Several MS-based methods of A domain substrate activation directly detect the PCP-bound thioester moiety. The first of these is the Ppant-loading assay, which is a bottom-up approach using either FTMS or matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry, measuring the mass shift observed when a Ppant-activated PCP is incubated with a pool of available substrates and then digested for analysis. This allows for an unbiased screening of A domain activity, but the composition of the substrate pool must be known in order to determine the identity of the activated monomer by mass shift alone. A top-down iteration of this method gave rise to the Ppant-ejection assay using either tandem MS capabilities (MS 2 or MS ) or collision induced dissociation (CID) to fragment an intact substrate-tethered PCP. Either FTMS or ion trap MS can be used in Ppant-ejection assays, and the detected mass corresponds to the substrate covalently bound to either the intact Ppant arm or a shortened, rearranged pantetheine prosthetic group. The move to a top-down workflow circumvents the need to proteolyze samples before analysis, and the validation of the technique on lower resolution instruments such as ion trap MS improves accessibility.