Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.
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Osaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
Japan Sci & Technol Agcy, Core Res Evolut Sci & Technol, Saitama 3320012, JapanOsaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
Fujita, Morihisa
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Maeda, Yusuke
Ra, Moonjin
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Univ Tokyo, Grad Sch Med, Tokyo 1130033, JapanOsaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
Ra, Moonjin
Yamaguchi, Yoshiki
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RIKEN ASI, Struct Glycobiol Team, Saitama 3510198, JapanOsaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
Yamaguchi, Yoshiki
Taguchi, Ryo
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Univ Tokyo, Grad Sch Med, Tokyo 1130033, Japan
Japan Sci & Technol Agcy, Core Res Evolut Sci & Technol, Saitama 3320012, JapanOsaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
Taguchi, Ryo
Kinoshita, Taroh
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Osaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
Osaka Univ, WPI Immunol Frontier Res Ctr, Suita, Osaka 5650871, Japan
Japan Sci & Technol Agcy, Core Res Evolut Sci & Technol, Saitama 3320012, JapanOsaka Univ, Res Inst Microbial Dis, Suita, Osaka 5650871, Japan
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Boston Univ, Ctr Biomed Mass Spectrometry, Dept Biochem, Boston, MA 02118 USABoston Univ, Ctr Biomed Mass Spectrometry, Dept Biochem, Boston, MA 02118 USA
Leymarie, Nancy
Zaia, Joseph
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Boston Univ, Ctr Biomed Mass Spectrometry, Dept Biochem, Boston, MA 02118 USABoston Univ, Ctr Biomed Mass Spectrometry, Dept Biochem, Boston, MA 02118 USA