Quantitative proteomic and lipidomic analysis of detergent-resistant membrane microdomain involved in signal transduction

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16570100
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: JUNTENDO UNIVERSITY
• Principal Investigator: YANAGIDA Mitsuaki Juntendo University, School of Medicine, Assistant Professor, 医学部, 講師 (80365569)
• Co-Investigator(Kenkyū-buntansha): KAGA Naoko Juntendo University, School of Medicine, Assistant, 医学部, 助手 (80338342) ; IWABUCHI Kazuhisa Juntendo University, School of Health Care and Nursing, Associate Professor, 医療看護学部, 助教授 (10184897)
• Project Period (FY): 2004 – 2005
• Keywords: proteomics / lipidomics / microdomain / mass spectrometry / stable-isotope labeling / innate immunity / phagocytosis

Research Abstract

Neutrophils, which share a major population of leukocytes, are responsible for killing of bacteria. Recent reports showed that plasma membrane microdomains (lipid rafts) of neutrophils mediated phagocytosis and that through phagocytosis, infectious microorganisms not only entered in the cells but also survived by use of microdomains. The membrane microdomains are sphingolipid- and cholesterol-rich domains distinct from other membrane surfaces and provide the characteristic regions where many specific proteins including signal-transducing molecules are clustered.
In order to elucidate the function of microdomains during the phagocytic process in neutrophils, we tried to obtain fundamental data of protein and lipid components related to this pathway. As a model of neutrophil, we used human promyelocytic leukemia cell line, HL-60, which could be differentiated into neutrophil-like cells by treatment with dimethylsulfoxide. We prepared the detergent-resistant membranes (DRMs) in HL-60 cells before and after differentiation, and performed proteomic analysis by nano-flow liquid chromatography-mass spectrometry (NanoLC-MS/MS).
NanoLC-MS/MS analysis identified 121 proteins, in which 41 and 25 proteins were mainly detected in untreated and DMSO-treated HL-60 DRMs, respectively. More than 30% of identified proteins were already identified membrane proteins. Otherwise, many G-proteins and cytoskeleton proteins were also detected. We thought that identified proteins that showed change during differentiation were candidate proteins involved in function of neutrophils. Quantitation with internal standard peptides labeled with stable 13C and 15N isotopes revealed that protein detection profile of transferrin receptor and flotillins corresponded with previously reported knowledge. We concluded that the quantitative proteomic analysis using stable isotopes were effective for elucidation of protein construction of microdomains.