Complete Tryptic Digestion of Membrane Protein Bacteriorhodopsin

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Ying-Qing Yu, Martin Gilar and John C. Gebler [Waters]
ISPPP 2003, Delray Beach, Florida, USA, November 9-12
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Bacteriorhodopsin, Membrane Proteins
Symmetry300(™) C18 300 Å 3.5 µm Steel 1.0 mm 150 mm Symmetry300(™) C18 300 Å 3.5 µm Steel 0.32 mm 150 mm
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Mass spectrometry peptide mapping of membrane proteins is complicated by the proteins limited solubility and proteolytic resistance. As a result, very limited peptide coverage is typically obtained. Surfactants used to improve the solubility disrupt enzyme activity and interfere with LC and MS analysis. We demonstrate that a complete peptide map of membrane protein (bacteriorhodopsin) can be achieved by using an enzyme compatible, acid-degradable ionic surfactant. Bacteriorhodopsin was digested overnight with trypsin with or without addition of surfactant. Trifluoracetic acid was added to stop the proteolysis and degrade acid-labile surfactant. Samples were analyzed by MALDI-MS, and capillary LC-MS. While current protocols for digestion of hydrophobic proteins (using urea or organic solvents) yielded only limited sequence coverage, a complete peptide map was obtained with the help of novel surfactant. Presumably, the surfactant effectively solubilizes and unfolds proteins, which enables trypsin to digest the hydrophobic transmembrane region. A high-quality peptide map was obtained from less than one microgram of protein. Overall sample handling prior to LC-MS analysis was substantially less laborious than earlier published methods (Hixson et al., Electrophoresis 2002, vol. 23, 3224).

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