Biotinylated peptides

A more recently introduced format is the AlphaScreen assay. The assay principal behind this technology has previously been described above. In the kinase format a biotinylated peptide is bound to a streptavidin donor bead, and a phopshospecific antibody is bound to the acceptor bead. When the substrate is phosphorylated, the beads come in close proximity and a signal is generated. An example using the assay for the detection of inhibitors of serine kinases is presented by Von Leo-prechting [26]. 

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. 

Girault, S. Chassaing, G. Blais, J. C. Brunot, A. Bolbach, G. Coupling of MALDI-TOF mass analysis to the separation of biotinylated peptides by magnetic strepta-vidin beads. Anal. Chem. 1996, 68, 2122-2126. 

Nare, B., Allocco, J.J., Kuningas, R., Galuska, S., Myers, R.W., Bednarek, M.A. and Schmatz, D.M. (1999) Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4. Analytical Biochemistry, 267, 390-396. 

Craft DV, Goss NH, Chandramouli N, Wood HG (1985) Purification of biotinidase from human plasma and its activity on biotinyl peptides. Biochemistry 24 2471 -2476 Grier RE, Heard GS, Watkins P, Wolf (1989) Low biotinidase activities in the sera of patients with impaired liver function evidence that the liver is the source of serum biotinidase. Clin Chim Acta 186 397-400... 

The probes of this type were shown to selectively label at least 75% of human kinases in crude cell lysates, thus demonstrating their selectivity and promiscuity for kinases [101]. As a follow up, the labeled kinases were subjected to proteolytic digestion, and the biotinylated peptides purified on avidin beads and analyzed by LC-MS/MS. This analysis demonstrated that the site of probe labeling was indeed the conserved active-site lysine as predicted. In contrast to the promiscuity demonstrated by the acyl phosphate probes, several selective covalent inhibitors of protein kinases have been used as ABPP probes. Wortmannin is a natural product derived from the fungus Penicillium funiculosum. It is a potent and specific covalent inhibitor of phosphoinositide 3-kinase (PI3K) and the PI3K-related kinase (PIKK) families [102, 103]. The use of natural products in relation to ABPP is covered by Breinbauer et al. [104]. 

Coester C, Kreuter J, Von Briesen H, Langer K (2000) Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA). Int J Pharm 196(2) 147-149... 

A product-capture strategy was also used to select improved subtiligases. Subtiligase is a double mutant of subtilisine that catalyses the ligation of peptides. A library of >109 variants involving 25 residues of the active site was constructed on the phage-displayed enzyme. Variants that ligated a biotinylated peptide on their own ex-... 

Schopfer, L.M., Champion, M.M., Tamblyn, N., Thompson, C.M., Lockridge, O. (2005). Characteristic mass spectral fragments of the organophosphorus agent FP-biotin and FP-biotinylated peptides from trypsin and bovine albumin (Tyr410). Anal. Biochem. 345 122-32. 

Biotin-avidin interaction. The cellular delivery capacity of several CPPs has been proven using complexes of biotinylated peptides with labeled avidin or strep-tavidin (23,27,37). Avidin could also serve as a scaffold for the assembly of molecules to be delivered into cells using biotinylated CPPs to facilitate the uptake process. 

Serial dilutions of CMV protease standard and DFP-treated protease samples (5-0.005 pM) were assayed for their cleavage activity of the biotinylated peptide substrate for 2 hours, as described in the legend of Table 1. The amount of intact substrate remaining after quenching the reactions were monitored on the BIAcore. 

A second example involves different evolved subtiligases. Subtilisins are proteolytic enzymes sutiligases are mutants that catalyzes the ligation of peptides. Evolved variants of subtiligase that ligate a biotinylated peptide onto their own extended N-termini were selectively captured, as shown in Figure 8.9. The reaction of the biotin-labeled peptide substrate with the evolved enzyme variant results in a... 

Biotin in the diet is largely protein bound, and digestion of these proteins by gastrointestinal enzymes produces biotinyl peptides, which may be further hydrolyzed by intestinal bio-tinidase to release biotin. Avidin, a protein found in raw egg whites, binds biotin tightly and prevents its absorption. The peptide biocytin (e-N-biotinyl lysine) is resistant to hydrolysis by proteolytic enzymes in the intestinal tract but together with biotin is readily absorbed. A biotin carrier, the sodium-dependent multivitamin transporter (SMVT)... 

To monitor protease activities in plant extracts, Wang etal.li8 generated biotinylated peptides that contained a /3-lactone reactive group. The probes labeled several enzymes in leaf proteomes of Arabidopsis thaliana. Interestingly, these studies led to the identification of a papain-like protease called RD21 that has the unexpected ability to ligate donor molecules such as peptides or lactones, probably through a thioester intermediate, to unmodified N termini of acceptor molecules. 

Proteolysis of biotin-containing enzymes releases -biotinyllysine, or biocytin. Biotinidase cleaves biocytin and biotinylated peptides, resulting from degradation of endogenous carboxylases, to biotin and lysine. Thus,... 

The peptides and peptide mixtures were cleaved from the resins and the side chains were deprotected with trifluoroacetic acid phenol ethanedithiol thioanisole water (96 2 1 2 1, v/w/v/v/v) (1 ml) within 3 h. The products were filtered from the resins, which were washed once with acetic acid (0.3 ml), and were precipitated at — 20 °C by the addition of cold n-heptane diethylether (1 1, v/v) (5 ml). The precipitates were washed twice by sonication with n-heptane diethylether (1 1, v/v) and were lyophilized from acetic acid water tert.-butyl alcohol (1 10 50, v/v/v), yielding individual peptides, the completely randomized peptide amide libraries Xn and 220 undecapeptide amide sublibraries O/X 0-NH2. The biotinylated peptide sequence (see below) was obtained by elongating the ApoB 100 peptide 2877-2894 with two spacer amino acids (Ahx), followed by biotin using the coupling method described above. 

Figure 2.4. The schematic shows the basis of a tyrosine kinase hTRF assay. The reaction (a) uses a biotinylated peptide substrate that becomes phosphorylated on tyrosine in the presence of kinase, ATP, and magnesium ions. The reaction product is then detected (b).

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