Harvard College

Sankyo Company, Limited

Kazuishi Kubota

Yasuteru Iijima

Steven P Gygi

Kaori Nakahara

Judit Villen

Ayako Hara

Yonghao Yu

Yohei Ozeki

A mass-spectrometry-based method and substrates are provided herein for large scale kinome activity profiling directly from crude lysates using 90 chemically synthesized peptide substrates with amino acid sequences derived from known phosphoproteins. Quantification of peptide phosphorylation rates was achieved via the use of stable isotope labeled synthetic peptides. A method and substrates for obtaining 90 simultaneous activity measurements in a single-reaction format were developed and validated. The kinome activity profiling strategy was successfully applied with lysates of: cells manipulated by combination of mitogen stimulation, pharmacological perturbation and siRNA-directed kinase knockdown; seven different breast cancer cell lines treated with gefitinib; and each of normal and cancerous tissue samples from renal cell carcinoma patients. This method concurrently measures multiple peptide phosphorylation rates to provide a diagnostic fingerprint pattern for activated kinases, protein phosphatases, modulators of these enzymes, and pathways (kinome) from as little starting material as a few cells.

Methods, compositions and kits for high throughput kinase activity screening using mass spectrometry and stable isotopes

Polypeptides having 2',5&#x2032;-oligoadenylate phosphodiesterase activity, antibodies against the polypeptides, polynucleotides coding for the polypeptides, recombinant plasmid DNAs into which the polynucleotides have been inserted, host cells transformed by the recombinant plasmid DNAs, processes for screening for 2&#x2032;,5&#x2032;-oligoadenylate phosphodiesterase activity inhibitor substances, and processes for screening for 2&#x2032;,5&#x2032;-oligoadenylate phosphodiesterase expression regulation inhibitor substances. The polypeptides, polynucleotides and host cells are useful for searching for therapeutic agents for viral infections and tumors.

2', 5&#x2032;-oligoadenylate phosphodiesterase

Growing community of inventors

Tokyo, Japan

Kazuishi Kubota