Microsequencing and amino acid analysis in the determination of protein structure
Novel applications of the Edman method of amino acid sequencing are being developed to allow the investigation of aspects of protein structure which were previously difficult to study. For example, a combination of proteolytic digestion, peptide isolation by HPLC, and gas phase sequencing has been used to elucidate the disulfide pairings in neurophysin. Methods are also being developed to overcome problems in sequencing the N-terminal residues of blocked proteins. One such method has allowed the determination of the complete amino acid sequence of prothymosin alpha and parathymosin, both of which possess an N-terminal acetylated serine residue. In addition, with new methods of electroblotting proteins from polyacrylamide gels onto polyvinylidene difluoride membranes, the amounts of protein needed for sequencing is constantly decreasing. It is now possible to obtain meaningful sequence information from only a few picomoles of protein and the sensitivity of the method is approaching the femtomole level.