We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5'-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated L-Phe-AMS (L-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates L-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes. (C) 2014 Elsevier Ltd. All rights reserved.