Accurate MALDI-TOF/TOF Sequencing of One-Bead-One-Compound Peptide Libraries with Application to the Identification of Multi-ligand Protein Affinity Agents Using In Situ Click Chemistry Screening

摘要

Combinatorial one-bead-one-compound (OBOC) peptide libraries are widely used for affinity screening, and the sequencing of peptides from hit beads is a key step in the process. For rapid sequencing, CNBr cleavage of the peptides from the beads, followed by de novo sequencing by MALDI-TOF/TOF is explored. We report on a semi-automated sequencing algorithm, and validate it through comparison against Edman degradation sequencing. The initial 44% sequencing success rate of the standard de novo sequencing software was improved to nearly 100%. The sequencing algorithm incorporates existing knowledge of amino acid chemistry, and a new strategy for differentiating isobaric amino acids. We tested the algorithm by using MALDI-TOF/TOF to identify a peptide biligand affinity agent against the protein bovine carbonic anhydrase II, starting from comprehensive one-bead-one-compound peptide libraries comprised of non-natural and artificial amino acid components, and using the strategy of in situ click/OBOC library screening.