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Quantifying Auxin Metabolites in Young Root Tissue of Medicago truncatula by Liquid Chromatography Electrospray-ionisation Quadrupole Time-of-flight (LC-ESI-QTOF) Tandem Mass Spectrometry
采用液相色谱-电喷射电离四极杆时间飞行(LC-SI-QTOF)串联质谱法定量测定蒺藜苜蓿幼苗根部中的生长素代谢物   

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Abstract

Auxins represent a major group of phytohormones controlling plant development. The spatio-temporal regulation of auxin gradients is essential for the initiation, growth and correct development of plant organs. Because auxins and their metabolites occur at trace levels in plant tissue, experiments requiring identification plus their selective and specific quantification can be most conveniently achieved using mass spectrometry (MS) and the associated chromatographic methods. With the advent of appropriate liquid-based ionisation techniques, emphasis has moved from the use of gas chromatography as the sample interface to the MS (GC/MS), with its concomitant need for derivatisation, to the more sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS). We describe an optimized liquid chromatography electrospray-ionisation quadrupole time-of-flight (LC-ESI-QTOF) methodology for the quantification of auxins. While the solvent extraction of young Medicago truncatula (M. truncatula) roots, as described herein, is relatively straightforward, older, woody or oily plant tissues may also be analyzed with appropriate modification to remove interferences and/or enhance extraction efficiency. In our hands, the analytical assay has proved sufficiently sensitive for the quantification of auxins to investigate their roles in various organogenic events, such as root nodulation in M. truncatula. Further increases in sensitivity can be expected with the use of the latest generation of instruments.

Keywords: Auxin(生长素), Nodulation(结瘤), Liquid chromatography(液相色谱法), Quadrupole time-of-flight(四极杆飞行时间), Masshunter(MassHunter)

Materials and Reagents

  1. Stainless steel balls, 3 mm diameter, sequentially washed with detergent, rinsed with tap water, Milli-Q water, and then sterilized in ethanol overnight prior to being air dried before use (AussieSapphire)
  2. 2 ml sterile non-autoclaved Eppendorf tubes (Sigma-Aldrich, catalog number: T2795 )
  3. 1.5 ml sterile non-autoclaved microtubes (Sigma-Aldrich, catalog number: SIAL311NZ1.5C )
  4. Nanosep® MF GHP 0.45 μm filter (Pall Life Sciences, catalog number: ODGHPC35 )
    Note: Currently, it is “VWR International, catalog number: ODGHPC35”.
  5. 2 ml amber glass autosampler vials (Pacific lab, catalog number: SV11AW )
  6. 250 μl inserts (Pacific lab, catalog number: IST0925N )
  7. 1 L Schott bottles (acid washed)
  8. Medicago truncatula seeds
  9. Liquid N2
  10. Methanol (99.8%) HPLC grade (Thermo Fisher Scientific, ACROS OrganicsTM, catalog number: 413770025 )
  11. Propanol, HPLC grade (Fisher Scientific, catalog number: A461-212 )
  12. Glacial acetic acid, HPLC grade (Fisher Scientific, catalog number: A113-50 )
  13. Formic acid, HPLC grade (Fisher Scientific, catalog number: A117-50 )
  14. Milli-Q water
  15. Ultra high purity N2 gas
  16. Indole-2, 4, 5, 6, 7-d5-3-acetic acid, D5-IAA (Cambridge Isotope Laboratories, catalog number: DLM-2926 )
    Note: It is also named “Indole-3-acetic acid (indole-D5, 97-98%)” on Cambridge Isotope Laboratories website.
  17. Indole-3-acetic acid, IAA (Sigma-Aldrich, catalog number: I3750 )
  18. Indole-3-butyric acid, IBA (Sigma-Aldrich, catalog number: I5386 )
  19. 4-chloro-indole-3-acetic acid, 4-Cl-IAA (OlChemIm Ltd., catalog number: 0031131 )
  20. Phenylacetic acid, PAA (Sigma-Aldrich, catalog number: P16621 )
  21. N-(3-indolylacetyl)-L-alanine, IAA-Ala (Sigma-Aldrich, catalog number: 345911 )
  22. N-(3-indolylacetyl)-DL-aspartate, IAA-Asp (Sigma-Aldrich, catalog number: 345938 )
  23. N-(3-indolylacetyl)-L-isoleucine, IAA-Ile (Sigma-Aldrich, catalog number: 347914 )
    Note: This product has been discontinued.
  24. N-(3-indolylacetyl)-L-leucine, IALeu (OlChemIm Ltd., catalog number: 0031611 )
  25. N-(3-Indolylacetyl)-L-phenylalanine, IAPhe (OlChemIm Ltd., catalog number: 0031623 )
  26. N-(3-indolylacetyl)-L-tryptophan, IATrp (OlChemIm Ltd., catalog number: 0031631 )
  27. N-(3-indolylacetyl)-L-valine, IAVal (OlChemIm Ltd., catalog number: 0031641
    Notes:
    1. Solvents and chemicals should be of the highest available purity.
    2. Auxin standards and internal standards are prepared in HPLC grade methanol. 1,000 mg/L stock solutions are prepared using acid washed volumetric flasks and aliquoted (1 ml) into 2 ml amber glass vials with crimped caps and stored at -80 °C until use.

