Mixed isotope photoaffinity

Fig. 10 Mixed isotope photoaffinity labeling strategy for determination of the labeling site. After binding and photocrosslinking the target enzyme with a 1 1 mixture of the light (Do) and heavy (D7) isotopically labeled A/BPs, the construct is purified and tryptically digested. LC-MS/MS analysis of the peptide pool allows discrimination between the labeled and unlabeled peptides. The modified fragment can easily be retrieved and identified by searching for the isotope signature...

Mixed isotope photoaffinity reagents have been developed to facilitate target identification by mass spectrometry. To demonstrate the approach, cyclosporine was both biotinylated and functionalised with a mixed isotope pho-toactivatable benzophenone probe (Figure 1.8). Irradiation of the probe in the presence of a mixture of proteins led to selective cross-linking to cyclophilin A. The biotin tag enabled easy separation of the cross-linked protein from other proteins in the mixture. Subsequently, after tryptic digestion, the mixed isotope tag facilitated the identification, by mass spectrometry, of the peptides that were cross-linked to the functionalised probe. 

Figure 1.8 A mixed isotope photoaffinity reagent in which cyclosporine has been functionalised with both biotin and a photoactivatable benzophenone probe.

The mixed isotope PAL strategy is still in development, but is believed to become a powerful tool in photoaffinity-based protein profiling strategies in the near future. With its aid, the photocrosslinking site(s) of an A/BP can more easily be identified, which will lead to a better understanding of protein structure and function in general. 

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