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Glucosinolates Determination in Tissues of Horseradish Plant
山葵植株组织的硫代葡萄糖苷的测定   

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Abstract

Glucosinolates (GLS) are secondary metabolites mainly found in plants belonging to the Brassicaceae family, including also horseradish (Armoracia rusticana G. Gaertn., B. Mey. & Scherb), a popular spice with a characteristic pungent flavor due to the abundance of GLS. Such compounds exhibit antibacterial, antifungal, and insecticidal activities, as well as human health properties. Therefore, it is very important to have a full understanding of their levels and profiles in plants. However, the characterization of GLS from horseradish crude extracts is a tough task, due to the complexity of the vegetal matrix and the occurrence of many GLS in trace amounts. Here we describe two alternative effective and rapid methods for GLS characterization in horseradish plants: Liquid chromatography coupled to high resolution mass spectrometry (LC-MS) for determination of intact GLS and HPLC-UV for determination of desulfo-GLS.

Materials and Reagents

  1. Horseradish tissue (hypogeous and epigeous portion) (see Note 1)
  2. Methanol (MeOH) (LC/MS grade) (Carlo Erba Reagents, catalog number: 414831 )
  3. Acetonitrile (ACN) (LC/MS grade) (Carlo Erba Reagents, catalog number: 412342 )
  4. Ultrapure Milli-Q water
  5. Liquid nitrogen
  6. Sinigrin hydrate from horseradish (99%) (Sigma-Aldrich, catalog number: S1647 )
  7. Rapeseed ERM certified Reference Material containing gluconapin, 4-hydroxyglucobrassicin, glucobrassicanapin, glucobrassicin and gluconaturtiin (Sigma-Aldrich, catalog number: ERMBC367 )
  8. Glucoiberin (C2 Bioengineering, catalog number: 10-JS 12-05-02 )
  9. Glucobarbarin (C2 Bioengineering, catalog number: 18-DM 19-10-99 )
  10. Glucotrapeolin (C2 Bioengineering, catalog number: 16-PM 19-10-99 )
  11. 70% MeOH (see Recipes)

In addition only for intact GLS determination

  1. Formic acid (gradient grade) (Sigma-Aldrich, catalog number: F0507 )
  2. 0.1% Formic acid (HCOOH) (see Recipes)

In addition only for desulfo-method

  1. DEAE-Sephadex A-25 (formiate form) obtained by using DEAE-Sephadex A-25 (chloride form) (Sigma-Aldrich, catalog number: A25120 ) and imidazole (Sigma-Aldrich, catalog number: 56750 )
  2. Sulfatase type H-1 (Sigma-Aldrich, catalog number: S-9626 )
  3. Sulfatase type H-1 (1 to 2.5) (see Recipes)
  4. DEAE-Sephadex A-25 (formiate form) (see Recipes)

Equipment

  1. Freeze Dry Systems (e.g. Labconco, model: Freezone 4.5 )
  2. Laboratory mill
  3. Disposable 50 ml and 15 ml polypropylene tubes
  4. 2 ml sample vials
  5. Water bath: beaker filled with water and placed on a heating device (electric hotplate or similar device).
  6. Thermometer
  7. Vortex mixer
  8. Refrigerated centrifuge (50 ml tubes) (e.g. Heraeus, model: Varifuge F )
  9. Glass Pasteur pipettes
  10. 0.22 µm nylon filter (Whatman)
  11. HPLC system with a photodiode array detector (e.g. Agilent, model: Agilent 1200 HPLC Liquid Chromatrography System)
  12. HPLC 2 ml glass vials (Phenomenex, model: AR1-3910-12 ) with caps (Phenomenex, model: AR0-8959-13-B )
  13. Liquid chromatography (LC) coupled with electrospray ionization (ESI) and high resolution mass spectrometry (MS) (e.g. Thermo Fisher Scientific, model: LC-ESI-FTICR MS )
  14. Intact glucosinolates Discovery C18 column, 250 x 4.6 mm, 5 µm particle size (pore size, 180Å) (Sigma-Aldrich, catalog number: 504971 ), with a Discovery C18 20 x 4 mm security guard cartridge (Sigma-Aldrich, catalog number: 505129 )
  15. Desulfoglucosinolates (Nucleodur C18 column, 125 mm x 3 mm) (MACHEREY-NAGEL, catalog number: MN760051.30 )

