Micro-Isolation of Polypeptides Precolumn Labeled with Hydrophobic Chromophore

Abstract

Detection of polypeptides is a crucial technique associated with every Chromatographie methods of peptide isolation. Conventionally, peptides are detected, after Chromatographie separation, by their reaction with reagents like ninhydrin [1, 2], fluorescamine [3, 4] or o-phthaldialdehyde [5, 6]. Alternatively, peptides can be detected through their intrinsic absorption of amide bonds at low UV region (200 nm–230 nm) [7,8]. This low UV detection system in combination with the use of reversed phase HPLC is at present the most versatile technique for the isolation of peptides. To enhance the sensitivity of detection, peptides can also be labeled with chromophore preceding Chromatographie separation. For instance, dansylated peptide mappings [9, 10]. We have developed two precolumn labeling techniques for micro-isolation of peptides. (1) A method using DABITC labeling for isolation of peptides with free N-termini [11]. (2) A method using DABIA labeling for selective isolation of cysteine containing peptides [12].