Equipment

  1. TissueLyser LT (QIAGEN, catalog number: 69980 )
  2. Sonicator bath (Cole-Parmer Instrument Company, model: 8545-4 )
  3. SpeedVac vacuum centrifuge (LabConco)
  4. Agilent 6530 High Resolution Accurate Mass LC-MS Q-TOF with Agilent Jetstream (AJS) ESI ion source interface
  5. Agilent Zorbax Eclipse high resolution XDB-C18 2.1 x 50 mm, 1.8 μm LC column

Software

  1. Agilent MassHunter software version B.05.00 for data acquisition and data analysis

Procedure

  1. This analytical assay can be applied to fresh frozen root tissue grown on plate media or soil. For our experiments, germinated M. truncatula seedlings (previously germinated overnight on water agar in a Petri dish) are grown on Fåhraeus media in Petri dishes for four days (Ng et al., 2015). Subsequently several analyses can thus be undertaken:
    1. To investigate the early stages of nodulation, a 4 mm segment around the inoculation site (not including the tip) is harvested.
    2. To determine auxin concentrations in nodules, whole root nodules are harvested.
    3. To measure the total root auxin concentration to compare between plant genotypes, species, etc., whole root tissues are harvested.
  2. M. truncatula roots are excised, collected in sterile, non-autoclaved 2 ml Eppendorf tubes and immediately snap-frozen in liquid N2. For auxin quantification in M. truncatula roots, 50-100 mg of tissue is sufficient. Frozen root tissues are crushed with stainless steel balls in a TissueLyser LT with a pre-cooled sample holder (stored at -20 °C).
    Notes:
    1. The stainless steel balls are left in the 2 ml Eppendorf tubes throughout the solvent extraction protocol.
    2. The TissueLyser LT frequency is set no higher than 40-45 Hz and for one min to sufficiently grind the young root tissue. Extended time may be necessary for tougher and/or older tissue.
    3. It is pertinent that the tissue is completely ground to maximize the solvent extraction efficiency for auxins (Figure 1).


      Figure 1. An example of Medicago truncatula root tissue before and after grinding by a stainless steel ball in a TissueLyser LT. Complete grinding of the tissue is essential for optimal metabolite extraction (i.e., tissue should be of a powder consistency).