Procedure

Intact GLS

  1. GLS extraction
    1. Clean plant material with distilled water and dry with paper towels. Separate roots from epigeous portion, weight and immediately freeze at -80 °C to inhibit myrosinase activity.
    2. Lyophilize the frozen tissues and grind to a fine powder using a laboratory mill. Chill roots in liquid nitrogen before the lyophilization to allow the crushing.
    3. Weigh 200 mg of frozen dry material in 50 ml polypropylene tubes and place in a water bath heated with an electric hotplate at 70-80 °C for 10 min. During this process, carry out the following steps: After 1 min add 2 ml of 70% methanol solvent and after 5 min mix with vortex for 20 sec.
    4. At the end of 10 min remove the polypropylene tube from the water bath and mix again with vortex for 20 sec.
    5. Centrifuge at 4 °C for 10 min at 2,400 x g.
    6. Collect the supernatant in a disposable 15 ml polypropylene tube by using glass Pasteur pipettes.
    7. Extract again the remaining pellet with 2 ml of 10% methanol in water bath heated with an electric hotplate at 70-80 °C for 10 min; after 5 min mix with vortex for 20 sec and the follow the same procedure from step A4-6.
    8. Combine the supernatants and vortex the extract for 20 sec.
    9. Filter through 0.22 µm nylon filter and transfer into 2 ml sample vials.

  2. GLS detection by LC MS
    1. Inject 25 µl of each sample in LC-ESI-FTICR MS system equipped with Discovery C18 column, 250 x 4.6 mm, 5 µm particle size (pore size, 180 Å), with a Discovery C18 20 x 4 mm security guard cartridge.
      Settings:
      Flow rate: 1 ml/min
      Column temperature: 25 °C
      Solvent gradient for chromatographic separation:

      Time (min)
      % Solvent A 
      0.1% HCOOH water
      % Solvent B
      ACN
      0
      90
      10
      10
      76
      24
      12
      40
      60
      15
      90
      10
      20
      90
      10


      Connect the LC system to the mass spectrometer by a laboratory-made splitter with a split ratio of 1:4 after the analytical column to allow 200 µl/min to enter the ESI source.
    2. Perform full-scan experiments in both the linear trap and the ICR cell. Collect data in profile mode in the range of m/z 50−1,000 by setting:
      Ionization mode: negative
      Resolution: 100.000 (FWHM) at m/z 400
      ESI needle voltage: -4.60 kV
      Capillary voltage: -22 V
      Temperature of the heated capillary: 350 °C
      Sheath gas (N2) flow rate: 80 (arbitrary units)
      In these conditions GLS elute with a retention time and molecular exact mass to charge ratio as reported in the following table (Agneta et al., 2012; Agneta et al., 2014):

      Chemical name
      Molecular formulae
      Monoisotopic exact value as [M-H]- (m/z)
      tR
      3-(Methylsulfinyl)propyl-GLS
      C11H21NO10S3
      422.02549
      4.3
      2-Propenyl-GLS
      C10H17NO9S2
      358.02720
      4.4
      2-Methylsulfonyl-oxo-ethyl-GLS
      C10H17NO12S3
      437.98402
      4.6
      3-Butenyl-GLS
      C11H19NO9S2
      372.04285
      5.5
      1-Methylpropyl-GLS
      C11H21NO9S2
      374.05850
      6.2
      2-Methylpropyl-GLS
      C11H21NO9S2
      374.05850
      6.4
      4-Mercaptobuthyl-GLS
      C11H21NO9S3
      406.03057
      6.5
      7-Methylsulfinylheptyl-GLS
      C15H29NO10S3
      478.08808
      7.3
      4-Hydroxyindol-3-ylmethyl-GLS
      C16H20N2O10S2
      463.04866
      7.4
      Unidentified isomer of 4-hydroxyindol-3-ylmethyl-GLS
      C16H20N2O10S2
      463.04866
      7.5
      2(S)-Hydroxy-2-phenylethyl-GLS
      and/or
      2(R)-Hydroxy-2-phenylethyl-GLS
      C15H21NO7S
      438.05341
      7.8
      4-Pentenyl-GLS
      C12H21NO9SO2
      386.05850
      7.8
      Benzyl-GLS
      C14H19NO9S2
      408.04285
      8.5
      Indol-3-ylmethyl-GLS
      C16H20N2O9S2
      447.05375
      10.3
      2-Phenylethyl-GLS
      C15H21NO9S2
      422.05850
      12.1
      4-Methoxyindol-3-ylmethyl-GLS
      C17H22N2O10S2
      477.06431
      12.7
      7-Methylthioheptyl-GLS   
      C15H29NO9S3
      462.09317
      16.7
      Note: It is possible to use the extracts to quantify GLS by preparing a calibration curve for each GLS using a series of dilution and calculating the area under the peak of each compound.


