Peptides, labelled

Peptide labeled at C-terminal Peptide labeled at N-terminal... 

Figure 7.17 The P829 peptide labeled with technetium-99m to prepare radiopharmaceutical 99mTc-P829, NeoTect , as discussed in reference 96.

Scheme 7. Preparation of tat-peptide labeled superparamagnetic iron oxide particles (In-DTPA-fluo-tat-S-S-CLIO) according to Weissleder et al. [96]...

Fig. 10. Structure of a tat-peptide labeled DOTA chelate [98] (M3+ = Dy3+, Gd3+ or inIn3+)...

Several BODIPY fluorophores with different excitation and emission maxima are available, and each can have alternative linkage chemistries. It is important to note that the linkage chemistry affects the fluorescence intensity. One cannot therefore quantitatively compare peptides labelled using different linkage chemistries. Once having selected a fluorophore/ linkage chemistry it is better to stick with it. 

Peptide labeled with fluorescein and coumarin is quenched until cleavage by protease, modification by phosphorylation, or dephosphorylation by kinase or phosphatase produces resistance to proteolytic cleavage... 

Limitations of the TR-FRET approach in terms of peptide sequence flexibility must be considered as well. The prerequisites and limitations for successful peptide labeling, for example, for the LANCE Ultra approach include (1) need for a cysteine in position zero, i.e., the peptide s N terminus, (2) no other free cysteine allowed in the peptide sequence, (3) overall charge different from zero, (4) no disulfide bonds in the peptide, (5) no tryptophan or histidine at the C terminal position, and (6) maximum length of 30 amino acids (personal communication). 

Various alternatives to the ICAT and ICPL technologies have been reported but these methods are consistently based on chemical tagging at the peptide level. In this chapter, only the most common and promising peptide labeling approaches will be discussed (bill, 2003 Moritz and Meyer, 2003). 

Proteolytic peptide labeling with isotopes was first reported by Schnolzer et... 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

In order to confirm the structure for the LO cofactor, model compounds mimicking the proposed enzymic structure were synthesized (13, 24). The UV-Vis abso tion spectrum of the model compound, with a A-max of 504 nm which is red-shifted compared to other TPQ-containing amine oxidases, corresponded almost exactly to that of native LO (13). Under the same conditions, the resonance Raman spectrum of the phenylhydrazine derivative of the model compound was found to be superimposable with that of the isolated LO peptide labeled with phenylhydrazine, yet very different from the phenylhydrazine derivative of a TPQ model compound (13). 

The Huisgen [3+2] cycloaddition between azides and alkynes is another bioorthognal ligation reaction for incorporation of probes into protein and peptide scaffolds. Two variants of this reaction have been developed using either copper(I) [218] or strained cyclooctyne molecules [219] to promote the reaction. As with the Staudinger ligation this method has found extensive use in protein and peptide labeling studies. 

D.F. Mason, D.C. Liebler, Quantitative analysis of modified proteins by CE-MS-MS of peptides labelled with phenyl isocyanate, J. Proteome Res., 2 (2003) 265. 

Thtee C57BL/6 mice pet gtoup wete injected inttadermally with 50 pg of liposomal plasmid DNA (A, C) ot 50 pg free plasmid DNA (B, D) followed by a second treatment after 48 h. Three control mice received 50 pg of an antiviral peptide antigen (gp33) in incomplete Freund s adjuvant (IFA) inttadermally. Nine days after the first immunization, spleen cells were re-stimulated for 5 days and analyzed in a Cr-release assay on peptide labeled and unlabeled EL-4 cells. The spontaneous Cr-release was <14% (22). 

The technique of isoelectric focusing has proven to be a useful tool in protein chemistry, and this too has been adapted to the microchip. Using 7-cm-long channels in glass microchips (200 [Xm wide and 10p,m deep) mixtures of Cy5-labeled peptides can be focused in less than 30 seconds. This same procedure has also been applied to plastic microchips made from EMMA by laser ablation and shown to focus mixtures of peptides labeled with rhodaraine green. Results for this type of microchannel isoelectric focusing are available in less than 5 minutes compared with traditional techniques that take over 1 hour. 

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