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New Tube-Gel Protocol Promises High- Throughput Prep for Membrane Proteins

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SAN ANTONIO — A new, high-throughput method for preparing membrane proteins for mass spectrometry analysis received a considerable amount of interest at last week's American Society for Mass Spectrometry conference here

The method, called the tube-gel digestion protocol, was developed by Haining Zhu, an assistant professor in the department of molecular and cellular biochemistry at the University of Kentucky.

Several speakers during a session on hydrophobic peptides and membrane proteins noted that membrane proteins are traditionally hard to analyze because they contain hydrophobic portions that can only be solubulized using detergents or organic solvents. Detergents must be removed from proteins and peptides before they enter a mass spec because they interfere with analysis. Organic solvents are compatible with mass spec analysis, but they can be tricky because they may require a larger volume of sample.

Zhu's new protocol is similar to a standard membrane protein preparation protocol in that it uses SDS detergent to solubulize membrane proteins. The trick is that hydrophobic proteins dissolved in detergent are mixed with acrylamide solution before the solution is polymerized, and then poured into a thin tube. Later, the detergent is washed out of the "tube gel," and the proteins are enzymatically digested inside the gel.

When compared to the traditional membrane protein protocol, which involves pouring an acrylamide slab gel, loading the solubulized proteins into lanes on the gel, running the gel, washing the gel, then slicing the gel into pieces before protein digestion inside the gel pieces, the new tube-gel method is much more high throughput, Zhu noted.

"You can go home and use [the protocol]. It does not cost anything extra," said Zhu, who spoke to ProteoMonitor after giving his talk.

To compare the efficiency of the tube-gel method to the traditional slab-gel membrane protein protocol, Zhu purified plasma proteins from a PC3 prostate cancer cell line using both methods, side-by-side. After mass spec analysis, he found that the two methods identified 115 proteins in common. In addition, the traditional membrane protein protocol identified 268 proteins that were not identified by the tube-gel method, while the tube-gel method identified 178 proteins that were not identified by the traditional method.

Zhu pointed out that while the traditional method did identify about 100 more proteins than the tube-gel method, it was also much slower and more laborious, requiring separate digestions of about 20 slices of gel, followed by the same number of mass spec runs. Because the tub-gel approach doesn't include an electrophoresis step, the protein separation is not a good as it would be using the traditional method, but Zhu said that the speed of the new protocol may overcome that drawback in some applications.

"The tube-gel digestion offers much higher-throughput analysis — it was one sample, one run," said Zhu.

Zhu also concluded that up to five percent SDS detergent could be used in the protocol without hurting base peak separation during liquid chromatography and mass spectrometry analysis.

Although membrane proteins are difficult to deal with, researchers who spoke during the ASMS session emphasized that they can not be ignored because they make up at least 25 percent of the human proteome, and they play very important roles in cell adhesion and transportation through ion channels, and as receptors and cell surface antigens.

A crowd of interested researchers surrounded Zhu after his talk to ask him about specific details of the protocol.

Christopher Haynes, a graduate student at the Georgia Institute of Technology who is studying the membrane protein serine palmitoyl transferase thanked Zhu for sharing his easy-to-use protocol with the audience.

"This is a great innovation that he has come up with," said Haynes. "I am going to try it."

Michael Glocker, the director of the proteome center at the University of Rostock in Germany said that Zhu's approach is interesting, but it needs to be proven with more than one protein.

Glocker said that he suspected that with Zhu's approach, more hydrophobic proteins may not be eluting from the gel after digestion, and that many of the proteins that are showing up after Zhu's mass spec analysis may in fact be the more soluble ones with hydrophilic portions.

Glocker's approach to analyzing hydrophobic proteins involves running an electrophoresis gel, blotting proteins onto a membrane, and then digesting the proteins on the membrane.

"I need just a few more examples to be convinced [about Zhu's protocol]," said Glocker.

Zhu said he had considered patenting his method, but ruled it out because it would be difficult to patent something that is so readily accessible.

"There are two issues with patenting," said Zhu. "The first is, how can you prevent other people from doing it? The second is I would like my method to be used by as many scientists as possible, [so why patent it]."

Zhu said he has written a paper describing the tube-gel method, and it is currently under review by the journal Molecular and Cellular Proteomics.

— Tien-Shun Lee ([email protected])