      Figure 1. Example of eXtracted Ion Chromatograms (XICs) using high resolution LC-ESI-FTICR MS acquired in negative ion mode of root crude extract of A. rusticana. The ions monitored are displayed in each trace (plots A-O) and correspond to the most abundant deprotonated molecules, [M−H], using a restricted window of ±0.0010 m/z unit centered around each selected ion. Peak numbers in bold correspond to: (1) 3-(methylsulfinyl)propyl-GLS; (2) 2-propenyl-GLS; (3)* 2-methylsulfonyl-oxo-ethyl-GLS; (4) 3-butenyl-GLS; (5)* 1-methylpropyl-GLS; (6)* 2-methylpropyl-GLS; (7)* 4-mercaptobuthyl-GLS; (8)* 7-methylsulfinylheptyl-GLS; (9) 4-hydroxyindol-3-ylmethyl-GLS; (10) unidentified isomer of 4-hydroxyglucobrassicin; (11) 2(S)-hydroxy-2-phenylethyl-GLS and/or 2(R)-hydroxy-2-phenylethyl-GLS; (12) 4-pentenyl-GLS; (13) benzyl-GLS; (14) indol-3-ylmethyl-GLS; (15) 2-phenethyl-GLS; (16) 4-methoxyindol-3-ylmethyl-GLS; (17)* 7‐methylthioheptyl-GLS (peaks with asterisk are tentatively assigned). The XIC signals of some peaks were acquired using a sample extract diluted 1:20 with the mobile phase. In bold are indicated also the molecular formula and the range of monoisotopic value as [M-H] ion (Δ m/z) (Agneta et al., 2014).

Desulfo GLS

  1. GLS extraction
    1. Follow the procedure of intact GLS step A1-8.
    2. Transfer 500 µl of the extract on the top of a 20 mg of Sephadex DEAE-A 25 (shortened Pasteur pipet) in the formiate form.
    3. Wash the column twice with 1 ml of deionized water
    4.  Add 100 µl of sulfatase type H-1 diluted 1 to 2.5 with water to achieve the desulfation and incubate overnight at 39 °C (minimum 16 h) (Möllers et al., 1999)
    5. Elute desulfatated GLS with 3 x 500 µl deionized water and collect in 15 ml polypropylene tubes.
    6. Vortex the sample for 20 sec.
    7. Filter through 0.22 µm nylon filter and transfer into 2 ml sample vials.

  2. GLS detection by HPLC
    1. Inject 25 µl of each sample in HPLC system equipped with Nucleodur C18 column, 125 mm x 3 mm.
      Settings:
      Flow rate: 0.6 ml/min
      Column temperature: 35 °C
      UV detection: 229
      Solvent gradient for chromatographic separation:
      Time(min)
      % Solvent A Water
      % Solvent B ACN
      0
      99
      1
      20
      80
      20
      25
      80
      20
      22
      99
      1

