Synthesis, Screening, and Sequencing of Cysteine-Rich One-Bead One-Compound Peptide Libraries

Abstract

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.

Determination of Hemin‐Binding Characteristics of Proteins by a Combinatorial Peptide Library Approach

“…In the past five years, in fact, only a few combinatorial library approaches in which cysteines have been included at partially randomized positions in peptide sequences have been described 79. 80, 84 Both sequencing methods gave the expected results, although the costs for automated Edman degradation hugely exceeded those for PED‐MS analyses, although the latter technique failed more often in complete identification of sequences, due to fragmentation or other effects during MS analysis.…”

Fine-tuning Nanocarriers Specifically toward Cargo: A Competitive Study on Solubilizing Related Photosensitizers for Photodynamic Therapy

“…The partitioning of the drugs in the library and the enrichment of the sensitizers at certain beads, which present suitable peptides, was followed by fluorescence microscopy at excitation of 668 and 674 nm for Ce6 and Pb a , respectively (SI, Figure S1). For both sensitizers, 40 positive beads were selected, the peptides were isolated after cleavage from the supports by cyanogen bromide, , and MALDI-ToF-MS/MS revealed amino acid sequences suitable for drug binding. A clear enrichment of aromatic Phe, hydrophobic Leu, and polar Gln residues is evident (SI Tables S1 and S2).…”

Combinatorial/Library Peptide Synthesis

“…The hydrogen-deuterium exchange can simplify interpretation of spectra obtained from electrospray MS [304]. Hits from cysteine-rich libraries were alkylated to transform cysteine residues into acetamidomethyl derivatives, cleaved from the beads, and the structure determined by an MS/MS experiment [305]. Another example of the direct application of MS/MS for structure determination of peptides from noncoded library of peptides was shown by Brown et al [306].…”