Biotin biotinylated peptide

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. 

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. 

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)... 

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,... 

Using the genetic approach the biotinylated obelin was obtained. A recombinant apoobelin capable of being biotinylated in vivo in E. coli cells with BirA was constructed by fusing in-frame a synthetic DNA-fragment encoding the artificial biotin acceptor peptide to the N-terminus of the obelin cDNA gene. The application of the fusion protein as a label in immunoassay was demonstrated. ... 

Tatsumi H, Fukuda S, Kikuchi M, Koyama Y. Construction of biotinylated firefly luciferase using biotin acceptor peptides. Anal Biochem 1996 243 176-80. 

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 12.15 Schematic representation of the switching of mixed TEGT-biotinylated peptide SAMs between a bioactive and bioinactive state. Depending on the electrical potential applied, the peptide can expose (-1-0.3 V) or conceal (—0.4 V) the biotin site and regulate its binding to neutravidin [33]. For a color version of this figure, see the color plate section. 

The intervention of gastrointestinal proteases and peptidases transforms the protein bound forms of dietary biotin into biocytin or biotinyl-L-lysine and biotin-short peptides. In humans, it was assumed that biotinyl proteins and peptides arose from only two sources protein bound in food and degradation of endogenous biotin-containing carboxylases. 

For biotin coming from endogenous biotinyl proteins, biotin-containing carboxylases are degraded to biotinyl peptides which leads to the formation of biocytin through sequential hydrolysis. The intervention of biotinidase releases lysine and biotin. 

Fig. 35 TEM image of peptide fibres coated with streptavidin-gold nanoparticles, (a, b) Peptides with biotin directly attached (using SAF-pl-biotin) particles are 10 nm, (c) Peptide fibre with biotinylated anti-FLAG antibody attached that was then bound to gold-labelled streptavidin particles are 5 nm. Reprinted with permission from Ryadnov and Woolfson [76]. Copyright 2004 American Chemical Society...

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Add a quantity of the biotin-PEG -amine solution to the solution containing the car-boxylate molecule to achieve the desired molar excess. For molecules containing a single carboxylate to be modified, a 1.5- to 2-fold molar excess may be sufficient. However, for proteins or peptides that also contain competing amines, a much larger excess of biotin compound should be used (e.g., 100-fold excess). For instance, for protein biotinylation, add 120 pi of the biotin-PEG -amine solution per ml of the solution prepared in Step 1. 

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