Direct microsequence analysis of polypeptides using an improved sequenator, a nonprotein carrier (Polybrene), and high pressure liquid chromatography

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Abstract
The use of a nonprotein carrier (Polybrene), high pressure liquid chromatography and modifications in Edman chemistry were combined with the improvements of a commercial spinning cup sequenator suggested by Wittmann-Liebold to analyze amino acid phenylthiohydantoins obtained from automated Edman degradation of microquantities of polypeptide directly without the use of radiolabel. This approach allowed the determination of the sequence of the N-terminal 47 residues of sperm whale myoglobin starting with 200 pmol of protein, 77 residues of an antibody light chain with 5 nmol of protein and 54 residues of an antibody heavy chain with 8 nmol of protein. In addition, a hydrophobic 14-residue peptide was completely sequenced at the 1.5 nmol level. This technique of direct analysis for microsamples is capable of providing routine, extended N-terminal sequence analysis for nanomole and subnanomole levels of polypeptides and proteins. It also is applicable to analysis of more classical sample quantities.