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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   

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参见作者原研究论文

本实验方案简略版
The Plant Journal
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. 1-week-old Arabidopsis T-87 cells (50 g fresh weight)
  2. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 5000006JA )
  3. 2 mM synthesized substrate peptide [e.g., (OAOSOT)3S] [using standard Fmoc solid-phase synthesis chemistry on a 431A peptide synthesizer (Life Technologies)]
  4. 2 mM Uridine 5’-diphosphogalactose disodium salt (Sigma-Aldrich, catalog number: U4500 )
  5. 1 M MOPS-KOH (pH 7.0)
  6. 10 mM MnCl2
  7. 10% TX-100
  8. 1% Formic acid
  9. Acetonitrile (HPLC grade) containing 0.1% formic acid
  10. Water (HPLC grade) containing 0.1% formic acid
  11. Tris-HCl (pH 7.0)
  12. MgCl2
  13. Dithiothreitol
  14. Leupeptin
  15. Phenylmethanesulfonyl fluoride
  16. Sucrose
  17. Extraction buffer (see Recipes)
  18. Suspension buffer (see Recipes)

Equipment

  1. Waring blender
  2. Miracloth (Merck Millipore Corporation, catalog number: 475855 )
  3. Ultracentrifuge
  4. 30 °C incubator
  5. Micro centrifuge
  6. Semi-micro HPLC system (JASCO International Co., model: Micro21LC )
  7. LCQ Deca XP-plus ESI ion-trap mass spectrometer (Thermo Fisher Scientific)
  8. TSK-gel Amide-80 (3 μm) column (2 x 150 mm) (Tosoh Bioscience LLC, catalog number: 21865 )

Procedure

  1. Preparation of Arabidopsis microsomal membranes
    1. Arabidopsis T-87 cells are maintained on a 1-week culture interval under continuous darkness at 22 °C with shaking at 120 rpm.
    2. Suspend 1-week-old Arabidopsis T-87 cell (50 g fresh weight) in 20 ml Extraction buffer.
    3. Cool off to 4 °C on ice.
    4. Homogenize at 20,000 rpm for 5 min at 4 °C in a Waring blender.
    5. Cool off to 4 °C on ice.
    6. Filter the slurry through two layers of Miracloth.
    7. Centrifuge the filtrate at 3,000 x g for 15 min at 4 °C.
    8. Centrifuge the supernatant at 100,000 x g for 30 min at 4 °C.
    9. Suspend the pellet (microsomal membranes: approximately 150 μg/μl) in 500 μl Suspension buffer by gentle pipetting.
    10. Determine the protein concentration by conventional Bradford assay according to the manufacturer’s protocol (Bio-Rad Protein Assay).

  2. Hyp O-galactosyltransferase activity assay
    1. Set up 20 μl HPGT assay reactions in 0.5 ml microcentrifuge tube as follows.

      HPGT assay components
      Amount per reaction
      1 M MOPS-KOH (pH 7.0)
      2 μl
      2 mM UDP-α-D-galactose
      5 μl
      10 mM MnCl2
      2 μl
      10% TX-100
      2 μl
      2 mM substrate peptide
      1 μl
      Arabidopsis T-87 microsomal membranes
      30 μg protein equivalent
      Water Total
      20 μl

    2. Incubate at 30 °C, 1 h.
    3. Add 2 μl 1% formic acid to stop reaction.
    4. Add 80 μl acetonitrile.
    5. Centrifuge at 20,000 x g for 5 min.

  3. LC/MS analysis
    10 μl aliquots of the assay solution were analyzed by LC-MS using a micro HPLC (high pressure liquid chromatography) system connected to an LCQ Deca XP-plus ESI ion-trap mass spectrometer. Chromatographic separation is performed by normal-phase HPLC on a TSK-gel Amide-80 (3 μm) column (2 x 150 mm).
    1. The mobile phase is composed of HPLC grade water containing 0.1% formic acid (eluent A) and HPLC grade acetonitrile containing 0.1% formic acid (eluent B). The column temperature is maintained at 25 °C.
    2. The HPLC flow rate is 100 μl/min, and the elution gradient was 60 to 30% B over 15 min.
    3. The HPLC eluate was introduced into an electrospray ionization (ESI) ion-trap mass spectrometer in the positive ionization mode.
    4. MS source parameters are as follows [e.g., (OAOSOT)3S peptide]:
      1. Capillary temperature: 200 °C
      2. Capillary voltage: 42 V
      3. Source voltage: 5 kV
      4. Source current: 8.5 μA
      5. Sheath gas flow: 50
      6. Aux gas flow: 0
      7. Sweep gas flow: 0
      8. The mass range: m/z 500-2,000
    5. The mass spectra are obtained by selected ion monitoring. [e.g., (OAOSOT)3S: m/z 951.1, Galactosylated product: m/z 1032.2] (Figure 2).


