Abstract
Screening of one-bead-one-compound (OBOC) libraries is a proven procedure for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The traditional peptide screening involves tedious steps such as affinity selection, bead picking, sequencing, and characterization. Herein, we present a high-throughput “all-on-one chip” system to avoid slow and technically complex bead picking steps. On a traditional glass slide provided with an electrically conductive tape, beads of a combinatorial peptide library are aligned and immobilized by application of a precision sieve. Subsequently, the chip is incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to nearly 100%. The new technique was validated by employing a FLAG-based model system, identifying new peptide binders for the monoclonal M2 anti-FLAG antibody, and was finally utilized to search for IgG-binding peptides. In the present format, more than 30,000 beads can be screened on one slide.
Highlights
Peptides represent a class of pharmaceutical compounds, molecularly poised between small molecules and proteins, yet biochemically and therapeutically distinct from both [1]
In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry, high-affinity binders are directly and unambiguously sequenced with high accuracy without picking of the positive beads
To circumvent the need for a fragmentation step by MALDI-TOF mass spectrometry (MS)/MS, which can result in incomplete sequence information, a simple encoding approach has been adapted for this purpose
Summary
Peptides represent a class of pharmaceutical compounds, molecularly poised between small molecules and proteins, yet biochemically and therapeutically distinct from both [1]. Among various chemical library formats, one-bead-one-compound (OBOC) libraries are a proven approach for the identification of protein-binding ligands [2]. By applying the split-and-mix method, introduced by Lam et al, millions of compounds can be readily synthesized in parallel [3]. When these libraries are created on resins that have a hydrophilic surface to suppress nonspecific reagent binding, they can be used directly in binding screenings for the identification of ligands for proteins or other targets [4]. The peptide will be released from the bead and, subsequently, its sequence is determined, either directly by mass spectroscopic or indirectly by encoded beads
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
