Mass Spectrometric Techniques for Label-free High-Throughput Screening in Drug Discovery Thomas P. Roddy,* ,† Christopher R. Horvath, † Steven J. Stout, † Kristin L. Kenney, † Pei-I Ho, † Ji-Hu Zhang, † Chad Vickers, ‡ Virendar Kaushik, ‡ Brian Hubbard, ‡ and Y. Karen Wang † Discovery Technologies, and Diabetes and Metabolism, Novartis Institute for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139 High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Tradi- tional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spec- trometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method develop- ment times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structur- ally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (∼175 000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spec- trometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrom- etry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384- well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demon- strated herein with screening data from two large-scale screens. High-throughput screening (HTS) is an important approach for finding active compounds, or “hits”, against a therapeutic target of interest. Functional activity-based HTS discovers hits by detecting a change in the activity of a target enzyme in the presence of compounds. Samples containing active compounds contain a relatively high or low concentration of product depending on whether an activator or an inhibitor is present. In either case, large numbers (from 50 000 to over 1 million) of compounds with diverse chemical characteristics are helpful in finding a variety of hits for the following lead compound selection process. As a result, fast analysis times and sample throughput are key to the success of HTS campaigns. Enzymatic functional assays typically use radioisotope or fluorescent labels, allowing rapid measurement of enzymatic reaction products in the sample wells of a high- density microtiter plate. However, labeled substrates may not function exactly the same as native substrates, and coupling reactions used to generate signal can also be affected by compounds. These added interactions can lead to false positives (compounds with active readout but no real activity) and false negatives (active compounds that are undetected). In addition, the synthesis of labeled substrates and the development of coupling reactions may significantly add to method development time and effort. Mass spectrometry (MS) detects enzymatic products without labeling or coupling reagents, potentially improving data quality and reducing method development efforts. Two published studies on acetylcholinesterase 1 and anthrax lethal factor 2 enzymatic targets have demonstrated the potential of this technique on a smaller scale, and O ¨ zbal, et. al from the acetylcholinesterase study have developed an MS-HTS service based on the concept (Biotrove, Woburn, MA). The acetylcholinesterase MS-based screen used a propriety microfluidic system which includes an online chromatographic step prior to introducing samples to an MS. In this study, 4608 compounds were screened in a total of 48 plates (96-well). The anthrax lethal factor study used a SAMDI (self-assembled monolayers for MALDI)-based assay to screen for inhibitors from a library of 10 000 compounds. There have been other label-free techniques which have shown promise for screening as well, including surface plasmon resonance (SPR), 3 desorption/ionization on silicon (DIOS), 4 frontal affinity chroma- * Author to whom correspondence should be addressed. E-mail: thomas.roddy@novartis.com. Phone: 617-871-3533. Fax: 617-871-4086. † Discovery Technologies. ‡ Diabetes and Metabolism. (1) O ¨ zbal, C. C.; LaMarr, W. A.; Linton, J. R.; Green, D. F.; Katz, A.; Morrison, T. B.; Brenan, J. H. Assay Drug Dev. Technol. 2004, 2, 373-381. (2) Min, D. H.; Tang, W. J.; Mrksich, M. Nat. Biotechnol. 2004, 6, 717-723. (3) Boozer, C.; Kim, G.; Cong, S.; Guan, H.; Londergan, T. Curr. Opin. Biotechnol. 2006, 17, 400-405. (4) Shen, Z.; Go, E. P.; Gamez, A.; Apn, J. V.; Fokin, V.; Greig, M.; Ventura, M.; Crowell, J. E.; Blixt, O.; Paulson, J. C.; Stevens, R. C.; Finn, M. G.; Siuzdak, G. ChemBioChem 2004, 5, 921-927. Anal. Chem. 2007, 79, 8207-8213 10.1021/ac062421q CCC: $37.00 © 2007 American Chemical Society Analytical Chemistry, Vol. 79, No. 21, November 1, 2007 8207 Published on Web 09/29/2007