Aldehyde-reactive probe

Analysis of M,G-dR in DNA by aldehyde reactive probe labeling and liquid chromatography tandem mass spectrometry. Chem. Res. Toxicol, 18, 51-60. 

Biotinylated aldehyde reactive probe (ARP) (Oxford Biomedical Research, Oxford, MI). 

Fig. 2. Quantitation of DNA damage. Borrelia burgdorferi B31A cells were grown in BSK-II under anaerobic conditions to a cell density of 5 x 10 cells/mL, treated with 0, 10, or 50 mM f-butyl peroxide (C, D, and E, respectively) for 4 h and DNA isolated. The DNA was then mixed with an aldehyde-reactive probe (Oxford Biomedical Research, Inc) labeled with biotin and detected with an HRP-streptavidin conjugate. The color development was monitored at 450 nm. The number of aldehyde-reactive probe (DNA base lesions)/10 bp DNA was determined using a standard curve. Escherichia coli TA4315 cells were grown in minimal media to ODgoo of 0.4, treated with 0 or 100 mM H2O2 for 30 min (A and B, respectively) and DNA isolated. The number of base lesions was determined as described above.

The ethylene diamine-dextran derivative may be used for the coupling of carboxylate-contain-ing molecules by the carbodiimide reaction, for the coupling of amine-reactive probes, or to modify further using heterobifunctional crosslinkers. The hydrazide-dextran derivative may be used to crosslink aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins. 

Purify the hydrazide-labeled oligo by gel filtration on Sephadex G-25 using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The hydrazide-containing probe now may be used to conjugate with a molecule containing an aldehyde reactive group. 

Succinimidyl esters are an excellent first choice to activate amine-reactive probes, but their low solubility has led to the alternative use of sulphonyl chlorides (Figure 4.17). The resultant sulphonamide link is extremely stable, even more stable than an amide link, and will survive even complete protein hydrolysis - a property that can be exploited in protein analysis. The disadvantage of sulphonyl chlorides is that they are unstable in aqueous buffers under mildly alkaline conditions (typically the pH required for the reaction with aliphatic amine ). Hence extreme care must be taken to perform bioconjugations with sulphonyl chlorides at low temperatures (approx 4 °C). Alternatively, amine-reactive probes may be equipped with isothiocyanate traps , from which thiourea links are formed post-reaction with amine functional groups, or with aldehydes, from which Schiff sbase links can be formed with amine functional groups (Figure 4.17). 

Finally, another group of derivatives are the aldehyde-/ketone-reactive probes. This group is based on the activation of a sulfonyl hydrazine group of carbon number 5. They are based on Lissamine and Texas Red structures and used to label aldehyde-/ketone-containing molecules (with sugars). The most common aldehyde-/ ketone-reactive probes are Lissamine rhodamine B hydrazine and Texas Red hydrazine. 

Generalized protocols for the use of hydrazine probes reactive toward aldehyde residues can be found in Section 1, this chapter. These procedures are directed at the labeling of cell-surface... 

Most immobilizahon chemistries for microarrays currently rely upon derivatization of the substrate with amine-reactive functional groups such as aldehydes, epoxides, or NHS esters. While we can choose from many available surface-reactive chemistries, it is important to keep in mind that they must be compatible with a printing process. Ideally, the biomolecule should react completely and rapidly with the substrate in order to achieve good spot formation. It is also critical that the probe remain or be recoverable in its active state following printing. If too reactive a chemistry is employed there is the possibility for excessive crosslinking that can hinder performance by reducing the number of rotatable bonds in the probe. 

---