  3. 20 ng of the internal standard (D5-IAA) is aliquoted to each sample and allowed to be absorbed by the root sample matrix (samples are always kept on dry ice).
    Notes:
    1. To add 20 ng internal standard, accurately pipette 20 μl of 1 mg/L internal standard stock solution to each sample.
    2. If dry ice is not available, keep samples on ice. The same applies to step 4.
  4. Next, 600 μl of the extraction solvent comprising of 20:79:1 methanol:propanol:glacial acetic acid (v/v/v) is aliquoted to each sample (samples still kept on dry ice or ice) and then vortexed vigorously for 5 sec.    
    Note: The extraction solvent is prepared by measuring volumes of the specified organic solvents above using an acid-washed 100 ml measuring cylinder. The solution is then transferred to an acid-washed 100 ml Schott bottle for safer storage. It is recommended that the extraction solution is made fresh prior to any new batch extraction to eliminate possible cross contamination.
  5. Auxin metabolites are extracted in an ultrasonic bath for 30 min at 4 °C.
  6. Samples are centrifuged at 16,100 x g for 15 min at 4 °C.
  7. The supernatant from each sample is transferred into new, sterile, non-autoclaved 1.5 ml microtubes.
  8. Repeat steps 4-6 and combine the supernatant from this second extraction with the first. The combined extract is reduced to dryness in a vacuum centrifuge (30 °C, ~20 min; Figure 2A).  
  9. Add 100 μl of methanol to each sample tube and then vortex vigorously, ensuring that the methanol comes into contact with the entire inner wall of the tube so that the auxin extract is adequately resuspended prior to transferring the solution to a Nanosep MF GHP 0.45 μm filter centrifugal device (Figure 2B) for sample filtration. An additional 100 μl of methanol is added into each sample tube, vortexed, and the solution combined in the corresponding Nanosep MF GHP 0.45 μm filter centrifugal device.  Samples are centrifuged at 16,100 x g for 1 min at room temperature to remove particulate matter that could otherwise block and dirty the LC narrow bore sample and sample lines; ESI nebuliser and chamber; and the Q-TOF glass capillary transfer line and skimmer.


    Figure 2. Consumables and equipment used for the extraction and quantification of auxins. A. Vacuum centrifuge used to concentrate the auxin and auxin metabolites in the samples by evaporating off the excess solvent. B. Centrifugal device used to filter samples prior to analysis. C. Amber autosampler glass vials with inserts used for the final resuspension of auxins prior to analysis. D. Agilent 6530 HPLC setup. E. Agilent High Resolution Accurate Mass LC-ESI-QTOF setup.

  10. The filtrate (containing soluble auxins) is transferred into amber (to minimise photo-oxidative degradation, as auxins are light sensitive) glass autosampler vials with 250 μl inserts (Figure 2C). Samples are reduced to dryness in a vacuum centrifuge and resuspended at a smaller volume (e.g., 50 μl) of methanol/water mixture (60:40, v/v) prior to analysis.
    Note: The vacuum centrifuge is set at 30 °C. For a 200 μl mixture, approximately 20 min is required to dry the samples.   
  11. Extracted auxins are analyzed immediately with the optimized LC-ESI-QTOF (Figure 2D-E). Alternatively, auxin extracts can be dried (freeze-dried or vacuum centrifuged) and stored for up to two weeks at -80 °C under an inert atmosphere (high purity N2 or Ar gas) prior to analysis.
  12. The concentrated, resuspended auxin extracts are injected (7 μl) onto an Agilent Zorbax Eclipse high resolution XDB-C18 2.1 x 50 mm, 1.8 μm LC column interfaced to an Agilent 6530 High Resolution Accurate Mass LC-ESI-MS Q-TOF system (Figure 2D-E). Solvent A consists of 99.9% water:0.1% formic acid and solvent B consists of 90% methanol:9.9% water:0.1% formic acid. Auxins are eluted from the column at a flowrate of 200 μl min-1 using the linear gradient described in Table 1.

    Table 1. Optimized linear gradient used for the elution of auxin metabolites 


  13. Samples are subjected to electrospray ionisation in both positive and negative ion polarities (greater instrument sensitivity was achieved for IAA, IBA and IAA-Ala in the positive ion mode. Other auxin metabolites were better detected in the negative ion mode). Optimized electrospray ionisation conditions are described in Table 2.

