Strong-cation-exchange sulfoethyl aspartamide chromatography for peptide mapping of Staphylococcus aureus V8 protein digests

Abstract

In two recent reports (D. L. Crimmins, J. Gorka, R. S. Thoma, and B. D. Schwartz (1988) J. Chromatogr. 443, 63-71; A. J. Alpert and P. C. Andrews (1988) J. Chromatogr. 443, 85-96) a sulfoethyl aspartamide column was shown to efficiently analyze peptides less than 25 residues in length which differ in the number of nominal positive charges at pH 3.0. In particular, the elution order for a series of distinct peptides ranging in nominal charge from +1 to +7 was found to be monotonic in nature indicating that separation was primarily via a cation-exchange mechanism. The present study employs this chromatographic system to isolate and characterize major fragments of proteolytic digests. Six commercially available proteins of known sequence (myoglobin, beta-casein, concanavalin A, carbonic anhydrase, lentil lectin, and enolase) were digested with Staphylococcus aureus V8 to generate peptide fragments. The resulting mixture was chromatographed on a sulfoethyl aspartamide column to isolate major fragments which were then subjected to amino acid analysis and N-terminal sequencing. With complete proteolysis (i.e., peptide fragments terminating in either an aspartic or a glutamic acid) separation of the fragments should result from the sum of histidine, lysine, and arginine residues contained in each fragment. Most of the peptide fragments eluted at the expected time on the sulfoethyl aspartamide column. Those fragments with anomalous behavior resulted from incomplete cleavage or cleavage at nonacidic residues or were greater than 35 residues in length. Each proteolytic digest was also analyzed by standard reverse-phase C4 chromatography to compare the peptide maps for these two distinct chromatographic modes.