Peptide and small molecule microarray for high throughput cell adhesion and functional assays

James R. Falsey, M. Renil, Steven Park, Shijun Li, Kit Lam

Research output: Contribution to journalArticle

310 Citations (Scopus)

Abstract

A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with Hcl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free Nα-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [γ33P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.

Original languageEnglish (US)
Pages (from-to)346-353
Number of pages8
JournalBioconjugate Chemistry
Volume12
Issue number3
DOIs
StatePublished - May 2001

Fingerprint

Cell adhesion
Microarrays
Cell Adhesion
Peptides
Assays
Throughput
Ligands
Molecules
Ligation
Thiazolidines
Microscopes
Oximes
Cell signaling
Biotin
Oligonucleotide Array Sequence Analysis
Sulfhydryl Compounds
Phosphorylation
Protein Binding
Biological Assay
Acids

ASJC Scopus subject areas

  • Chemistry(all)
  • Organic Chemistry
  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Peptide and small molecule microarray for high throughput cell adhesion and functional assays. / Falsey, James R.; Renil, M.; Park, Steven; Li, Shijun; Lam, Kit.

In: Bioconjugate Chemistry, Vol. 12, No. 3, 05.2001, p. 346-353.

Research output: Contribution to journalArticle

Falsey, James R. ; Renil, M. ; Park, Steven ; Li, Shijun ; Lam, Kit. / Peptide and small molecule microarray for high throughput cell adhesion and functional assays. In: Bioconjugate Chemistry. 2001 ; Vol. 12, No. 3. pp. 346-353.
@article{01e6539df1534d5a819eebd3555fd0b3,
title = "Peptide and small molecule microarray for high throughput cell adhesion and functional assays",
abstract = "A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with Hcl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free Nα-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [γ33P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the {"}one-bead one-compound{"} combinatorial library method.",
author = "Falsey, {James R.} and M. Renil and Steven Park and Shijun Li and Kit Lam",
year = "2001",
month = "5",
doi = "10.1021/bc000141q",
language = "English (US)",
volume = "12",
pages = "346--353",
journal = "Bioconjugate Chemistry",
issn = "1043-1802",
publisher = "American Chemical Society",
number = "3",

}

TY - JOUR

T1 - Peptide and small molecule microarray for high throughput cell adhesion and functional assays

AU - Falsey, James R.

AU - Renil, M.

AU - Park, Steven

AU - Li, Shijun

AU - Lam, Kit

PY - 2001/5

Y1 - 2001/5

N2 - A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with Hcl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free Nα-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [γ33P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.

AB - A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with Hcl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free Nα-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [γ33P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.

UR - http://www.scopus.com/inward/record.url?scp=0035017737&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035017737&partnerID=8YFLogxK

U2 - 10.1021/bc000141q

DO - 10.1021/bc000141q

M3 - Article

VL - 12

SP - 346

EP - 353

JO - Bioconjugate Chemistry

JF - Bioconjugate Chemistry

SN - 1043-1802

IS - 3

ER -