      In these conditions GLS elute with a retention time as reported in the following table:
      Chemical name
      tR
      Relative response factora
      3-(Methylsulfinyl)propyl-GLS
      2.8
      1.07
      2-Propenyl-GLS
      4.3
      1.00
      3-Butenyl-GLS
      7.3
      1.11
      1-Methylpropyl-GLS and/or
      8.9
      1.00b
      2-Methylpropyl-GLS
      8.9
      1.00b
      2(S)-Hydroxy-2-phenylethyl-GLS and/or 2(R)-Hydroxy-2-phenylethyl-GLS
      10.1
      0.98c
      4-Pentenyl-GLS
      10.9
      1.15
      Benzyl-GLS
      11.8
      0.95
      Indol-3-ylmethyl-GLS
      13.9
      0.29
      2-Phenylethyl-GLS
      16.1
      0.95
      4-Methoxyindol-3-ylmethyl-GLS
      16.5
      0.25
      aThe response factors values used for the desulfated glucosinolates quantification as reported in EN ISO 9167-1 (1992), with except of following desulfated molecules: 1-methylpropyl-GLS, 2-methylpropyl-GLS and 2(S)-hydroxy-2-phenylethyl-GLS and/or 2(R)-Hydroxy-2-phenylethyl-GLS
      bResponse factor of 2-propenyl-GLS as desulfated molecules used for the relative quantification
      cExperimental response factor determinate by using the 2(S)-hydroxy-2-phenylethyl-GLS in comparison to 2-propenyl-GLS as desulfated molecules (see Note 2)

Notes

  1. Plant tissues were collected from a field collection of the Institute of Plant Genetics, National Research Council, Thematic Centre for the Preservation of Mediterranean Biodiversity, located in Policoro (MT) (40° 17′ 30″ N, 16° 65′ 16″ E), where many accessions of horseradish, previously collected from various villages of the internal areas of the Basilicata region, are maintained and vegetatively propagated (for details see Sarli et al., 2012).
  2. It is possible to use the extracts to quantify desulfo GLS by preparing a calibration curve for each GLS by using a series of dilution and calculating the area under the peak of each compound.
    It is also possible to quantify desulfo GLS by using the response factors reported in the table above, if you add an internal standard after step A3 (Procedure A), and by applying the following formula:
    µmol     Areadg x RFdg x n
    ------- = ------------------------
    g          Areast x RFst X m
    Wherein:
    Areadg is the peak area of the desulfoglucosinolate, Areast is the peak area of internal standard, RFdg is the response factor of the corresponding desulfoglucosinolate, RFst is the response factor of the internal standard, n is the quantity, in µmol, of the internal standard added to the sample, m is the mass, in g, of the sample.

Recipes

  1. 70% MeOH
    70 ml of MeOH
    30 ml of Milli-Q water
  2. 0.1% Formic acid (HCOOH)
    1 ml of Formic acid
    999 ml of Milli-Q water
  3. Sulfatase type H-1 (1 to 2.5)
    1 ml of sulfatase type H-1
    1.5 ml of Milli-Q water
  4. DEAE-Sephadex A-25 (formiate form)
    Convert the chloride form to the formiate form by adding for each column 500 µl of 6 mM imidazole formiate (dissolve 40 g imidazole in 100 ml 30% formic acid) and then rinsing the column with water.

References

  1. Agneta, R., Lelario, F., De Maria, S., Möllers, C., Bufo S. A. and Rivelli A. R. (2014). Glucosinolate profile and distribution among plant tissues and phenological stages of field-grown horseradish. Phytochem 106: 178-187.
  2. Agneta, R., Rivelli, A. R., Ventrella, E., Lelario, F., Sarli, G. and Bufo, S. A. (2012). Investigation of glucosinolate profile and qualitative aspects in sprouts and roots of horseradish (Armoracia rusticana) using LC-ESI-hybrid linear ion trap with Fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation. J Agric Food Chem 60(30): 7474-7482.
  3. EN ISO 9167-1 (1992). Rapeseed-Determination of glucosinolates content-Part 1: Method using high-performance liquid chromatography.
  4. Möllers, C., Nehlin, L., Glimelius, K. and Iqbal M. C. M. (1999). Influence of in vitro culture conditions on glucosinolate composition of microspore-derived embryos of Brassica napus. Physiol Plantarum 107(4): 441-446.
  5. Sarli, G., De Lisi, A., Agneta, R., Grieco, S., Ierardi, G., Montemurro, F., Negro, D., Montesano, V. (2012). Collecting horseradish (Armoracia rusticana, Brassicaceae): local uses and morphological characterization in Basilicata (Southern Italy). Genet Resour Crop Ev 59(5): 889-899.

