Peptide characterization with a sulfoethyl aspartamide column

Abstract

A strong cation-exchange (SCX) high-performance liquid chromatography column (sulfoethyl aspartamide, 200 × 4.6 mm) was used to analyze more than 50 peptides, ranging in length from 5 to 20 residues. These data show that the elution positions of the peptides increase monotonically with the number of positively charged residues. [A 60-min linear gradient of 0 to 100% eluent B at 1 ml/min was used, where eluent A is 5 m M phosphate (pH 3.0)-acetonitrile (75:25) and eluent B is eluent A + 0.5 M sodium chloride.] A comparison of SCX with a standard C 18 reversed-phase (RP) column [60-min linear gradient of 0 to 60% B at 1 ml/min, where eluent A is 0.1% trifluoroacetic acid (TFA), and eluent B is 0.095% TFA-acetonitrile (10:90)] further demonstrates the utility of SCX in peptide characterization. SCX separated an (Arg) 3-containing peptide from the Arg-deleted peptide while RP could not. In addition, SCX and RP resolved the methionine oxidation products of ACTH (4–10) (RP: Met [O]<Met [O 2]<Met; SCX: Met [O]<Met<Met [O 2]), suggesting a mixed-mode mechanism for the ion-exchange system. Finally, SCX separated the sulfated and non-sulfated forms of cholecystokinin (26–33) and Leu-enkephalin as well as the N-terminal acetylated forms of neurotensin (8–13) and angiotensinogen (1–14) from the respective unmodified peptides.