LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity
基于LC/MS法检测羟脯氨酸-O-半乳糖苷转移酶的活性
发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1711 浏览次数: 7349
评审: Renate WeizbauerHarrie van ErpAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
Abstract
Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins regulating a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. The initial Hyp O-galactosylation is mediated by Hyp O-galactosyltransferase (HPGT) that catalyzes the transfer of a D-galactopyranosyl residue to the hydroxyl group of Hyp residues of peptides from the sugar donor UDP-α-D-galactose (Figure 1). Here we describe a LC/MS-based method for the detection of HPGT activity in vitro.
Figure 1. Reaction scheme for Hyp galactosylation by HPGT. HPGT catalyzes the addition of a D-galactopyranose from an UDP-α-D-Gal to the hydroxylgroup of Hyp residues.
Materials and Reagents
- 1-week-old Arabidopsis T-87 cells (50 g fresh weight)
- Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006JA )
- 2 mM synthesized substrate peptide [e.g., (OAOSOT)3S] [using standard Fmoc solid-phase synthesis chemistry on a 431A peptide synthesizer (Life Technologies)]
- 2 mM Uridine 5’-diphosphogalactose disodium salt (Sigma-Aldrich, catalog number: U4500 )
- 1 M MOPS-KOH (pH 7.0)
- 10 mM MnCl2
- 10% TX-100
- 1% Formic acid
- Acetonitrile (HPLC grade) containing 0.1% formic acid
- Water (HPLC grade) containing 0.1% formic acid
- Tris-HCl (pH 7.0)
- MgCl2
- Dithiothreitol
- Leupeptin
- Phenylmethanesulfonyl fluoride
- Sucrose
- Extraction buffer (see Recipes)
- Suspension buffer (see Recipes)
Equipment
- Waring blender
- Miracloth (Merck Millipore Corporation, catalog number: 475855 )
- Ultracentrifuge
- 30 °C incubator
- Micro centrifuge
- Semi-micro HPLC system (JASCO International Co., model: Micro21LC )
- LCQ Deca XP-plus ESI ion-trap mass spectrometer (Thermo Fisher Scientific)
- TSK-gel Amide-80 (3 μm) column (2 x 150 mm) (Tosoh Bioscience LLC, catalog number: 21865 )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Ogawa-Ohnishi, M. and Matsubayashi, Y. (2016). LC/MS-based Detection of Hydroxyproline O-galactosyltransferase Activity. Bio-protocol 6(2): e1711. DOI: 10.21769/BioProtoc.1711.
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分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 糖类 > 糖蛋白
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