简介

硫代葡萄糖苷(GLS)是次要代谢物,主要在属于十字花科的植物中发现,包括辣根(Armoracia rusticana G.Gaertn,B.Mey。&Scherb),由于GLS丰度而具有特征刺激性风味 。 这种化合物表现出抗菌,抗真菌和杀虫活性以及人类健康性质。 因此,对其在植物中的水平和谱的完全理解是非常重要的。 然而,由于植物基质的复杂性和痕量的许多GLS的出现,来自辣根粗提取物的GLS的表征是艰难的任务。 在这里我们描述两种替代的有效和快速的方法在辣根植物的GLS表征:液相色谱耦合高分辨率质谱(LC-MS)测定完整的GLS和HPLC-UV测定脱硫-GLS。

材料和试剂

  1. 辣根组织(下牙和上皮部分)(见注1)
  2. 甲醇(MeOH)(LC/MS级)(Carlo Erba Reagents,目录号:414831)
  3. 乙腈(ACN)(LC/MS级)(Carlo Erba Reagents,目录号:412342)
  4. 超纯Milli-Q水
  5. 液氮
  6. 来自辣根的水合物(99%)(Sigma-Aldrich,目录号:S1647)
  7. 含有葡萄糖酸钙,4-羟基葡萄糖脑苷脂,葡萄糖脑苷脂,葡萄糖脑苷脂和葡萄糖酸钙(Sigma-Aldrich,目录号:ERMBC367)的油菜ERM认证的参考材料。
  8. Glucoiberin(C2Bioengineering,目录号:10-JS 12-05-02)
  9. Glucobarbarin(C2Bioengineering,目录号:18-DM19-10-99)
  10. 葡萄糖降胆胺(C2生物工程,目录号:16-PM 19-10-99)
  11. 70%MeOH(参见配方)

此外仅适用于完整的GLS确定

  1. 甲酸(梯度级)(Sigma-Aldrich,目录号:F0507)
  2. 0.1%甲酸(HCOOH)(参见配方)

此外,仅适用于desulfo-method

  1. 通过使用DEAE-Sephadex A-25(氯化物形式)(Sigma-Aldrich,目录号:A25120)和咪唑(Sigma-Aldrich,目录号:56750)获得的DEAE-Sephadex A-25(甲酸盐形式)
  2. 硫酸酯酶型H-1(Sigma-Aldrich,目录号:S-9626)
  3. 硫酸酯酶型H-1(1到2.5)(参见配方)
  4. DEAE-Sephadex A-25(甲酸盐形式)(参见配方)

设备

  1. 冻干系统(例如Labconco,型号:Freezone 4.5)
  2. 实验室磨机
  3. 一次性50ml和15ml聚丙烯管
  4. 2 ml样品瓶
  5. 水浴:烧杯装满水并置于加热装置(电热板或类似装置)上
  6. 温度计
  7. 涡流搅拌器
  8. 冷冻离心机(50ml管)(例如Heraeus,型号:Varifuge F)
  9. 玻璃巴斯德移液器
  10. 0.22μm尼龙过滤器(Whatman)
  11. 具有光电二极管阵列检测器(例如Agilent,型号:Agilent1200HPLC液相色谱系统)的HPLC系统。
  12. HPLC具有盖(Phenomenex,型号:AR0-8959-13-B)的2ml玻璃小瓶(Phenomenex,型号:AR1-3910-12)
  13. 与电喷雾离子化(ESI)和高分辨率质谱(MS)(例如Thermo Fisher Scientific,型号:LC-ESI-FTICR MS)偶联的液相色谱(LC)
  14. 完整葡糖异硫氰酸盐Discovery C 18柱,250×4.6mm,5μm粒径(孔径,180Å)(Sigma-Aldrich,目录号:504971),Discovery C 18 sub> 20×4mm保安柱(Sigma-Aldrich,目录号:505129)
  15. 脱硫葡糖苷酸酯(Nucleosur C18 C18柱,125mm×3mm)(MACHEREY-NAGEL,目录号:MN760051.30)

