Affinity Selection from Peptide Libraries to Determine Substrate Specificity of Protein Tyrosine Phosphatases Gregory Huyer,* , † ,1 John Kelly,‡ Jason Moffat,* Robert Zamboni,‡ Zongchao Jia,§ Michael J. Gresser,* , † and Chidambaram Ramachandran* ,2 *Department of Biochemistry and Molecular Biology; ‡Department of Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire–Dorval, Que ´bec H9R 4P8, Canada; †Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montre ´al, Que ´bec H3G 1M8, Canada; and §Department of Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada Received September 15, 1997 Affinity selection from peptide libraries is a pow- erful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phos- phatases using peptide libraries containing a non- hydrolyzable phosphotyrosine analog, difluorophos- phonomethylphenylalanine. A size-exclusion method is used to separate enzyme–peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column ap- proach. In addition, the feasibility of using mass spec- trometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individ- ual peptides and comparing their affinity for enzyme with the predictions from the affinity selection pro- cess. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional in- sights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY  1 position (immediately N-terminal to the phos- photyrosine), as well as a broad pY  1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries. © 1998 Academic Press The reversible phosphorylation of proteins on tyrosine residues regulates many cellular processes (1– 4). The tyrosyl phosphorylation status of a protein is controlled by the competing activities of the protein tyrosine kinases (PTKs) 3 and the protein tyrosine phosphatases (PTPs). Over 100 PTKs and 50 PTPs have been identified, and it is predicted that the human genome contains up to 500 of each (3, 4). Clearly one of the major challenges in the study of these enzymes is to determine the signaling pathway(s) in which they participate, i.e., to identify their in vivo targets and substrates. One approach to determining potential substrates of PTKs and PTPs has been to study their in vitro catalytic efficiency toward purified proteins or synthetic peptides. For PTPs, the use of synthetic pY-containing peptide sub- strates in particular has been informative, and a number of groups have characterized various PTPs in this way in an attempt to define recognition elements (5–12). However, it is very difficult to carry out anything more than alimited study with individual peptides because the process is costly, time consuming, and laborious. The use of peptide libraries provides a means to overcome some of these drawbacks. Songyang et al. 1 Present address: T Cell Laboratory, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. 2 To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Ther- apeutic Research, P.O. Box 1005, Pointe-Claire–Dorval, QC, Canada H9R 4P8. Fax: (514) 695-0693. 3 Abbreviations used: PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; EGF, epidermal growth factor; pY, phospho- tyrosine; SH2, src homology 2; F 2 Pmp, difluorophosphonomethyl- phenylalanine; FDP, fluorescein diphosphate; FMP, fluorescein monophosphate; DTT, dithiothreitol; BSA, bovine serum albumin; GST, glutathione S-transferase; CZE, capillary-zone electrophoresis; IPTG, isopropyl--D-thiogalactopyranoside; TFA, trifluoroacetic acid; MAP, mitogen-activated protein. 0003-2697/98 $25.00 19 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ANALYTICAL BIOCHEMISTRY 258, 19 –30 (1998) ARTICLE NO. AB972541