17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||14:30 – 16:00|
Jin Young Kim1, Jeong Hwa Lee1, Yeong Hee Ahn2, Kun Cho1, Gun Wook Park2, Kyung-Hoon Kwon2, Kyung Hee Byun3, Sung Min Ahn3, Bong Hee Lee3, Jong Shin Yoo21 Proteomics Team, Korea Basic Science Institute, Daejeon, Korea
Correspondence address: Jin Young Kim, Korea Basic Science Institute, Proteomics Team, 52 Yeoeun-Dong Yusung -Gu, Daejeon, 305-333 Korea.
Keywords: Isotope, Stable; MS/MS, Liquid Chromatography; Phosphorylation; Quantitative Analysis.
Novel aspect: Proteome and phosphoprotein analysis of HMO6 activated by LPS using SILAC.
HMO6 is an immortalized human microglial cell line from human embryonic telecephalon tissue and exhibits various properties similar to those documented in human microglia. HMO6 cells can be activated by the treatment of Amyloid-beta and lipopolysaccharide (LPS), having considerable utility as an in vitro model for the studies of human microglia in many central nervous system diseases such as Alzheimer’s disease. In this study, we aim to characterize proteomic changes in HMO6 cells upon activation by LPS. For the quantitative analysis of differentically expressed protein from HMO6 cell with and without LPS treatment, we used stable isotope labeling by amino acids in cell culture, that metabolically labels the entire proteome, making it distinguishable by MS analysis. Two populations of HMO6 were grown in medium containing distinct forms of lysine – normal 12C (Lys 0) and isotopic variants 13C (Lys 6). The combined proteins from two cellular lysates were resolved on 1DE and proteolytically digested. The resulting peptide mixture was analyzed by Nano-LC/MS/MS coupled to LT/FT/MS. Particularly, to effectively compare the change of phosphorylation in proteins, we have developed phosphopeptide analysis method by guanidine ethane thiol (GET) – tagging with phosphopeptide enrichment by TiO2, and applied for the signaling protein analysis in HMO6 changed by LPS.