Retention behaviour of a template-assembled synthetic protein and its amphiphilic building blocks on reversed-phase columns

The retention behaviour of a six-helix bundle template-assembled synthetic protein (TASP) molecule and its amphiphilic building blocks was investigated. The TASP consists of a circular template, cyclo(1-12)[KG]6, and six identical potentially alpha-helical peptides of the sequence KLALKLALKALKLALKLA. As an alpha-helix. this peptide is amphiphilic along the axis of its helix. Based on this sequence, the retention times of a set of acetylated peptides containing from seven to twenty amino acids on a Nucleosil C18 column were compared with another set of peptides with the same amino acid composition but a non-amphiphilic structure. Peptide elution was effected with linear trifluoroacetic acid (TFA) - water to TFA - acetonitrile gradients. The difference in retention times increased with peptide length; the 9-mers eluted at the same time, but there was a difference of 3.5 min for the 13-mers and 22.3 min for the 20-mer, indicating the induction of secondary structure on binding to the stationary phase, The same pair of 20-mers on Vydac C18, C4 and biphenyl columns gave differences in retention times of 23.2, 16.7 and 12.3 min, respectively. The TASP molecule was irreversibly adsorbed to C18 stationary phases, whereas it was eluted from C4 and biphenyl columns as a single sharp peak. Several side-products resulting from the synthesis of the TASP molecule were identified by matrix-assisted laser desorption ionization mass spectroscopy. A comparison of the retention times of these side-products and the results of pre-column denaturation experiments indicated that the tertiary structure of the TASP molecule is maintained on binding to biphenyl and C, columns

ANALYSIS OF SYNTHETIC PEPTIDES USING MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS-SPECTROMETRY

Matrix-assisted laser desorption ionization mass spectroscopy (LDI MS), a novel method for analysis of large molecules, has been used for characterization of synthetic peptides and their by-products. The potential of LDI MS is demonstrated by analyzing crude synthetic peptides representing typical members of newly designed peptides and proteins. In the first case, a fragment condensation reaction yielding a highly hydrophobic six-helix bundle template-assembled synthetic protein (TASP) is monitored. Then, a crude 19-mer peptide designed to adopt an amphiphilic alpha-helical structure and its by-products from SPPS are identified. Finally, analysis of crude hirulog-1, a 20-mer peptide designed as a thrombin inhibitor, using C18 reversed phase high performance liquid chromatography (RP HPLC), capillary electrophoresis (CE) ahd LDI MS, manifests the potential of the latter method