程序

完整GLS

  1. GLS提取
    1. 用蒸馏水清洗植物材料,用纸巾擦干。 分离根部与表皮部分,重量并立即冻结 -80℃以抑制黑芥子酶活性
    2. 冻干冻结 组织并使用实验室研磨机研磨成细粉末。 寒冷根 在液氮中冻干前进行粉碎。
    3. 称取200毫克冷冻干材料在50毫升聚丙烯管中   置于用电热板在70-80℃加热的水浴中 10分钟。 在此过程中,执行以下步骤:1分钟后 加入2ml的70%甲醇溶剂,5分钟后用涡旋混合20分钟 秒。
    4. 在10分钟结束时,从水浴中取出聚丙烯管,并再次涡旋混合20秒
    5. 在4℃以2,400×g离心10分钟。
    6. 收集上清液在一次性的15毫升聚丙烯管通过使用玻璃巴斯德移液器
    7. 用2ml 10%甲醇再次萃取剩余的沉淀 用电热板在70-80℃加热水浴10分钟; 后   5分钟混合,涡旋20秒,并遵循相同的程序   步骤A4-6
    8. 合并上清液并旋转提取液20秒。
    9. 过滤通过0.22μm尼龙过滤器,并转移到2ml样品瓶中。

  2. LC MS检测GLS
    1. 在配有LC-ESI-FTICR MS系统的样品中注入25μl样品 Discovery C 18柱,250×4.6mm,5μm粒度(孔径,180μm) Å),Discovery C 18×20×4mm安全保护盒。
      设置:
      流速:1ml/min
      柱温:25℃
      用于色谱分离的溶剂梯度:

      时间(分钟)
      %溶剂A
      0.1%HCOOH水
      %溶剂B
      ACN
      0
      90
      10
      10
      76
      24
      12
      40
      60
      15
      90
      10
      20
      90
      10


      通过实验室制造将LC系统连接到质谱仪 分流器分流比为1:4后分析柱允许 200μl/min,进入ESI源
    2. 执行全扫描实验   在线性陷阱和ICR细胞。 在中收集数据 通过设置:
      ,在 m/z 50-1,000的范围内选择轮廓模式 电离模式:负
      分辨率:100.000(FWHM)在m/z 400
      ESI针电压:-4.60kV
      毛细管电压:-22 V
      加热毛细管的温度:350℃
      护套气体(N 2级)流量:80(任意单位)
      在这些条件下,GLS以保留时间和分子量精确洗脱   质量/电荷比,如下表所示(Agneta等人,   2012; Agneta等人,2014):

      化学名称
      分子式
      单同位素精确值为[M-H] - ( m/z )
      t R
      3-(甲基亚磺酰基)丙基-GLS
      C 11
      422.02549
      4.3
      2-丙烯基-GLS
      C 10 17 NO 9 2
      358.02720
      4.4
      2-甲基磺酰基 - 氧代 - 乙基-GLS C 10 17 NO 3
      437.98402
      4.6
      3-丁烯基-GLS
      C 11
      372.04285
      5.5
      1-甲基丙基-GLS
      C 11
      374.05850
      6.2
      2-甲基丙基-GLS
      C 11
      374.05850
      6.4
      4-Mercaptobuthyl-GLS
      C 11
      406.03057
      6.5
      7-Methylsulfinylheptyl-GLS
      C 15 29 NO 10 S 3
      478.08808
      7.3
      4-羟基吲哚-3-基甲基-GLS
      C 16 20 2 10 2
      463.04866
      7.4
      4-羟基吲哚-3-基甲基-GLS的未知异构体
      C 16 20 2 10 2
      463.04866
      7.5
      2(S) - 羟基-2-苯乙基-GLS
      和/或
      2(R) - 羟基-2-苯乙基-GLS
      C 15 H 21 NO 7 438.05341
      7.8
      4-戊烯基-GLS
      C 12 21 NO 9 2
      386.05850
      7.8
      Benzyl-GLS
      C 14 19 NO 9 2
      408.04285
      8.5
      吲哚-3-基甲基-GLS
      C 16 20 N O 2
      447.05375
      10.3
      2-苯乙基-GLS
      C 15
      422.05850
      12.1
      4-甲氧基吲哚-3-基甲基-GLS
      C 17 22 N O 10 2
      477.06431
      12.7
      7-甲硫基庚基-GLS   
      C 15
      462.09317
      16.7
      注意:可以使用提取物通过准备a来量化GLS   每个GLS的校准曲线使用一系列稀释和 计算每个化合物峰下的面积。


