Identifying Ca2+-Binding sites in proteins by liquid chromatography-mass spectrometry using Ca2+-Directed dissociations

Here we describe a new method to identify calcium-binding sites in proteins using high-resolution liquid chromatography- mass spectrometry in concert with calcium-directed collision-induced dissociations. Our method does not require any modifications to the liquid chromatography- mass spectrometry apparatus, uses standard digestion protocols, and can be applied to existing high-resolution MS data files. In contrast to NMR, our method is applicable to very small amounts of complex protein mixtures (femtomole level). Calcium-bound peptides can be identified using three criteria: (1) the calculated exact mass of the calcium containing peptide; (2) specific dissociations of the calcium-containing peptide from threonine and serine residues; and (3) the very similar retention times of the calcium-containing peptide and the free peptide.

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