Shotgun proteomics targets membrane proteins

The team that introduced perfluorooctanoic acid into proteomics. (From left to right: Drs. Masura Miyagi, Sara E. Tomechko, and Chandra Sekhar Rao Kadiyala). Image courtesy of Miyagi’s lab.

01/13/2011

Vincent Shen

Using a detergent from the chemical industry, researchers may have improved membrane protein isolation and analysis.

Case Western University scientists have isolated membrane proteins using a detergent from the chemical industry. In a recent paper published at PLoS One, they detail how this detergent could help researchers to analyze membrane proteins and other proteins from scarce tissue samples with shotgun proteomics.

Led by Masura Miyagi, an assistant professor at Case Western University’s Center for Proteomics and Bioinformatics, the team used a volatile detergent called perfluorooctanoic acid (PFOA)—which is used by the chemical industry in the manufacturing process of non-stick cooking pans, stain-resistant carpeting and clothing, and food contact paper—to release proteins that were deeply embedded in the cell membrane. The researchers were then able to analyze these proteins with their shotgun proteomic approach, which uses liquid chromatography-tandem mass spectrometry (MS) to identify protein sequences.

“Currently, it is very difficult to analyze any membrane protein,” said Chandra Sehkar Kadiyala, the first author of the paper and research associate of Miyagi’s lab. Hydrophobic transmembrane domains trap proteins in the insoluble fraction during protein purification. This decreases the recovery of membrane proteins, preventing protein identification.

While conventional detergents can increase the recovery of these proteins, these detergents also hamper subsequent protein identification by MS. Researchers use MS to measure each protein fragment’s unique mass spectrum, identifying the proteins in a sample. The detergents, however, interfere with mass spectrum measurements, undermining accurate protein identification.

Unlike conventional detergents, PFOA can be easily removed from samples through evaporation. After several months of adjusting variables such as pressure, solvent, and acidity, Miyagi’s team managed to achieve 99.9% PFOA-free samples before MS analysis.

“By using [PFOA], we can solubilize the membrane protein and find [its] sequence information,” said Kadiyala. After MS analysis, the researchers were able to identify 75 proteins in their sample. About two-thirds of these proteins were membrane proteins.

Although the method provides researchers with a new way to access membrane proteins, it remains time-consuming. Currently, the PFOA requires six hours to vaporize. In addition to applying the method to other tissues and organisms, the team is hoping to reduce the time needed for PFOA vaporization.

The paper, “Perfluorooctanoic acid for shotgun proteomics,” was published Dec. 30 at PLoS One.