High-performance liquid chromatographic purification of extremely hydrophobic peptides: transmembrane segments

Transmembrane peptides of integral membrane proteins often exhibit extremely high hydrophobicity. Therefore, the solubility of such peptides in solvents commonly used in HPLC is usually very low and the interaction with generally applied stationary phases such as silica gel or C18 reversed phases appears to be extremely strong, which makes the characterization and purification of these peptides difficult. The analytical characterization and preparative separation of the synthesized M1 transmembrane sequence of the inhibitory glycine receptor M(r) 48,000 subunit and some of its fragments is shown. M1 and its larger fragments could be dissolved in a dichloromethane-hexafluoro-2-propanol mixture containing a trace amount of pyridine for their separation on a C4 phase by employing linear two-component gradients of formic acid-2-propanol and formic acid-water with ratios up to 4:1 (v/v). Conditions to avoid formylation of the peptides are indicated.