Peptides method modifications

Genove, E., Semino, C., and Zhang, S. Self-Assembling Peptides Incorporating Modifications, Method for Preparation and Use as Scaffolds in Tissue Engineering, 2004-US20549... 

Solid-phase synthesis (SPS) of peptides containing at their C-termini the usual carboxylic acid or carboxamide functionalities is a well-established process the peptide is traditionally attached to the resin through the a-carboxyl group of the C-terminal residue, and synthesis proceeds in the C A direction (1,2). However, synthetic peptides containing modifications at the C-termini are often desired because of their potential therapeutic properties and/or synthetic significance as intermediates in peptide and protein chemistry. Therefore, effective solid-phase methods are needed for the preparation of these peptide targets (3). The present chapter describes backbone amide linker (BAL) strategies (4)... 

This chapter aims to present an overview on the main peptide bond modifications which have been proposed in the recent years. The solution- and solid-phase synthesis of the new synthetic oligomers, the methods for their characterization, and their biological and folding properties will be discussed. Particular emphasis will be given to those modifications which are suitable for combinatorial chemistry applications and developments. 

While the first 20-30 residues of a peptide can readily be determined by the Edman method, most polypeptides contain several hundred amino acids. Consequently, most polypeptides must first be cleaved into smaller peptides prior to Edman sequencing. Cleavage also may be necessary to circumvent posttranslational modifications that render a protein s a-amino group blocked , or unreactive with the Edman reagent. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

The second method also relies on site-specific chemical modification ofphosphoproteins (Oda et al., 2001). It involves the chemical replacement of phosphates on serine and threonine residues with a biotin affinity tag (Fig. 2.7B). The replacement reaction takes advantage of the fact that the phosphate moiety on phosphoserine and phosphothreonine undergoes -elimination under alkaline conditions to form a group that reacts with nucleophiles such as ethanedithiol. The resulting free sulfydryls can then be coupled to biotin to create the affinity tag (Oda et al., 2001). The biotin tag is used to purify the proteins subsequent to proteolytic digestion. The biotinylated peptides are isolated by an additional affinity purification step and are then analyzed by mass spectrometry (Oda et al., 2001). This method was also tested with phosphorylated (Teasein and shown to efficiently enrich phosphopeptides. In addition, the method was used on a crude protein lysate from yeast and phosphorylated ovalbumin was detected. Thus, as with the method of Zhou et al. (2001), additional fractionation steps will be required to detect low abundance phosphoproteins. 

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