    Table 2. Optimized electrospray ionisation conditions in both positive and negative ion polarities


  14. The Q-TOF is run in targeted MS/MS mode (developed specifically for quantification purposes) with collision-induced dissociation (N2 collision gas supplied at 18 psi) and a 1.3 m/z (mass-to-charge) isolation window. The Q-TOF is run in an extended dynamic range (2 Hz) with the MS mode set at 100-1,000 m/z at an acquisition rate of 3 spectra s-1, whereas the MS/MS mode at 50-1,000 m/z and at an acquisition rate of 3 spectra s-1 (Ng et al., 2015). Optimized collision energies and signature product ions for individual auxin analytes are listed in Table 3. Note that these auxin metabolites are targeted in our experiments because they were hypothesized to be present in M. truncatula. For the quantification of auxins in other plant tissues, species or to answer other biological questions, additional auxin or auxin-like compounds could also be targeted using this MS/MS acquisition method.

    Table 3. Optimized collision energy and signature product ions for individual analytes in the positive (upper panel) and negative (lower panel) ion modes. Extracted from Ng et al. (2015).


  15. Data are analyzed with the Agilent MassHunter software version B.05.00. For each run, authentic calibration and quality control (QC) reference standards with internal standard (D5-IAA) are used for unbiased identification and quantification of auxin metabolites in real samples. The retention time, precursor and signature product ions of authentic standards are used to confirm putative positive hits in real samples.
    An example is given below in Figure 2 (using Extracted Ion Chromatogram (EIC) in the MassHunter software). Authentic standards are always run in conjunction with real samples as a quality control check for system performance and to account for any minor change in parameters (e.g., retention time shifts) that might occur between experiments resulting from possible HPLC column batch variation, dirty LC-MS and/or mobile phase preparation.
    Note: The auxin QC standard mixture is prepared with all auxin compounds at 1 mg/L and internal standard at 0.4 mg/L.    
  16. The auxins and auxin metabolites would ideally be quantified against their own stable isotope (e.g., 13C- or 2H-labelled internal standard). However, these are often either not available or prohibitively expensive. While not yielding ideal, absolute quantitative values, the use of a related internal standard (in this case 2H5-IAA), will readily enable quantitative comparisons between different samples.
  17. The auxins and auxin metabolites are validated over a calibration range (Ng et al., 2015) with the internal standard fixed at a known concentration. Calibration curves are graphed for each analyte to derive linear regression equations and correlation coefficients for quantification, and to ascertain their respective limits of detection (LODs) and lower limits of quantification (LLOQs).
  18. To calculate relative concentration for each auxin metabolite (e.g., ng/g tissue), the following equation was used:

    The extraction ion corresponds to the most abundant product ion (quant ion) for each analyte (see Figure 3). The peak area is divided by extracted quant ion peak area for the internal standard to obtain an area ratio. 20 ng is the concentration of the internal standard added to each root tissue sample. Concentrations are reported on a fresh weight basis, but can also be expressed as dry weight if necessary.


    Figure 3. Identification of IAA in a Medicago truncatula root sample. Authentic IAA standard (A, C) is used as a reference to compare with a putative IAA hit in a real sample (B, D). The retention time of the IAA compound identified in the sample (B) is close to that of the standard (A). The precursor and product ions of the putative IAA compound in the sample (D) closely match those found in the authentic IAA standard (C). The fragmentation product ion m/z 130.06 is used as the quantification (quant) ion as it is most abundant and the other signature product ions are qualifier (confirmation) ions.

Acknowledgments

This protocol was adapted from Müller and Munné-Bosch (2011) and Buer et al. (2013) and was performed by Ng et al. (2015). This work was supported by an Australian Research Council Future Fellowship awarded to Ulrike Mathesius (FT100100669).

References

  1. Buer, C. S., Kordbacheh, F., Truong, T. T., Hocart, C. H. and Djordjevic, M. A. (2013). Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture. Planta 238 (1): 171-189.
  2. Muller, M. and Munné-Bosch, S. (2011). Rapid and sensitive hormonal profiling of complex plant samples by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Plant Methods 7: 37.
  3. Ng, J. L., Hassan, S., Truong, T. T., Hocart, C. H., Laffont, C., Frugier, F. and Mathesius, U. (2015). Flavonoids and auxin transport inhibitors rescue symbiotic nodulation in the Medicago truncatula cytokinin perception mutant cre1. Plant Cell 27(8): 2210-2226.

