Construction of a Protein-Detection System Using a Loop Peptide Library with a Fluorescence Label

Abstract

Construction of a novel protein-detection system was carried out using a designed peptide library with fluorescent labels based on loop structures. As a basic model study, detection of alpha-amylase using fluorescent-labeled peptides derived from an active loop of tendamistat was examined. The detection methods for proteins with immobilized peptides as well as peptides in solution have been successfully established. Based on these results, a loop peptide library that has various turn sequences grafted on a stable loop structure has been constructed. Various proteins with recognition patterns corresponding, for instance, to "protein fingerprints" could be detected using an immobilized peptide library. The present results suggest that the system can be applied to the development of a peptide microarray that behaves as a protein chip.

Protein “fingerprinting” in complex mixtures with peptoid microarrays

“…This finding is critical in further development of these arrays as bioanalytical tools, because selective chemical labeling of a protein of interest in a complex mixture can rarely be achieved. A small amount of analytical work has been done to distinguish purified proteins with simpler arrays in other configurations (14)(15)(16). However, this work provides a previously undescribed demonstration that a glass slide-based SMM can ''fingerprint'' a native protein in a complex mixture.…”

“…However, little work has focused on the development of SMMs as analytical tools for biological research (14)(15)(16), with the notable exception of peptide arrays as tools for determining the substrate preferences for proteases and other proteinmodifying enzymes (12,(17)(18)(19)(20)(21) and the binding preferences of antibodies (22)(23)(24). We hypothesized that hybridization of any particular protein to a SMM with thousands of features is likely to provide a unique pattern of binding to the array, allowing that factor to be identified by virtue of this ''molecular fingerprint.''…”

Cationic Antimicrobial Peptides

“…Thus, the activities of C-terminally immobilized peptides might result not only from the right sequences, but also from minor contributions of deficient sequences. N terminus and side chain immobilization of KLAL and MK5E on TentaGel S NH 2 using the thioalkylation [150] and oxime-forming ligation strategies [151] were performed with HPLC-purified sequences (Fig. 27).…”

Protein‐Detecting Microarrays: Current Accomplishments and Requirements

“…[51] Synthetic peptides containing a free cysteine amino acid residue were also immobilized covalently onto plastic surfaces modified with bromoacetyl groups through the layers of poly(l-lysine) and dextran coatings to detect protein-peptide interactions in an array format. [52,53] Chemical ligation of N-terminal cysteine-containing biomolecules to slides containing thioester moieties has also been successfully performed. [54] Although these are quite useful methods to attach synthetic peptides and sugars to the solid surfaces, there are still bottlenecks in immobilizing proteins, due to the characteristic localization of free cysteine residues in each protein.…”