      Figure 2. Selected ion chromatogram of substrate peptide and the galactosylated product. Substrate peptide was incubated with solubilized membrane fractions in the presence of UDP-α-D-galactose, then analyzed by LC-MS with selected ion monitoring of the substrate (m/z 951.1) and the galactosylated product (m/z 1032.2).

Recipes

  1. Extraction buffer (prepare freshly and keep on ice)
    25 mM Tris-HCl (pH 7.0)
    10 mM MgCl2
    2 mM dithiothreitol
    2 μM leupeptin
    2 mM phenylmethanesulfonyl fluoride
    250 mM sucrose
  2. Suspension buffer
    10 mM Tris-HCl (pH 7.0)
    250 mM sucrose

Acknowledgments

This is the detailed protocol for the detection of HPGT activity described by Ogawa-Ohnishi and Matsubayashi (2015). This research was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science, and Technology (No. 25221105).

References

  1. Ogawa-Ohnishi, M. and Matsubayashi, Y. (2015). Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis. Plant J 81(5): 736-746.
  2. Ogawa-Ohnishi, M., Matsushita, W. and Matsubayashi, Y. (2013). Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana. Nat Chem Biol 9(11): 726-730.

简介

阿拉伯半乳聚糖蛋白(AGP)是在生长和发育期间调节多种过程的植物特异性胞外糖蛋白。 AGP生物合成包括羟脯氨酸(Hyp)残基的O - 半乳糖基化,随后复合糖链的逐步延伸。 最初的Hyp O O - 半乳糖基化由Hyp O - 半乳糖基转移酶(HPGT)介导,其催化D-吡喃半乳糖残基转移到肽的Hyp残基的羟基上 糖供体UDP-α-D-半乳糖(图1)。 在这里,我们描述了用于在体外检测HPGT活性的基于LC/MS的方法。


图1. HPGT对Hyp半乳糖基化的反应方案。 HPGT催化从UDP-α-D-Gal向Hyp残基的羟基添加D-吡喃半乳糖。

材料和试剂

  1. 1周龄拟南芥T-87细胞(50g鲜重)
  2. Bio-Rad蛋白测定(Bio-Rad Laboratories,目录号:5000006JA)
  3. 2mM合成的底物肽[例如(OAOSOT)3 S] [使用431A肽合成仪(Life Technologies)上的标准Fmoc固相合成化学]]
  4. 2mM尿苷5'-二磷酸半乳糖二钠盐(Sigma-Aldrich,目录号:U4500)
  5. 1 M MOPS-KOH(pH 7.0)
  6. 10mM MnCl 2
  7. 10%TX-100
  8. 1%甲酸
  9. 含有0.1%甲酸的乙腈(HPLC级)
  10. 含有0.1%甲酸的水(HPLC级)
  11. Tris-HCl(pH 7.0)
  12. MgCl 2
  13. 二硫苏糖醇
  14. 亮肽素
  15. 苯基甲磺酰氟
  16. 蔗糖
  17. 提取缓冲液(参见配方)
  18. 悬浮缓冲液(参见配方)

设备

  1. 韦林搅拌机
  2. Miracloth(Merck Millipore Corporation,目录号:475855)
  3. 超速离心机
  4. 30℃培养箱
  5. 微量离心机
  6. 半微量HPLC系统(JASCO International Co.,型号:Micro21LC)
  7. LCQ Deca XP-plus ESI离子阱质谱仪(Thermo Fisher Scientific)
  8. TSK-gel Amide-80(3μm)柱(2×150mm)(Tosoh Bioscience LLC,目录号:21865)

