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Pressured proteins: A little pressure in proteomics squeezes 4-hour step into a minute
Date:7/9/2008

efore dying from the duress.

To determine if pressure would help in proteomics, the team used the protein albumin. The researchers incubated albumin with the enzyme trypsin at several different pressures for one minute each, then counted how many pieces into which trypsin cut albumin. At 10,000 pounds-per-square-inch up to 35,000 psi, trypsin appeared to maximally cleave albumin. For these tests, the team used facilities at the DOE's Environmental Molecular Sciences Laboratory on the PNNL campus.

The team then tested whether this process could be used for proteomics, in which large numbers of proteins are studied at once. So, they extracted the proteins from a bacterial culture and subjected half to the traditional overnight approach and half to 35 kpsi for one minute.

Overall, the resulting collection of peptides looked very similar between the two methods. The pressure method generated about 10 percent more unique peptides but cut slightly fewer total bonds than the traditional method. The researchers concluded that the benefit of time gained by the pressure method and the additional unique peptides outweighed the dip in total bonds.

Additionally, the team wondered how pressure sped up the enzyme digestion. To determine if pressure caused bacterial proteins to unfold and make their bonds more vulnerable to trypsin, the team turned to myoglobin, a compact glob of a protein that packs a second, loose molecule inside. When they put myoglobin under 35 kpsi in the absence of trypsin, myoglobin lost its molecular parcel, suggesting that pressure opens up or denatures proteins, baring their bonds and giving trypsin more room to work.

Now, the PNNL researchers are integrating the pressurized digestion into their proteomics protocol where appropriate. In other work, Lopez-Ferrer and colleagues are testing whether ultrasound would also speed up this first step. They believe pressure and ultrasound might bring different advanta
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Contact: Mary Beckman
mary.beckman@pnl.gov
509-375-3688
DOE/Pacific Northwest National Laboratory
Source:Eurekalert

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