Biotin tagged

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. 

NHS-LC-biotin can be used to add a biotin tag to monoclonal antibodies directed at certain tumor antigens. The biotinylated monoclonals are allowed to bind to the tumor cell surfaces in vivo, and subsequent administration of an avidin or streptavidin conjugate can form the basis for inducing cytotoxic effects or creating traceable complexes for use in imaging techniques (Hnatowich et al., 1987). 

Figure 17.2 Maleimide groups provide good dienophiles for a Diels-Alder reaction. Biotin-PEG2-maleimide can react with an oligo-diene molecule to form a covalent cycloaddition product, which adds the biotin tag to the oligo.

Figure 18.14 NHS-SS-PEG4-biotin can be used to label a primary antibody molecule that has specificity for a protein or interest. Incubation of the biotinylated antibody with a sample, such as a cell lysate, allows the antibody to bind to its target. Capture of the antibody-antigen complex on an immobilized streptavidin reagent effectively isolates the targeted protein from the other proteins in the sample. The disulfide linkage in the spacer arm of the biotin tag permits elution of the immune complex from the streptavidin support using DTT and without using the strong denaturing condition typically required to break the streptavidin-biotin interaction.

Figure 28.13 A sulfo-SBED-captured protein interaction can be released using DTT to cleave the disulfide within the cross-bridge leading to the bait protein. The result transfers the biotin label to the unknown interacting protein. The biotin tag thus allows the interacting protein to be detected or isolated using (strept)avidin reagents.

Fig. 11. Comparison of the peptidyl transfer reaction in the ribosome and in the selected peptidyltransferase ribozyme. The ribosome contains a binding site for the peptidyl-tRNA (P-site) and for the aminoacyl-tRNA (A-site). In the selected ribozyme the binding site for the AMP-Met-Bio substrate would be analogous to the P-site. The attacking a-amino group which is bound in the A-site in the ribosome is covalently attached to the 5 -end in the ribozyme. Catalytically active RNAs preferentially attach the biotin tag onto themselves and can thus be separated from the inactive ones...

Miee treated with a nontoxie dose of a biotin-tagged OP called FP-biotin had FP-biotinylated albumin in blood and... 

---