Peptide mapping using combinations of size-exclusion chromatography, reversed-phase chromatography and capillary...

Recombinant extracellular superoxide dismutase was proteolytically degraded by trypsin and the digest was thereafter separated using three different separation techniques. The size differences of the obtained fragments were exploited by size-exclusion chromatography (SEC) on a highly efficient column for peptide separation. Collected fractions representing different size groups of peptides were then separated on the basis of hydrophobicity on reversed-phase liquid chromatography (RPLC). This led to simplified RPLC-chromatograms where collected peaks were considerably more pure than if the digest was separated direct on RPLC and the identity of eluted peaks could easily be determined by amino acid analysis. Finally the digest was run on capillary electrophoresis for separation based on charge differences. For the identification of the different peaks in the electropherogram, the total digest was spiked with known peptides purified by the two other separation techniques. This technique proved to be very powerful for peak identification in an electropherogram from a digest composed of many fragments eluting closely together.