Poster Presentations
Day 3, June 12（Fri.） Room P (5F 501+502)
- 3P-07
Identification of Drug Target Protein Using Site-Specific Limited Proteolysis Mass Spectrometry (S-Lip-MS)
(Tsumura)
oMasashi Hiramoto, Takashi Matsumoto
To understand the mechanisms of action of drugs, the identification of target proteins is crucial. Methods that do not require derivatization of specific compounds are preferable for medicines derived from natural products. One such method is Lip-MS ; however, our investigation did not yield sufficient reproducibility. Therefore, we hypothesized that using a substrate-specific enzyme for limited digestion could improve reproducibility, and we devised S-Lip-MS, which employs Arg-C for limited digestion and Lys-C for complete digestion. This method has the advantage of allowing peptide sequence analysis under tryptic digestion conditions.
In the concept confirmation experiment, we used Methotrexate, Rapamycin, and Staurosporin as reference compounds. Each drug and vehicle were added to commercially available mouse liver cytosol, followed by Arg-C digestion under non-denaturing conditions, and then Lys-C digestion after boiling denaturation. The resulting peptides were analyzed using NanoLC-MSMS. Many peptide fragments of DHFR were altered, and by statistically comparing the groups with vehicle, Rapamycin, and Staurosporin, it was revealed that Methotrexate could be reliably selected as a candidate target with reproducibility. This method shows promise as a drug target identification technique.