title: SORTCERY-A High-Throughput Method to Affinity Rank Peptide Ligands.
abstract: Uncovering the relationships between peptide and protein sequences and binding properties is critical for successfully predicting, re-designing and inhibiting protein-protein interactions. Systematically collected data that link protein sequence to binding are valuable for elucidating determinants of protein interaction but are rare in the literature because such data are experimentally difficult to generate. Here we describe SORTCERY, a high-throughput method that we have used to rank hundreds of yeast-displayed peptides according to their affinities for a target interaction partner. The procedure involves fluorescence-activated cell sorting of a library, deep sequencing of sorted pools and downstream computational analysis. We have developed theoretical models and statistical tools that assist in planning these stages. We demonstrate SORTCERY's utility by ranking 1026 BH3 (Bcl-2 homology 3) peptides with respect to their affinities for the anti-apoptotic protein Bcl-xL. Our results are in striking agreement with measured affinities for 19 individual peptides with dissociation constants ranging from 0.1 to 60nM. High-resolution ranking can be used to improve our understanding of sequence-function relationships and to support the development of computational models for predicting and designing novel interactions.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address: kuranes@mit.edu.
authors.forename: Lothar Luther
authors.initials: LL
authors.lastname: Reich
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address: sdutta@mit.edu.
authors.forename: Sanjib
authors.initials: S
authors.lastname: Dutta
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address: keating@mit.edu.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Keating
mesh_terms: D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D000072224:Bcl-2-Like Protein 11; D003627:Data Interpretation, Statistical; D005434:Flow Cytometry; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020539:Sequence Analysis, Protein / Q000379:methods*; D015003:Yeasts; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D051017:Apoptosis Regulatory Proteins; C474007:Bax protein (53-86); D000072224:Bcl-2-Like Protein 11; D008024:Ligands; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D010455:Peptides; D011518:Proto-Oncogene Proteins; D051020:bcl-X Protein
keywords: Bcl-2 family; deep sequencing; high-throughput assay; protein interaction; yeast display