Separation of Peptides Having Structures and Derivatives of Mimosine, a Nonproteinogenic Amino Acid, by a Novel Reverse-Phase HPLC Column Packed With Wide-Pore Silica

Abstract

& To elucidate the mechanisms of the self-assembly and understand the molecular basis of the pathogenesis of conformational changes of proteins, b-amyloid or prion protein related peptides are indispensable materials. Hence, these peptides are known to have b-sheet rich structures that tend to cause difficulty in syntheses, as well as characterization by the conventional solid-phase assembly with reversed-phase columns. In general, reversed-phase HPLC combined with on-line mass spectrometry (LCMS) is routinely used for analyses and purification of resulting crude peptides and is carried out using preparative scale HPLC-columns. However, for such peptides, conventional C18 columns often give broad-peaks. We have improved separation procedures using a novel column packed with wide-pore silica having hydrophilic properties that gave separation of structural conformers with improved profiles. Thus, we have prepared several peptides for conformational studies. The same column also offers advantages for the chiral analysis of mimosine, a nonproteinogenic amino acid, which cannot be separated by the conventional C18 columns.

Rapid octreotide separation from synthetic peptide crude mixtures by chromatography on poly(styrene–co-divinylbenzene)-based reversed phases

“…On the other hand, the shape of the narrow chromatograms became broad and peak tailing occurred. This chromatographic retention behavior was in accordance with the feature of silica-based stationary phase in reversed-phase HPLC [16][17][18]. Where the HPLC material usually exhibited non-specific interactions, such as was the case with residual silanol groups on the surface of silica, or the case with the benzene ring on the surface of PSt, these interactions can be suppressed by the use of TFA.…”

“…Although reversed-phase HPLC can be used for the purification of peptides with a range of physicochemical properties, the technique has some limitations. Extremely hydrophilic peptides may bind only very weakly or not at all to the columns, and extremely hydrophobic peptides and proteins may be recovered only in low yield [11,16]. Silica beads are instable under high pH condition, and the instability constantly limits the application and requires neutral pH mobile phases.…”

Characterization of peptides obtained from digests of bovine brain which accelerate structural conversions of the recombinant bovine prion protein

“…Digestion has been optimized with a two-hour incubation time using trypsin, chymotrypsin and pepsin; longer incubation times did not improve the peptide-mapping patterns. Each digest was chromatographed on a recently developed HPLC column (HiPep-Intrada) which had been designed to give high resolution suitable for peptides and proteins with structures such as amyloid Ab or prion fragments [7]. The chromatogram of the tryptic digests is shown in Fig.…”