Signaling proteins exemplified by calmodulin usually bind cooperatively to multiple ligands. Intermediate states and allosteric behavior are difficult to characterize. Here we extend a recently reported mass spectrometry (MS)-based method named LITPOMS (ligand titration, fast photochemical oxidation of proteins and mass spectrometry) that characterizes complex binding systems typically found as signaling proteins. As reported previously, calmodulin's response to binding four Ca2+ can be determined by LITPOMS to reveal binding sites, binding order, and most importantly composite binding behavior. Modeling this behavior provides site-specific binding affinities. In this article, we dissect the composite, peptide-level conformational changes at several regions either by digestion with a different protease or by tandem MS of LITPOMS behavior at the amino-acid residue level. Such dissection greatly elevates spatial resolution and increases the confidence of binding-order assignment. These complementary views of complex protein conformational change recapitulate the cumulative understanding via a single approach, providing new insights on poorly understood yet important allostery and underpin an approach applicable for exploring other signaling systems.