      图  1.使用高分辨率的离子色谱图(XIC)示例 LC-ESI-FTICR MS以A的根粗提取物的负离子模式获得。 。监测的离子显示在每个轨迹中 (图A-O),并且对应于最丰富的去质子化分子, [M-H] - ,使用以0.0010m/z为单位的约束窗口为中心 每个选定的离子。粗体的峰数对应于:(1) 3-(甲基亚磺酰基)丙基-GLS; (2)2-丙烯基-GLS; (3)* 2-甲基磺酰基 - 氧代 - 乙基-GLS; (4)3-丁烯基-GLS; (5)* 1-甲基丙基-GLS; (6)* 2-甲基丙基-GLS; (7)* 4-巯基丁基-GLS; (8)* 7-甲基亚磺酰基庚基-GLS; (9)4-羟基吲哚-3-基甲基-GLS; (10)  4-羟基葡萄糖脑苷脂的未知异构体; (11) 2(S) - 羟基-2-苯乙基-GLS和/或2(R) - 羟基-2-苯乙基-GLS; (12)4-戊烯基-GLS; (13)苄基-GLS; (14)吲哚-3-基甲基-GLS; (15) 2-苯乙基-GLS; (16)4-甲氧基吲哚-3-基甲基-GLS; (17)* 7-甲硫基庚基-GLS(具有星号的峰被暂时分配)。 使用样品提取物获得一些峰的XIC信号 用流动相稀释1:20。 黑体字也表示 分子式和单同位素值的范围为[M-H] - 离子(Δ m/z)(Agneta等人,2014)。

Desulfo GLS

  1. GLS提取
    1. 按照完整GLS步骤A1-8的程序。
    2. 将500μl提取物转移到20mg的甲酸盐形式的Sephadex DEAE-A 25(缩短的巴斯德吸管)的顶部。
    3. 用1ml去离子水洗涤柱子两次
    4.  加入100μl用水稀释至1〜2.5的硫酸酯酶型H-1   脱硫并在39℃(最少16小时)孵育过夜(Mullers等人,1999)。
    5. 用3×500μl去离子水洗脱脱硫GLS,并收集在15ml聚丙烯管中
    6. 涡旋样品20秒。
    7. 过滤通过0.22μm尼龙过滤器,并转移到2ml样品瓶中

  2. 通过HPLC进行GLS检测
    1. 将25μl每种样品注入配备有Nucleodur C18s column,125mm×3mm的HPLC系统中。
      设置:
      流速:0.6ml/min
      柱温:35℃
      紫外检测:229
      用于色谱分离的溶剂梯度:
      时间(分)
      %溶剂A水
      %溶剂B ACN
      0
      99
      1
      20
      80
      20
      25
      80
      20
      22
      99
      1

      在这些条件下,GLS洗脱,保留时间如下表所示:
      化学名称
      t R
      相对响应因子 a
      3-(甲基亚磺酰基)丙基-GLS
      2.8
      1.07
      2-丙烯基-GLS
      4.3
      1.00
      3-丁烯基-GLS
      7.3
      1.11
      1-甲基丙基-GLS和/或
      8.9
      1.00 b
      2-甲基丙基-GLS
      8.9
      1.00 b
      2(S) - 羟基-2-苯乙基-GLS和/或2(R) - 羟基-2-苯乙基-GLS 10.1
      0.98 c
      4-戊烯基-GLS
      10.9
      1.14
      Benzyl-GLS
      11.8
      0.95
      吲哚-3-基甲基-GLS
      13.9
      0.29
      2-苯乙基-GLS
      16.1
      0.95
      4-甲氧基吲哚-3-基甲基-GLS
      16.5
      0.25
      a   用于脱硫硫代葡萄糖异硫氰酸酯的响应因子值 定量,如EN ISO 9167-1(1992)中报告的,除了 随后的脱硫分子:1-甲基丙基-GLS,2-甲基丙基-GLS 和2(S) - 羟基-2-苯乙基-GLS和/或2(R) - 羟基-2-苯乙基-GLS b 2-丙烯基-GLS作为用于相对定量的脱硫分子的响应因子
      c 实验   响应因子通过使用2(S) - 羟基-2-苯乙基-GLS测定   与作为脱硫酸分子的2-丙烯基-GLS相比(参见注释2)

