title: Process automation toward ultra-high-throughput screening of combinatorial one-bead-one-compound (OBOC) peptide libraries.
abstract: With an aim to develop peptide-based protein capture agents that can replace antibodies for in vitro diagnosis, an ultra-high-throughput screening strategy has been investigated by automating labor-intensive, time-consuming processes that are the construction of peptide libraries, sorting of positive beads, and peptide sequencing through analysis of tandem mass spectrometry data. Although instruments for automation, such as peptide synthesizers and automatic bead sorters, have been used in some groups, the overall process has not been well optimized to minimize time, cost, and efforts, as well as to maximize product quality and performance. Herein we suggest and explore several solutions to the existing problems with the automation of the key processes. The overall process optimization has been done successfully in orchestration with the technologies such as rapid cleavage of peptides from beads and semiautomatic peptide sequencing that we have developed previously. This optimization allowed one-round screening, from peptide library construction to peptide sequencing, to be completed within 4 to 5 days. We also successfully identified a 6-mer ligand for carcinoembryonic antigen-cell adhesion molecule 5 (CEACAM 5) through three-round screenings, including one-round screening of a focused library.
authors.affiliation: Institute of Bioengineering and Nanotechnology, Singapore.
authors.forename: Junhoe
authors.initials: J
authors.lastname: Cha
authors.forename: Jaehong
authors.initials: J
authors.lastname: Lim
authors.forename: Yiran
authors.initials: Y
authors.lastname: Zheng
authors.forename: Sylvia
authors.initials: S
authors.lastname: Tan
authors.forename: Yi Li
authors.initials: YL
authors.lastname: Ang
authors.forename: Jessica
authors.initials: J
authors.lastname: Oon
authors.forename: Mei Wei
authors.initials: MW
authors.lastname: Ang
authors.forename: Jingjing
authors.initials: J
authors.lastname: Ling
authors.forename: Marcus
authors.initials: M
authors.lastname: Bode
authors.forename: Su Seong
authors.initials: SS
authors.lastname: Lee
mesh_terms: D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000819:agonists / Q000737:chemistry; D002272:Carcinoembryonic Antigen / Q000737:chemistry; D020650:Combinatorial Chemistry Techniques / Q000295:instrumentation / Q000379:methods; D003933:Diagnosis; D058851:GPI-Linked Proteins / Q000819:agonists / Q000737:chemistry; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D008863:Microspheres; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D019151:Peptide Library; D020539:Sequence Analysis, Protein / Q000379:methods; D053719:Tandem Mass Spectrometry
chemical_list: D014408:Biomarkers, Tumor; C527875:CEACAM5 protein, human; D002272:Carcinoembryonic Antigen; D058851:GPI-Linked Proteins; D008024:Ligands; D010446:Peptide Fragments; D019151:Peptide Library