Peptide and small molecule microarray for high throughput cell adhesion and functional assays
Peptide and small molecule microarray for high throughput cell adhesion and functional assays
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DOI：
10.1021/bc000141q
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发表时间：
2001-05-01
影响因子：
4.7
通讯作者：
Lam, KS
中科院分区：
文献类型：
--
作者：
Falsey, JR;Renil, M;Lam, KS
制备了一类新型的化学微芯片，用于通过肟键或噻唑烷环连接反应将小分子配体与多肽共价连接。将市售显微镜载玻片彻底清洁并用（3-氨丙基）三乙氧基硅烷（APTES）衍生化。然后通过两种不同的途径将氨基载玻片转化为甘氨酰衍生物：（1）Fmoc-Ser偶联，然后脱保护和氧化，或（2）与受保护的乙醛酸偶联，最后用HCl脱保护。在羧基末端用4，7，10-三氧杂-1，13-十三烷二胺琥珀酰亚胺酸接头和氨基-氧基或1，2-氨基-硫醇基衍生的生物素或肽配体（例如，具有游离N-α-氨基的半胱氨酸）仅仅使用DNA微阵列点样器印刷到这些载玻片上。化学连接后，用三种不同的生物学测定法分析固定化配体的微阵列：（1）荧光检测的蛋白结合测定法，（2）使用[γ P-33]-ATP和特异性蛋白激酶标记肽底物点的功能磷酸化测定法，和（3）与完整细胞的粘附测定法。在细胞粘附试验中，我们不仅可以确定肽对不同细胞系的结合特异性，我们还可以使用免疫荧光技术在微芯片上原位确定附着细胞的功能性细胞信号传导。这种化学微芯片系统使我们能够快速分析我们从“一珠一化合物”组合文库方法中鉴定的众多配体的功能特性。
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 N-alpha-amino group) mere 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 [gamma P-33]-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.