[9] Primary structure of bacteriorhodopsin: Sequencing methods for membrane proteins

Publisher This chapter discusses the sequencing methods for membrane proteins. For sequencing, separation procedure is required. Separation of the short-sized fragments achieved by gel permeation in organic solvents using Sephadex LH-20. The corresponding problem of the separation of C-1 fragments containing larger and more hydrophobic fragments is solved by a combination of reverse-phase HPLC 1° and gel permeation using Sephadex LH-60 in organic solvents. Amino acid sequencing of the fragments is performed by a combination of stepwise (Edman) degradation methods and gas chromatographic mass spectrometry. Edman degradations are carded out both in solution phase (Beckman sequencer) and in solid phase after attachment of the peptides to a derivatized glass support. Some fragments could not be completely sequenced directly and further enzymatic cleavages were required. These fragments are derivatized with hydrophilic reagents—such as 4-sulfophenylisothiocyanate (SPITC)—to render them soluble under the appropriate aqueous conditions.