程序

  1. 拟南芥微粒体膜的制备
    1. 拟南芥将T-87细胞在22℃,连续黑暗中以120rpm振荡保持1周培养间隔。
    2. 将1周龄的拟南芥T-87细胞(50g鲜重)悬浮于20ml提取缓冲液中。
    3. 在冰上冷却至4℃。
    4. 在Waring搅拌器中在4℃下以20,000rpm匀化5分钟
    5. 在冰上冷却至4℃。
    6. 通过两层Miracloth过滤浆料。
    7. 在4℃下以3,000×g离心滤液15分钟。
    8. 在4℃下将上清液以100,000xg离心30分钟
    9. 通过轻轻吹打使悬浮在500μl悬浮缓冲液中的沉淀(微粒体膜:约150μg/μl)。
    10. 通过常规Bradford测定法确定蛋白质浓度 根据制造商的方案(Bio-Rad蛋白测定)。

  2. Hyp O O - 半乳糖基转移酶活性测定
    1. 在0.5ml微量离心管中设置20μlHPGT测定反应如下
      HPGT测定组件
      每次反应金额
      1 M MOPS-KOH(pH 7.0)
      2微升
      2mM UDP-α-D-半乳糖 5微升
      10mM MnCl 2
      2微升
      10%TX-100
      2微升
      2mM底物肽 1微升
      拟南芥 T-87微粒体膜
      30μg蛋白质当量
      水总量
      20微升

    2. 在30℃,1小时孵育。
    3. 加入2μl1%甲酸以终止反应
    4. 加入80μl乙腈
    5. 以20,000×g离心5分钟。

  3. LC/MS分析
    通过LC-MS使用连接到LCQ Deca XP-plus ESI离子阱质谱仪的微HPLC(高压液相色谱)系统分析10μl等分的测定溶液。色谱分离通过在TSK-凝胶Amide-80(3μm)柱(2×150mm)上的正相HPLC进行。
    1. 流动相由含0.1%甲酸的HPLC级水组成 (洗脱液A)和含有0.1%甲酸的HPLC级乙腈 (洗脱液B)。柱温度保持在25℃
    2. HPLC流速为100μl/min,洗脱梯度在15分钟内为60至30%B
    3. 将HPLC洗脱液引入电喷雾电离(ESI) ?离子阱质谱仪在正电离模式下
    4. MS源参数如下[例如(OAOSOT) 3 S肽]:
      1. 毛细管温度:200℃
      2. 毛细管电压:42 V
      3. 源电压:5 kV
      4. 源电流:8.5μA
      5. 鞘气流量:50
      6. 辅助气流量:0
      7. 扫气流量:0
      8. 质量范围:m/z <500> 2,000
    5. 质谱通过选择离子监测获得。 [例如(OAOSOT) 3 S: m/z 951.1,半乳糖基化产物:m/z 1032.2]图2)。


      图2.底物肽的选择离子色谱图 半乳糖基化产物。将底物肽与溶解的 ?膜部分在UDP-α-D-半乳糖存在下进行分析 通过具有选定的底物离子监测的LC-MS(m/z 951.1)和 半乳糖基化产物(m/z <1032.2)。

食谱

  1. 提取缓冲液(新鲜制备并保存在冰上)
    25mM Tris-HCl(pH7.0) 10mM MgCl 2/
    2mM二硫苏糖醇 2μM亮抑蛋白酶 2mM苯基甲磺酰氟 250mM蔗糖
  2. 暂停缓冲区
    10mM Tris-HCl(pH7.0) 250mM蔗糖

致谢

这是Ogawa-Ohnishi和Matsubayashi(2015)描述的用于检测HPGT活性的详细方案。这项研究由教育,文化,体育,科学和技术部的科学研究资助(S)支持(No. 25221105)。

参考文献

  1. Ogawa-Ohnishi,M。和Matsubayashi,Y。(2015)。 在拟南芥中鉴定三种有效的羟脯氨酸O - 半乳糖基转移酶。 Plant J 81(5):736-746。
  2. Ogawa-Ohnishi,M.,Matsushita,W。和Matsubayashi,Y。(2013)。 在拟南芥中鉴定三个羟脯氨酸O - 阿拉伯糖基转移酶。 Nat Chem Biol 9(11):726-730。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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