Biotin labelled peptides

Key Words Spot synthesis peptide array screening detection probing HRP POD biotin-labeling peptide-protein interaction. 

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... 

A more recent separation technique uses the so-called isotope coded affinity tags (ICAT Applied Biosystems, Foster City, CA). In this technique, a tag comprising a stable isotopically labeled marker and a biotin molecule is reacted with the protein extract from a perturbed sample whilst an unlabelled tag is used to mark proteins from a control sample. Following purification on an affinity column to isolate the biotin labeled peptides the biotin is cleaved leaving a mixture of tagged peptides. These either have a labeled (clinical sample) or unlabelled marker (control). The mixture is subject to LC-MS(/MS)... 

Figure 30 Fibrous scaffolds produced by Kres et al. through incubation of the designed peptides with metal, (a) With Zn(II) (b) With Cu(n) (c) With Co(II) (d) With Ni(II) (e) A bright-held image of the network when the biotin-labeled peptide is incorporated and bound to fluorescently labeled streptavidin (f) A fluorescence microscopy image of (e). (Scale bars in images (a-d) = 5 tm and (e, f) = 200 pm.) (Reproduced from Ref. 94. Wiley-VCH, 2009.)...

Fig. 34 Functionalisation of bait peptides. I Gold-labelled streptavidin is attached directly. 2 Biotin-labelled anti-FLAG antibody is bound to the FLAG on the peptide (reaction b) and this is used to attach gold-labelled streptavidin (reaction d). Reprinted with permission from Ryadnov and Woolfson [76]. Copyright 2004 American Chemical Society...

Figure 3.3. Structure of the ICAT reagent. The reagent contains a biotin affinity tag that is used to isolate ICAT-labeled peptides. The reagent also contains a linker that exists in a heavy (where X= deuterium) or light form (X= hydrogen) and a reactive group with specificity towards the thiol groups of cysteine residues. Figure adapted from Gygi et al. (1999).

To make these substrates suitable for biological assays, the introduction of functional groups that can be traced with the proper analytical techniques is essential. The use of radio-, fluorescent-, and biotin-labeled lipidated peptides has been reported. The synthesis of fluorescent substrates is chemically straightforward and allows for production of larger quantities than the enzymatic synthesis used for radiolabeled peptides and is thus preferred over the use of radioactive compounds. [1 21] Common fluorescent probes can be introduced by conjugation to a free functional group present in the peptide. The fluorescent moiety is... 

Figure 6.4 The structure of the ICAT reagent that consists of three elements an affinity tag (biotin), which is used to isolate ICAT-labeled peptides a linker that can incorporate stable isotopes and a reactive group with specificity toward thiol groups (cysteines). The reagent exists in two forms heavy (contains eight deuteriums) and light (contains no deuteriums). (Reprinted with permission from Gygi et al., 1999. Copyright 1999 Nature Publishing Group.)...

Lamos and colleagues reported the modification of cyclophilin A (CypA) by binding immunosuppressive cyclosporin A with a benzophenone-Dj j and a biotin moiety (compound 89, Fig. 11a) [91]. As a proof of principle, they used a 1 1 mixture of this TIP and its nondeuterated isoster for the selective PAL and pulldown of CypA among three other proteins. Subsequent tryptic digestion of the elutes and LC-MS/MS analysis allowed the identification of 11 CypA characteristic peptides, two of which were modified with the probe, as evidenced from the double, 11 Da separated, peaks in the mass spectra. The large 11-Da mass difference allowed easy visual recognition of labeled peptides in the mass spectra, which makes this a powerful method for determination of the modification site after PAL however, application in more complex systems still remains to be done. 

Scintillation proximity (FlashPlate, SPA) Product of reaction is a 33P labeled peptide biotin that can be captured on a detection bead that scintillates from proximity to 33P dephosphorylation by phosphatases can High-throughput, relatively artifact free in imaging-based systems universal readout for kinases versatile Radioactive waste disposal may be less sensitive than TR-FRET Park (1999) Sills (2002) von Ahsen (2006)... 

The availability of a new version of isotopically labeled ATP, [33P]ATP, provided benefits of safety and longer half-life. The lowered energy was also better suited for scintillation proximity assays (SPAs). The SPA was a major step forward because it eliminated the need for wash steps by capturing the [33P]-labeled peptide on a functionalized scintillating crystal, usually via a biotin-streptavidin interaction. 

Though biotin-avidin complexation has been used widely to isolate specific targets, it is notorious for generating false-positive identifications. To mle out the possibility that our results were an artifact we set out to find the OP-labeled peptide. We reasoned that convincing proof for OP labeling required identification of the labeled peptide and the labeled amino acid. 

During years of mass spectrometry analysis, we had consistently identified OP-labeled tubulin in mouse brain. Therefore, we studied pure bovine tubulin (Cytoskeleton, Inc.) by treating it with soman, sarin, FP-biotin, DFP, chlorpyrifos oxon, and dichlorvos. We isolated the OP-labeled tryptic peptides and analyzed them by fragmentation in the QTRAP 2000 and QTRAP 4000 mass spectrometers. We identified five OP-labeled peptides in tubulin (Grigoryan et al, 2008). In every peptide, the OP was covalently attached to tyrosine (Table 56.1). 

The isotope-coded affinity tag (ICAT) technique involves differential labeling of two different protein populations on the side chain of reduced cysteinyl residues using one of two chemically identical but isotopically different ICAT reagents (19). By incorporating a biotin affinity tag into the ICAT reagents, selective isolation and purification of labeled peptides substantially reduces sample complexity. The ICAT approach has been applied successfully to the systematic identification and quantification of proteins contained in the microsomal fraction of cells... 

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. ... 

Biotin acceptor peptides >15 Biotin Biotin ligase, ATP Covalent and irreversible Intracellular, cell surface Allows use of derivatized streptavidins to label cell surface proteins [31]... 

Biotin acceptor peptides >15 Keto isostere of biotin Biotin ligase, ATP, hydrazide derivatives Covalent and reversible Cell surface Two-step labeling required [32]... 

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