Adenine nucleotides are small, abundant molecules that bind numerous proteins involved in pivotal cellular processes. These nucleotides are co-factors or substrates for enzymes, regulators of protein function, or structural binding motifs. The identification of nucleotide-binding sites on a proteome-wide scale is tempting in view of the high number of nucleotide-binding proteins, their large in vivo concentration differences, and the various functions they exert. Here, we report on a functional, chemical, gel-free proteomics technology that allows the identification of protein adenine nucleotide-binding site(s) in cell lysates. Our technology uses a synthetic ATP analogue, 5'-rho-fluorosulfonylbenzoylad-enosine (FSBA), as an affinity/activity-based probe for nucleotide-binding sites. When applied on a cellular level, 185 different FSBA-labeled sites in a human Jurkat cell lysate were identified. Functional and structural aspects of the use of FSBA on a proteome-wide scale are discussed.