team’s research, there were some related studies from other people in the field suggesting the link of proteins to dirigent protein function and the cell wall structure, but further exploration was necessary to understand the molecular mechanisms.  

“The studies show that the protein colocalizes with specific structures in the plant,” he said. “They were found in very specific cell wall structures in plants. That is how people have connected the dots to say that proteins might be doing something to create that specific cell wall structure. That is the strongest link we have right now.” 

Paired with their discoveries, Zhou and his team also made a serendipitous discovery of several homolog proteins that were not identified before. Homologs are proteins that are related to the base protein—they came from the same ancestor. While similar in structure, they perform different activities. 

“Plants can have a variety of different homologs, and there are a lot of unknowns,” he said. “It is not easy to predict what they do.” 

Zhou said homologs can bind to each other and form heterocomplexes. Using the native mass spectrometry method, they were able to detect this important protein interaction in the plant. 

“This work still needs a lot of validation, as we are still in the discovery phase,” he said. “There are a lot of homologs present in the plant, but not many studies out there to understand what their functions are … We are continuing the work along this line to try and purify those proteins from different plants and make those proteins in the lab.”