Analysis of Phosphatidylinositol 3,4,5-Trisphosphates of PTEN Expression on Mammalian Cells

• Main Authors: Nusrat Jahan, Taeseong Park, Young Hwan Kim, Dongsun Lee, Hackyoung Kim, Kwangmo Noh, Young Jun Kim
• Format: Article
• Language: English
• Published: Korean Society for Mass Spectrometry 2013-09-01
• Series: Mass Spectrometry Letters
• Subjects: Phosphoinositides; Lipidomics; PTEN; Mass Spectrometry; Enzymatics
• ISSN: 2233-4203; 2093-8950

The goal of this study is to find an experimental condition which enables us to perform enzymatic studies on the cellular behavior of PTEN (phosphatase and tensine homolog) through identification of molecular species of phosphatidylinositol 3,4,5- trisphosphates and their quantitative analysis in a mammalian cell line using mass spectrometry. We initially exployed a two-step extraction process using HCl for extraction of phosphatidylinositol 3,4,5-trisphosphates from two mammalian cell lines and further analyzed the extracted phosphatidylinositol 3,4,5-trisphosphates using tandem mass spectrometry for the identification of them. We finally quantified the concentration of phosphatidylinositol 3,4,5-trisphosphates using internal standard calibration. From these observation, we found that HEK 293-T cells is a good model to examine the enzymatic behavior of PTEN in a cell, and the minimum amount of phosphatidylinositol 3,4,5-trisphosphates is more than 50 pmol for quantification in a mass spectrometer. These results suggest that the well-optimized experimental conditions are required for the investigation of the cellular PTEN in terms of the catalytic mechanism and further for the detailed identification of cellular substrates