describe the indirect measurement of PPi exchange by initiating A domain assays with γ-18 O4-ATP, which will release its labeled γ-phosphate as PPi in the forward adenylation reaction. As with the radiolabeled 32 P[PPi] assay, this technique relies on the reverse reaction, incorporating unlabeled PPi to form γ-16 O4-ATP. The rate of γ-16 O4-ATP formation and γ-18 O4-ATP consumption can therefore be observed by mass shift, and the integrated peak ratio of γ-16 O4-ATP to all ATP species present allows for quantification of enzyme activity. This assay can be performed on either a MALDI-ToF instrument or electrospray ionization (ESI) LC-MS instruments, but it is a discontinuous method to probe reactions.