Due to the spectroscopically-silent nature of calcium and its physiological abundance, determination of the calcium binding capability of proteins is challenging. First, most experimental methods such as dialysis are only sensitive to the total calcium content. In addition, overcoming the persistent background contamination of calcium during the preparation of calcium-free sample for proteins with strong calcium binding affinities is a non-trivial task. Further, since most calcium binding proteins contain multiple calcium binding sites that cooperatively bind calcium resulting in induced conformational change (e.g., CaM) ( Figure 2 ), obtaining site-specific calcium binding affinity is limited by complication from contributions from cooperativity and conformational entropy [23] . Hence, understanding the molecular mechanism of biological function related to calcium is largely hampered by the lack of site specific information about the calcium-binding properties, especially for the ubiquitous EF-hand calcium-binding motif. Progress in understanding the molecular mechanism of calcium modulated biological process requires us to answer several important questions. First, what are the site-specific calcium binding affinities of calcium binding proteins, particularly those that utilize multiple coupled calcium binding sites to respond to sharp changes in cellular calcium concentration? Next, how can we predict or identify calcium binding sites in proteins using genomic and structural information? Finally, how can we verify calcium binding capabilities in the bacteria and virus genomes?