Jaehi Kim

Automated Maskless Photolithography System for Peptide Microarray Synthesis on a Chip

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.

Facile method for preparation of silica coated monodisperse superparamagnetic microspheres

This paper presents a facile method for preparation of silica coated monodisperse superparamagnetic microsphere. Herein, monodisperse porous polystyrene-divinylbenzene microbeads were prepared by seeded emulsion polymerization and subsequently sulfonated with acetic acid/H2SO4. The as-prepared sulfonated macroporous beads were magnetized in presence of Fe2+/Fe3+ under alkaline condition and were subjected to silica coating by sol-gel process, providing water compatibility, easily modifiable surface form, and chemical stability. FE-SEM, TEM, FT-IR, and TGA were employed to characterize the silica coated monodisperse magnetic beads (∼7.5 µm). The proposed monodisperse magnetic beads can be used as mobile solid phase particles candidate for protein and DNA separation.

Substrate screening of protein kinases: Detection methods and combinatorial peptide libraries

The study of protein kinases has become a matter of great importance in the development of new drugs for the treatment of diseases, including cancer and inflammation. Substrate screening is the first step in the fundamental investigation of protein kinases and the development of inhibitors for use in drug discovery. Towards this goal, various studies have been reported regarding the development of phospho‐peptide detection methods and the screening of phosphorylated peptide sites by protein kinases. This review introduces the detection methods for phosphorylation events using the reagents with (γ32P)ATP, ligand‐linked ATP, phospho‐peptide‐specific antibodies and metal chelating compounds. Chemical modification methods using β‐elimination for the detection of phospho‐Ser/Thr peptides are introduced as well. In addition, the implementations of combinatorial peptide libraries for screening peptide substrates of protein kinases are discussed. The phage display approach has been suggested as an alternative method of using synthetic peptides for screening the substrate specificities of protein kinase. However, a solid phase assay using a peptide library‐bound polymer resin or a peptide‐arrayed glass chip is preferred for high throughput screening (HTS).

Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.

Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the ‘high‐throughput phosphorylation profiling (HTPP)’ method was developed on the basis of a fully randomized one‐bead one‐compound (OBOC) combinatorial ladder type peptide library and MALDI‐TOF MS. The OBOC ladder peptide library was constructed by the ‘split and pool’ method on a HiCore resin. The peptide library sequence was Ac‐Ala‐X‐X‐X‐Ser‐X‐X‐Ala‐BEBE‐PLL resin. The substrate specificity of murine PKA (cAMP‐dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co‐activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1‐dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay. Copyright

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on β-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.

Preparation of tri(ethylene glycol) grafted core-shell type polymer support for solid-phase peptide synthesis

A core‐shell type polymer support for solid‐phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on‐bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core‐shell type polymer supports (TEG SURE) for efficient solid‐phase peptide synthesis and on‐bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on‐bead bioassays.

Thin silica shell coated Ag assembled nanostructures for expanding generality of SERS analytes

Surface-enhanced Raman scattering (SERS) provides a unique non-destructive spectroscopic fingerprint for chemical detection. However, intrinsic differences in affinity of analyte molecules to metal surface hinder SERS as a universal quantitative detection tool for various analyte molecules simultaneously. This must be overcome while keeping close proximity of analyte molecules to the metal surface. Moreover, assembled metal nanoparticles (NPs) structures might be beneficial for sensitive and reliable detection of chemicals than single NP structures. For this purpose, here we introduce thin silica-coated and assembled Ag NPs (SiO2@Ag@SiO2 NPs) for simultaneous and quantitative detection of chemicals that have different intrinsic affinities to silver metal. These SiO2@Ag@SiO2 NPs could detect each SERS peak of aniline or 4-aminothiophenol (4-ATP) from the mixture with limits of detection (LOD) of 93 ppm and 54 ppb, respectively. E-field distribution based on interparticle distance was simulated using discrete dipole approximation (DDA) calculation to gain insight into enhanced scattering of these thin silica coated Ag NP assemblies. These NPs were successfully applied to detect aniline in river water and tap water. Results suggest that SiO2@Ag@SiO2 NP-based SERS detection systems can be used as a simple and universal detection tool for environment pollutants and food safety.

SERS-Based Flavonoid Detection Using Ethylenediamine-β-Cyclodextrin as a Capturing Ligand

Ethylenediamine-modified β-cyclodextrin (Et-β-CD) was immobilized on aggregated silver nanoparticle (NP)-embedded silica NPs (SiO2@Ag@Et-β-CD NPs) for the effective detection of flavonoids. Silica NPs were used as the template for embedding silver NPs to create hot spots and enhance surface-enhanced Raman scattering (SERS) signals. Et-β-CD was immobilized on Ag NPs to capture flavonoids via host-guest inclusion complex formation, as indicated by enhanced ultraviolet absorption spectra. The resulting SiO2@Ag@Et-β-CD NPs were used as the SERS substrate for detecting flavonoids, such as hesperetin, naringenin, quercetin, and luteolin. In particular, luteolin was detected more strongly in the linear range 10−7 to 10−3 M than various organic molecules, namely ethylene glycol, β-estradiol, isopropyl alcohol, naphthalene, and toluene. In addition, the SERS signal for luteolin captured by the SiO2@Ag@Et-β-CD NPs remained even after repeated washing. These results indicated that the SiO2@Ag@Et-β-CD NPs can be used as a rapid, sensitive, and selective sensor for flavonoids.

Corrigendum: Direct Identification of On-Bead Peptides Using Surface-Enhanced Raman Spectroscopic Barcoding System for High-Throughput Bioanalysis

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.