Several sophisticated methods have been invented in the past to enrich or globally monitor protein families as well as changes in protein expression level under different physiological conditions. Activity-based chemical proteomics provides tailor-made molecular tools and, hence, is a powerful method to enrich, tag, or characterize protein families, especially those of low abundance and with distinct functions in different cellular compartments. ABPs comprising irreversible inhibitors have been proven to be suitable molecular tools for the tagging of enzyme targets but also for profiling of enzyme activity. Probes relying on reversibly binding ligands may be employed either for inhibitor affinity purification or—in combination with an additional reactive site (e.g., a photoreactive group)—for tagging the target proteins to suitable reporter groups. Proteins for which irreversible inhibitors are unknown can be addressed with this latest generation of ABPs. Kinases are a typical example for such large groups of protein families with functions in signaling and sensoring of physiological processes. In plant proteomics, it is of particular interest to identify subfamilies of kinases in chloroplasts, mitochondria, and the cytosol as a first step to elucidate their role in environmental adaptation processes and in inter-organelle communication. The first successful steps toward the semiquantitative tagging and identification of Ser/Thr
FIGURE 4.6. Retrieval of spiked creatine kinase from thylakoid membrane preparations after photoaffinity labeling. Lane 1: Silver stain. Lane 2: Silver stain, with additional 150 ng creatine kinase. Lane 3: Fluorescence image. Lane 4: Fluorescence image, with additional 150ng creatine kinase (see arrow). (Figure reproduced from reference 29.)
protein kinases within chloroplasts of Chlamydomonas underscore the impact of this method for elucidating their cellular function.
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