Peptide purification, displacement

II. PURIFICATION OF AMINO ACIDS AND PEPTIDES BY DISPLACEMENT CHROMATOGRAPHY... 

Complex peptide mixmres can now be analyzed without prior purification by tandem mass spectrometry, which employs the equivalent of two mass spectrometers linked in series. The first spectrometer separates individual peptides based upon their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses determined. As the sensitivity and versatility of mass spectrometry continue to increase, it is displacing Edman sequencers for the direct analysis of protein primary strucmre. 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Tetrabenzo[a,c,g,i]fluorene has been used to selectively link synthetic intermediates to charcoal, for the purpose of their purification. In polar solvents, the tetrabenzo-fluorene is strongly adsorbed by charcoal this enables efficient separation of the intermediate from reagents. After centrifugation and washing, the intermediate is displaced from the charcoal and released into solution by addition of a non-polar solvent, and a new synthetic operation in solution can be conducted (Figure 3.43). Tetrabenzo-fluorene has also been used for the purification of peptides [849] and oligonucleotides [850], and for the synthesis of quinolones [851]. 

Two case studies will be shown here to demonstrate the development of purification processes in both overload and resolution based separations 51]. The first example summarizes the purification of a synthetic peptide by overload chromatography, or more accurately described as sample self displacement chromatography The techniques applied to this separation are applicable to any molecule and can be applied to all modes of chromatography, with the exception of size exclusion chromatography. 

The self-displacement approach to purification of a synthetic peptide containing 25 amino acids is shown below[51]. The analytical HPLC trace (Figure 5.1) shows the impurity peak (A), which is displaced by the main component (B) in the demonstration. 

Now some detail—and we will discuss the Merrifield version of peptide synthesis. Spherical cross-linked polystyrene beads of about 50 pm in diameter are used and attached to various spacers of which the simplest is just a CH2 group from the chloromethylated polystyrene we have just discussed. The caesium (Cs) salt of the amino acid is used to displace the chloride as it is a better nucleophile than the Na or K salts. A better alternative is Pam (shown in the margin). It can be used as the nucleophile to displace the chloride first. The amino acid is then added after purification. No chloromethyl groups can remain on the polymer with this spacer. 

Hodges, R. S., Burke, T. W. L., and Mant, C. T. (1988). Preparative purification of peptides by reversed phase chromatography—sample displacement versus gradient elution modes. ]. Chromatogr. 444, 349-362. 

When the identify of the inhibitory activity present in a sample is unknown (as is frequently the case when RRAs are used to screen for novel substances), the absolute amount of the substance cannot be determined from an RRA. Nevertheless, RRAs are useful to detect the presence of unidenhfied substances m tissue extracts, to monitor the success of purification techniques used to concentrate or isolate an unknown agent, or to detect the dilution or loss of the inhibitory activity during separation or purification attempts Even in instances when the identity of the inhibitory activity present in a sample is known, as is the case for the RRA for endogenous opiates (Childers et al., 1977, Simantov et al., 1977), the concentration of the substance may remain indeterminant, since this assay does not discriminate between the various opioid peptides that occur in brain. In either of these latter situations, the difficulty m determining the absolute amounts of the inhibitory activity present in the initial sample is circumvented by expressing the displacement of specifically bound ligand in arbitary units of equivalence relative to a reference compound. 

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