笔记

  1. 植物组织从植物遗传研究所,国家研究委员会,地中海生物多样性保护专题中心,位于波利科罗(MT)(40°17' 30"N,16°65'16"E),其中先前从巴西利卡塔地区内部各个村庄收集的许多辣根种子被保持并且无性繁殖(详情参见Sarli等,/em>,2012)。
  2. 可以使用提取物通过使用一系列稀释和计算每种化合物的峰下面积来制备每个GLS的校准曲线来定量脱硫GLS。
    如果在步骤A3(程序A)后添加内标,并应用以下公式,也可以使用上表中报告的响应因子对脱硫GLS进行定量:
    μmol    区域 dg x RF dg x n
    ------- = ------------------------
    g          Area st x RF st X m
    其中:
    区域dg是去唾液酸葡萄糖苷酸的峰面积,面积是内标的峰面积,RF sub是相应物质的响应因子其中内标物的响应因子,n是加入到样品中的内标物的量,以μmol计,m是样品的质量,g。 br />

食谱

  1. 70%MeOH
    70ml MeOH
    30ml Milli-Q水
  2. 0.1%甲酸(HCOOH)
    1ml甲酸
    999 ml Milli-Q水
  3. 硫酸酯酶H-1(1〜2.5)
    1ml硫酸酯酶型H-1
    1.5ml Milli-Q水
  4. DEAE-Sephadex A-25(甲酸盐形式)
    通过向每个柱中加入500μl的6mM咪唑甲酸盐(将40g咪唑溶解在100ml 30%甲酸中),然后用水冲洗该柱,将氯化物转化为甲酸盐形式。

参考文献

  1. Agneta,R.,Lelario,F.,De Maria,S.,Möllers,C.,Bufo S.A.和Rivelli A.R.(2014)。 硫代葡萄糖苷在野生型辣根的植物组织和物候阶段之间的分布和分布。 em> Phytochem 106:178-187
  2. Agneta,R.,Rivelli,A.R.,Ventrella,E.,Lelario,F.,Sarli,G.and Bufo,S.A。(2012)。 调查辣根芥中的芥子油苷和根的硫代葡萄糖苷特征和定性方面(使用具有傅立叶变换离子回旋共振质谱和红外多光子解离的LC-ESI-混合线性离子阱。使用具有傅立叶变换离子回旋共振质谱和红外多光子解离的LC-ESI-杂化线性离子阱。
  3. EN ISO 9167-1(1992)。 油菜籽 - 硫代葡萄糖苷含量的测定 - 第1部分:使用高效液相色谱法的方法。/a>
  4. Möllers,C.,Nehlin,L.,Glimelius,K。和Iqbal M.C.M。(1999)。 影响体外培养条件 上 (Brassica napus)的小孢子衍生胚胎的芥子油苷组合物。 Physiol Plantarum 107(4):441-446。
  5. Sarli,G.,De Lisi,A.,Agneta,R.,Grieco,S.,Ierardi,G.,Montemurro,F.,Negro,D.,Montesano,V.(2012)。 收集辣根( Armoracia rusticana ,十字花科):local 用途和形态学特征在巴斯利卡塔(意大利南部)。 Genet Resour Crop Ev 59(5):889-899。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lelario, F., De Maria, S., Agneta, R., Mӧllers, C., Bufo, S. A. and Rivelli, A. R. (2015). Glucosinolates Determination in Tissues of Horseradish Plant. Bio-protocol 5(16): e1562. DOI: 10.21769/BioProtoc.1562.
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