Utilizing advanced top-down proteomics approaches, Northwestern Proteomics researchers and collaborators in the Jewett Lab identified a proteoform responsible for increasing the speed of a well-known metabolic enzyme, Triosephosphate Isomerase (TPI) that drives energy production in the cell. While the traditional bottom-up approach breaks down and degrades proteins into units for further analysis, the top-down approach enables systematic discovery of the modification landscape of whole multi-proteoform complexes providing deeper insights into their cellular function.

“We found a significant amount of phosphorylation on the TPI that was unexpected,” says Luis Schachner, the study’s first author. Phosphorylation is a molecular change, a post-translational modification (PTM) of the cell that is different from what is genetically encoded. “Our approach detects proteoforms directly and tells us how they are changing.”

The researchers hypothesized that phosphorylation made the enzyme able to catalyze reactions more quickly, providing a way for the cell to increase enzyme activity without increasing the amount of enzyme in the cell.

To prove their hypothesis, the Kelleher Lab researchers approached Ben De Soye, a postdoctoral scholar in the Jewett Lab at the time, to synthesize TPI proteoforms with the specific phosphorylations needed to study its activity and biophysical structure.

“My contribution was making it easier to incorporate unnatural amino acids into proteins, by engineering the cell to make it less capable of opposing these efforts,” said Des Soye.

Utilizing the Jewett Lab’s designer protein technology, Schachner and team then employed top-down proteomics to better understand the relationships between phosphorylation of TPI and its activity.

“By bringing both technologies together, we realized that we could achieve much higher performance than if we didn’t have that visibility into how this protein was decorated,” said says Michael Caldwell, Scientific Officer for Northwestern Proteomics.

From these findings, Schachner identified a supercharged version of TPI with up to four times the efficiency of its unedited original counterpart. Their work, says Schachner, provided an ideal test bed that answered a very interesting structural question about the function of proteoforms in the body.

“TPI is actually one of the best-studied enzymes in the whole world. It was very compelling to discover something new about this enzyme,” says Schachner. “This was the first time someone identified a proteoform using proteomics, made it synthetically because the technology didn’t exist before, and found that its function speeds up the enzyme process validating our hypothesis that it was potentially increasing metabolism.”

Main image: Phosphorylation of TPI turbo charges an engine of human metabolism.

By Chelsea Zhao and Lisa La Vallee