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Deinococcus Diva Melds New Tech to Map Proteome

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Mary Lipton was in the right place at the right time. Continuing an interest in proteins stemming from her PhD days at the University of South Carolina, she moved out for a postdoc position at the Pacific Northwest National Laboratory and was in the perfect spot, she says, “when the proteomics field really erupted.”

After nine years at PNNL, the last five as a senior research scientist, Lipton’s protein curiosity resulted in her being the lead author on a paper announcing the protein map of Deinococcus radiodurans, the bacterium known for its radiation resistance, bioremediation capability, and capacity to repair its own DNA. It’s the most complete protein map published to date, according to the lab, and Lipton credits the success of the project largely to the lab’s Fourier-transform ion cyclotron resonance high-throughput mass spectrometer.

Lipton was part of a team looking to use the FTICR in a pilot project to study its proteomics prowess. “We wanted to focus on an organism that was relevant to a DOE mission,” she says, since the team was requesting funding from the agency. Deinococcus, which had just been sequenced and annotated, seemed an obvious choice. “We had to start from scratch [for the project],” Lipton says. The pieces to analyze the organism’s proteome were all in place at PNNL, but had not been used together before.

Because Deinococcus is such a tough critter, part of looking at its proteins involved studying it under various stresses, such as heat and cold shock, exposure to DNA-damaging chemicals and radiation, and starvation.

Key to the project was the AMT, or accurate mass tag, database that Lipton and colleagues built from identified peptides used as biomarkers for proteins, which drastically reduced the amount of time spent on traditional tandem mass spec.

Thanks to the AMT process, Lipton says, after exposing Deinococcus to radiation, the team could identify the proteins expressed just four to five hours afterward. “If we didn’t have that database, we’d have to take the cell and put it back through tandem mass spec, and … it would take a week,” she says.

Though FTICR is only used at PNNL, Lipton says the lab is currently working with manufacturers, so the improvements made at the lab may become commercially available in the next couple of years. The mass spec has increased sensitivity, can find low-abundance proteins, and accurately quantifies protein levels, according to Lipton. She and her team are looking at ways to make the mass spec just like a quantitative DNA microarray: “We’re developing methods using the FTICR to get [that kind of] information out of a protein.”

— Meredith Salisbury

 

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