International Journal of Mass Spectrometry xxx (2004) xxx–xxx An affinity selection–mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries Discovery of a novel antagonist of E. coli dihydrofolate reductase D. Allen Annis a,∗ , John Athanasopoulos a , Patrick J. Curran a , Jason S. Felsch a , Krishna Kalghatgi a , William H. Lee a , Huw M. Nash a , Jean-Paul A. Orminati a , Kristin E. Rosner a , Gerald W. Shipps Jr. a , G.R.A. Thaddupathy b , Andrew N. Tyler a,1 , Lev Vilenchik a , Carston R. Wagner b , Edward A. Wintner a a NeoGenesis Pharmaceuticals, 840 Memorial Drive, Cambridge, MA 02139, USA b Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA Received 1 October 2003; accepted 22 November 2003 Abstract The NeoGenesis Automated Ligand Identification System (ALIS), an affinity selection–mass spectrometry (AS–MS) process consisting of a rapid size-exclusion chromatography stage integrated with reverse-phase chromatography, electrospray mass spectrometry, and novel data searching algorithms, was used to screen mass-encoded, 2500-member combinatorial libraries, leading to the discovery of a novel, bioactive ligand for the anti-infective target Escherichia coli dihydrofolate reductase (DHFR). Synthesis of the mass-encoded, ligand-containing library, discussion of the deconvolution process for verifying the structure of the ligand through independent synthesis and screening in a small mixture (sub-library) format, and ALIS–MS/MS techniques to assign its regioisomeric connectivity are presented. ALIS-based competition experiments between the newly discovered ligand and other, known DHFR ligands, and biological activity assessments with stereo- and regioisomers of the hit compound confirm its DHFR-specific biological activity. The method described requires no foreknowledge of the structure or biochemistry of the protein target, consumes less than 1 g protein to screen >2500 compounds in a single experiment, and enables screening of >250,000 compounds per system per day. These advantages highlight the potential of the ALIS method for drug discovery against genomic targets with unknown biological function, as well as validated targets for which traditional discovery efforts have failed. © 2004 Elsevier B.V. All rights reserved. Keywords: Affinity selection–mass spectrometry; Combinatorial chemistry; Multidimensional chromatography 1. Introduction The number of human and bacterial proteins identified as possible targets for small molecule therapy of human disease is increasing profoundly, partly as a result of whole genome sequencing efforts and advances in proteome analysis. At the ∗ Corresponding author. Tel.: +1 617 588 5143. E-mail address: aannis@neogenesis.com (D.A. Annis). 1 Present Address: ArQule Inc., 19 Presidential Way, Woburn, MA 01801, USA. same time, new paradigms in chemical synthesis are inspir- ing unparalleled creativity and technical sophistication in the construction of libraries of candidate therapeutic compounds [1]. As the number of targets implicated in disease processes grows and the ingenuity of combinatorial chemists evolves, the need for generic and efficient techniques to identify bio- logically active lead compounds from large chemical libraries is becoming urgent [2]. Affinity selection–mass spectrometry (AS–MS) has emerged as an attractive technique for studying protein– ligand interactions and screening biomolecular receptors 1387-3806/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ijms.2003.11.022