Affinity-based probe

Fig. 3 Basic principle of photoaffinity labeling (PAL), which requires an affinity-based probe (A/BP) and UV irradiation...

Scheme 3 Affinity-based probes targeting MMPs, from studies by Dive et al. [54]. (a) Photoreactive A/BP containing a tritium label. ZBG zinc binding group, (b) Possible constructs formed between hMMP-12 and 21 after photolysis proposed by Dive et al. [55]. (c) Structures of A/BPs with or without a photophore for pull-down of active MMPs...

Another approach to study kinase activity was introduced by Hagenstein et at 2 who designed an affinity-based probe equipped with a benzophenone photocross-linker for covalent modification of plant kinases. 

Figure 16.9 Structures of chemical components of activity/affinity probes The activity/affinity-based probe consists of reactive group and tag connected by a linker (L indicates the points of connection to the linker). Specific examples of each of the chemical components are shown. Two types of tags are commonly employed. The affinity tag for isolation/purification and the flnorescent or radiolabel tags for identification. Taken from Jeffery and Bogyo (2003)...

Ofir and colleagues developed a microarray system using an affinity-based probe immobilization strategy. They fused the exceptionally stable family-3a CBM, from the ceUulosome of C. thermocellum, with antibodies or peptides. The recombinant proteins were immobilized on cellulose surfaces by specific adsorption and used for serodiagnosis of human immunodeficiency virus patients [168,170]. 

Hybridization can also be performed in solution phase. Since the capture probe is in solution, the kinetics of hybridization is faster than when the capture probe is immobilized. In the solution phase hybridization format, the capture probe is labeled with an affinity label such as biotin that captures the sample target sequence. The labeled probe then binds to the sample target sequence to form the sandwich. After the hybridization is complete, the sandwich is transferred to an affinity support such as avidin or streptavidin, which will capture the sandwich through the biotin-labeled capture probe. Sandwich hybridization performed in solution followed by capture on an affinity support has been referred to as affinity-based hybrid collection (Kl). 

Chan EW, Chattopadhaya S, Panicker RC et al (2004) Developing photoactive affinity probes for proteomic profiling hydroxamate-based probes for metalloproteases. J Am Chem Soc 126 14435-14446... 

The term activity-based protein profiling implies mechanism-based probe/ target reactivity. Photoaffinity labelling approaches represent a complementary technique to mechanism based APBB probes. The use of these photoreactive affinity-based protein profiling probes in proteomic studies are reviewed by Overkleeft et al. 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

We have developed an analogous, but more robust system which is not necessarily constraint by the aforementioned limitations. The obvious extension has been to couple an affinity-based separation with mass spectrometry. Hutchens et al. have shown that affinity probe surfaces can be ust to capture specific protein ligands allowing detection by laser desorption mass spectrometry (. The limitations to their technique have been that the surface area for ligand capture is quite small and salt (or detergent) contaminants are still problematic. Perfusive affinity resins, on the other hand, provide a tremendous surface area for binding. The nature and composition of the solvents required for affinity chromatography, however, are not directly compatible with mass spectrometric analysis. 

Affinity Enrichment ChIP-based methods have been mostly combined with microarrays. These affinity-based techniques are now rapidly shifting to analysis by the next-generation techniques (56,66). As it has been described earlier, DNA immunoprecipitated with the specific methylcytosine antibody is de-cross-linked and defragmented. After ligation with specific probes, the sample is applied for on-site deep sequencing. 

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

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