Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries

Abstract

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Evolving a Peptide: Library Platforms and Diversification Strategies

“…Positional sub-libraries are assayed in parallel to gather information on the optimal residue for each diversity position, consequently identifying the fittest member [47]. Other hit identification techniques include Edman degradation [48] and mass spectrometry (MS)-based strategies [49,50]. Mass spectrometry is highly sensitive and very fast, and is the method of choice when it comes to generating sequence data in the femtomolar range.…”

Site‐Selective Cysteine–Cyclooctyne Conjugation

“…Each bead in the library has a single peptide species, which enables each peptide to react with a probe without interference from other library members. The GLLKG sequence was used to promote retention of library members on the column for de novo sequencing of hits using liquid chromatography tandem mass spectrometry (LC–MS/MS) . The library was reacted with DBCO‐(PEG) 4 ‐biotin ( 1 ), and the beads containing reacted species were isolated by fluorescence‐activated bead sorting (FABS) after staining with a streptavidin‐conjugated fluorophore (Figure S2).…”

Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting

“…In a search for new xenoprotein therapeutics, Bradley Pentelute (MIT, USA) described a high‐throughput approach for the synthesis of xenoprotein/peptide libraries (i.e., proteins and peptides that contain artificial amino acids) by fully automated flow‐based peptide synthesis . Starting from a small cysteine‐knot protein (knottin) with a rigid molecular scaffold, peptide libraries with diversified knottin loops containing unnatural building blocks were synthesized, tested for their binding to Ebola glycoproteins and identified through high‐throughput de novo sequencing by tandem mass spectrometry …”

“…[40] Starting from as mall cysteine-knot protein (knottin) with ar igid molecular scaffold, peptidel ibraries with diversified knottin loops containing unnatural buildingb locks were synthesized, tested for their binding to Ebola glycoproteins and identified through high- ChemBioChem 2018, 19,1 15 -120 www.chembiochem.org throughput de novo sequencing by tandem mass spectrometry. [41] Peptide/ProteinLabeling and Bioorthogonal Chemistries to Manipulate ProteinsinV itro and in Vivo Methods for chemoselectively labeling biomolecules have become indispensable tools in modern biology,a nd recent years in particularh ave seen ab oost in the development and design of new approaches that exploit either uniquee nzymatic reactions or bioorthogonal chemistries. [42] Christian Hackenberger (Leibniz FMP and Humboldt University Berlin, Germany) described his lab's recent progress in developing new chemoselectiver eactions for labeling proteins anda ntibodies.…”