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Detection of Hydroxyproline O-galactoside by LC/MS
采用 LC/MS检测羟脯氨酸-O-半乳糖苷   

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Abstract

Hydroxyproline (Hyp) O-galactosylation is a plant-specific post-translational modification found in extracellular glycoproteins such as arabinogalactan proteins (AGPs). 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-Gal. Here we describe an LC/MS-based method for the detection of Hyp O-galactoside.

Materials and Reagents

  1. Cotton
  2. 1 ml Micropipette tip
  3. Hyp O-galactosylated peptides or proteins
  4. 0.22 M Ba(OH)2
  5. 0.32 M sulfuric acid
  6. 1 M NaOH
  7. 1 M HCl
  8. 10% aqueous ammonia
  9. 80% acetonitrile containing 0.1% formic acid
  10. 99.9% acetonitrile (HPLC grade) containing 0.1% formic acid
  11. Water (HPLC grade) containing 0.1% formic acid

Equipment

  1. Heat block
  2. Centrifugal evaporator
  3. BT AG 50W-X8 Resin (100-200 mg resin, H+ form) (Bio-Rad Laboratories, catalog number: 143-5441 )
  4. Micro centrifuge
  5. Micro HPLC system (JASCO International Co., model: micro21 LC-01 )
  6. LCQ Deca XP-plus ESI ion-trap mass spectrometer (Thermo Fisher Scientific)
  7. TSK-gel Amide-80 (3 μm) column (2.0 x 150 mm) (Tosoh Bioscience LLC, catalog number: 21865 )

Procedure

  1. Ba(OH)2 hydrolysis
    1. Dissolve galactosylated peptide in 500 μl 0.22 M Ba(OH)2 in a glass vial with cap.
    2. Incubate at 105 °C, 6 h.
    3. Incubate on ice for 5 min.
    4. Add 500 μl of 0.32 M sulfuric acid on ice.
    5. Centrifuge at 20,000 x g for 5 min.

  2. Partial purification of Ba(OH)2 hydrolysate (Figure 1)
    1. Plug a 1 ml micropipette tip with a small amount of cotton.
    2. Pack 200 mg AG 50W-X8 resin into the tip column.
    3. Wash the column with 1 ml of 1 M NaOH by gravity flow.
    4. Wash the column with 1 ml of 1 M HCl by gravity flow.
    5. Wash the column with 1 ml of water by gravity flow.
    6. Apply supernatant of the Ba(OH)2 hydrolysate of the galactosylated peptide to the tip column.
    7. Wash the column with 1 ml of water by gravity flow.
    8. Elute with 1 ml of 10% aqueous ammonia by gravity flow.
    9. Evaporate the sample to dryness.
    10. Dissolve in 100 μl 80% acetonitrile containing 0.1% formic acid.


      Figure 1. Partial purification of Ba(OH)2 hydrolysate. Supernatant of the Ba(OH)2 hydrolysate of the galactosylated peptide was applied to the tip column.

  3. LC/MS analysis
    10 μl aliquots of the assay solution will be 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 40% B over 10 min.
    3. Subject the HPLC eluate to coupled electrospray ionization (ESI) in the positive ionization mode.
    4. MS source parameters are as follows:
      1. Capillary temperature: 200 V
      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 500-2000


        Figure 2. Detection of Hyp O-galactoside in Ba(OH)2 hydrolysates of in vitro galactosylated AGP14 by LC-MS. The sample was analyzed by selected ion monitoring of Hyp (m/z 132.1) and Gal-Hyp (m/z 294.1). Ba(OH)2 hydrolysis yields a diastereomeric pair of amino acids.

    5. The mass spectra are obtained by selected ion monitoring in zoom scan mode (Hyp: m/z 132.1, Gal-Hyp: m/z 294.1).

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

羟脯氨酸(Hyp)O - 半乳糖基化是在细胞外糖蛋白例如阿拉伯半乳聚糖蛋白(AGP)中发现的植物特异性翻译后修饰。 Hyp 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来自糖的肽的Hyp残基的羟基上 供体UDP-α-D-Gal。 在这里,我们描述了基于LC/MS的Hyp O /α-半乳糖苷的检测方法。

材料和试剂

  1. 棉花
  2. 1 ml微量吸头
  3. Hyp O O - 半乳糖基化肽或蛋白质
  4. 0.22 M Ba(OH)2
  5. 0.32M硫酸
  6. 1 M NaOH
  7. 1 M HCl
  8. 10%氨水
  9. 含有0.1%甲酸的80%乙腈
  10. 含有0.1%甲酸的99.9%乙腈(HPLC级)
  11. 含有0.1%甲酸的水(HPLC级)

设备

  1. 热块
  2. 离心蒸发器
  3. BT AG 50W-X8树脂(100-200mg树脂,H +型)(Bio-Rad Laboratories,目录号:143-5441)
  4. 微量离心机
  5. 微HPLC系统(JASCO International Co.,型号:micro21 LC-01)
  6. LCQ Deca XP-plus ESI离子阱质谱仪(Thermo Fisher Scientific)
  7. TSK-gel Amide-80(3μm)柱(2.0×150mm)(Tosoh Bioscience LLC,目录号:21865)

程序

  1. Ba(OH)2水解
    1. 将溶解于500μl0.22M Ba(OH)2中的半乳糖基化的肽溶解在带盖的玻璃小瓶中。
    2. 在105℃,6小时孵育。
    3. 在冰上孵育5分钟。
    4. 在冰上加入500μl0.32M硫酸
    5. 以20,000×g离心5分钟。

  2. 部分纯化Ba(OH)2水解产物(图1)
    1. 用少量棉花插入1ml微量吸头。
    2. 将200 mg AG 50W-X8树脂装入吸头柱。
    3. 通过重力流动用1ml 1M NaOH洗涤柱
    4. 通过重力流动用1ml 1M HCl洗涤柱子。
    5. 通过重力流量用1ml水洗涤柱子。
    6. 将半乳糖基化肽的Ba(OH)2水解产物的上清液应用于尖端列。
    7. 通过重力流量用1ml水洗涤柱子。
    8. 通过重力流动用1ml 10%氨水洗脱
    9. 将样品蒸发至干。
    10. 溶于100μl含有0.1%甲酸的80%乙腈中

      图1. Ba(OH)2水解产物的部分纯化。上清液 将半乳糖基化肽的Ba(OH)2水解产物应用于 ?尖柱。

  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等分试样的测定溶液。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10分钟内为60至40%B
    3. 使用正离子模式的HPLC洗脱液进行耦合电喷雾电离(ESI)。
    4. MS的源参数如下:
      1. 毛细管温度:200 V
      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 500-2000


        图2.在水溶液中检测Hyp em O - 半乳糖苷在Ba(OH)2水解产物中的 通过LC-MS分析体外半乳糖基化AGP14。 通过选择分析样品 ?Hyp(m/z <132.1)和Gal-Hyp(m/z <294.1)的离子监测。 Ba(OH)2水解产生非对映体对的氨基酸

    5. 质谱通过在缩放扫描模式中选择的离子监测(Hyp:m/z 132.1,Gal-Hyp:m/z 294.1)获得。

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)。 在拟南芥中鉴定三种羟脯氨酸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). Detection of Hydroxyproline O-galactoside by LC/MS. Bio-protocol 6(2): e1710. DOI: 10.21769/BioProtoc.1710.
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