Isolation of peptides

Bennett, H.P.J. Browne, C.A. Goltzman, D. and Solomon, S. Isolation of peptide hormones by reverse-phase high pressure liquid chromatography. In Gross, E., and Meinenhofer, J., ed, Pepti des. Structure and Bioloai cal Function. Rockford,... 

Hydrophobic Effects and Solvophobic Considerations for the Isolation of Peptides by Reversed-Phase Chromatography Methods... 

Harding, H. W., and Rogers, G. E. (1976). Isolation of peptides containing citrulline and the cross-link, epsilon-(gamma-glutamyl) lysine, from hair medulla protein. 

This approach offers a number of significant advantages. Since whole cells are used as the affinity support, the receptors are likely to be in their native conformation, allowing the isolation of peptide ligands directed against conformationally structured epitopes [70, 71]. Moreover, the technique leads to the identification of cell surface markers which distinguish otherwise similar cell types such as antigens, epitopes, or receptors, without the need to either identify or purify a particular receptor in advance [38, 42, 43, 72]. 

The contributions of Richard Block to the serum protein problem originated from the hypothesis of Kossel. From recent data on the amino acid composition of the proteins found in animal sera, a formulation is derived which reflects the properties of a continuous system of molecular species originating from a common biosynthetic pathway, as if from mixed polymers of monomeric peptides of lower molecular weight. Indirect evidence of this is found in the amino acid interrelationship, and direct evidence is limited to the isolation of peptides of common composition, whose primary structures are still under investigation. These findings suggest that undifferentiated proteins may be continuous systems rather than discrete molecular species. 

Ono, S., Hosokawa, K., Miyashita, K., and Takahasi, K. 2003. Isolation of peptides with angiotensin I-converting enzyme inhibitory effect derived from hydrolysate of upstream chum salmon muscle. J. Food Sci., 68,1611-1613. 

Native Hez-PBAN underwent oxidation of its two methionines during the course of its isolation and purification. Oxidation of methionine to methionine sulfoxide is known to occur during the isolation of peptides and proteins from natural sources (15, 16). It was observed that purified samples of synthetic Hez-PBAN were easily oxidized to a mixture of oxidation products resulting from the oxidation of Mer and/or Met to their respective sulfoxides. These observations are consistent with the isolation of Hez-PBAN from the PBAN II zone. We speculate that the continuum of biologically-active HPLC fractions observed between 44 and 52 min after RP-HPLC Step A, represents Hez-PBAN (PBAN III), a mixture of Hez-PBAN monosulfoxides (PBAN II), and Hez-PBAN disulfoxide (PBAN I), all of which were biologically active. 

After the actions of PCl/3 and PC2 for cleavage at the C-terminal side of paired basic residues (Fig. 3), the resulting peptide products contain basic residues at the C-termini that are then removed by carboxypeptidase E (CPE). Studies of ijjgj contain mutant, inactive CPE, showed that these animals show altered neuropeptide production (49-55). Studies of CPE peptide substrates in the fat/fat mice have been facilitated with specific isolation of peptides with C-terminal basic residues by the anhydrotrypsin affinity column for enrichment of CPE substrates (49-53). Analyses of such CPE substrates have demonstrated that numerous neuropeptides use CPE for their biosynthesis (49-55). 

An early, reliable strategy for obtaining internal sequences involved electroblotting proteins to nitrocellulose membranes, in situ protease digestion, and subsequent isolation of peptides by reverse phase HPLC (7). This method has been employed in our laboratory for more than three years with a success rate of >95%. The two major limitations of this approach are 1) the blotting efficiency using nitrocellulose tends to be highly variable and frequently recovery at this step is low, and 2) the multiple step nature of this procedure further reduces overall recoveries. Even when recoveries are optimized at each step of the procedure, it is usually necessary to start with at least 5 to 10 times more protein than the amount required for direct N-terminal sequence analysis. 

This was first foimd by Sanger et al. (1955) in a peptide from insulin and was observed with other peptides by Hirs et al. (1956) and Smyth et al. (1962). The reaction appears to occur when acidic buffers or dilute acids are employed for isolation of peptides. Conversion of the cyclic pyrrolidone carboxyl residue to a glutamyl residue is obtained on mild hydrolysis in dilute acids or alkalies. The cyclization reaction leads to difficulties when sequence methods are used which proceed from the amino-terminal end of a peptide. In addition, this reaction can occur when an internal glutamine residue becomes amino-terminal in the course of stepwise sequence analysis under acidic conditions, as in the Edman methods. An incorrect sequence for a peptide from ribonuclease was deduced as the result of cyclization of amino-terminal glutamine and acidic destruction of serine and threonine in the same peptide (Smyth et al., 1962). 

The isolation of peptides fully protected according to the Fmoc strategy is rather challenging, as the choice of aUyl scavengers is limited to those whose basicity is sufficiently low to avoid competitive removal of the N-terminal Fmoc group (essentially HOBt, N-methylanUine, or tributyltin hydride). The problem is more acute than for the selective deprotection of simple allyl or Aloe groups because allylic linkers with their disubstituted... 

White, S. J., Simmonds, R. E., Lane, D. A., Baker, A. H. (2005). Efficient isolation of peptide ligands for the endothelial cell protein C receptor (EPCR) using candidate receptor phage display biopanning. Peptides, 26, 1264-1269. 

The fluorometric and chromatographic techniques described in this chapter can be valuable in all phases of peptide chemistry and biochemistry. For quantitative analysis, they can provide a degree of sensitivity and specificity not always attainable with bioassay or immunoassay. They allow preparative isolations of peptides from tissue extracts to be carried out at a level that is orders of magnitude lower than is possible by classical means. They... 

The novel method of isolation of peptide amides [129] yielded an additional gastrointestinal peptide with biological activities. Galanine, H-Gly-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-His-Ala-Ile-Asp-Asn-His-Arg-Ser Phe-HiS Asp Lys-Tyr-Gly-Leu-Ala-NH2 raises the blood sugar level and contracts the isolated rat ileum. Synthesis of the (porcine) peptide followed [133] its isolation and sequence determination within a short time. 

---