简介

生长素运输能力的测量提供了涉及植物中生长素运输的生理机制的定量数据。 这种技术很容易执行,并给出快速的结果。 将放射性标记的生长素(吲哚-3-乙酸)通过琼脂块加入到Medic藜苜蓿的根部。 使用液体闪烁计数器测量根中放射性标记的生长素摄取的最终放射性。 在这里,我们描述了生长素运输能力周围的结瘤敏感区在年轻幼苗根的测量。 truncatula 响应根瘤菌接种。 类似的测定可以适用于其他植物物种并回答其他生物学问题。...

关键字:生长素, 结瘤, 液相色谱法, 四极杆飞行时间, MassHunter

材料和试剂

  1. 不锈钢球,直径为3mm,依次用洗涤剂洗涤,用自来水,Milli-Q水漂洗,然后在乙醇中灭菌过夜,然后在使用前空气干燥(澳大利亚沙飞)。
  2. 2ml无菌非高压灭菌Eppendorf管(Sigma-Aldrich,目录号:T2795)
  3. 1.5ml无菌非高压灭菌微管(Sigma-Aldrich,目录号:SIAL311NZ1.5C)
  4. Nanosep MF GHP0.45μm过滤器(Pall Life Sciences,目录号:ODGHPC35)
    注意:目前,它是"VWR国际,目录号:ODGHPC35"。
  5. 2 ml琥珀色玻璃自动进样器小瓶(Pacific lab,目录号:SV11AW)
  6. 250μl插入物(Pacific lab,目录号:IST0925N)
  7. 1升Schott瓶(酸洗)
  8. Medic属truncatula 种子
  9. 液体N <2>
  10. 甲醇(99.8%)HPLC级(Thermo Fisher Scientific,ACROS Organics TM ,目录号:413770025)
  11. 丙醇,HPLC级(Fisher Scientific,目录号:A461-212)
  12. 冰醋酸,HPLC级(Fisher Scientific,目录号:A113-50)
  13. 甲酸,HPLC级(Fisher Scientific,目录号:A117-50)
  14. Milli-Q水
  15. 超高纯N 2气体
  16. 吲哚-2,4,5,6,7-d5-3-乙酸,D5-IAA(Cambridge Isotope Laboratories,目录号:DLM-2926)
    注意:在剑桥同位素实验室网站上也称为"吲哚-3-乙酸(吲哚-D5,97-98%)"。
  17. 吲哚-3-乙酸,IAA(Sigma-Aldrich,目录号:I3750)
  18. 吲哚-3-丁酸,IBA(Sigma-Aldrich,目录号:I5386)
  19. 4-氯 - 吲哚-3-乙酸,4-Cl-IAA(OlChemIm Ltd.,目录号:0031131)
  20. 苯乙酸,PAA(Sigma-Aldrich,目录号:P16621)
  21. N-(3-吲哚基乙酰基)-L-丙氨酸,IAA-Ala(Sigma-Aldrich,目录号:345911)
  22. N-(3-吲哚基乙酰基)-DL-天冬氨酸,IAA-Asp(Sigma-Aldrich,目录号:345938)
  23. N-(3-吲哚基乙酰基)-L-异亮氨酸,IAA-Ile(Sigma-Aldrich,目录号:347914)
    注意:此产品已停产。
  24. N-(3-吲哚基乙酰基)-L-亮氨酸,IALeu(OlChemIm Ltd.,目录号:0031611)
  25. N-(3-吲哚基乙酰基)-L-苯丙氨酸,IAPhe(OlChemIm Ltd.,目录号:0031623)
  26. N-(3-吲哚基乙酰基)-L-色氨酸,IATrp(OlChemIm Ltd.,目录号:0031631)
  27. N-(3-吲哚基乙酰基)-L-缬氨酸,IAVal(OlChemIm Ltd.,目录号:0031641)< br /> 注意:
    1. 溶剂和化学品应具有最高的可用纯度
    2. 在HPLC级甲醇中制备生长素标准品和内标。 使用酸洗的容量瓶制备1,000mg/L储备溶液,并等分(1ml)到2ml琥珀色玻璃小瓶并置于-80°C储存,直到使用。

设备

  1. TissueLyser LT(QIAGEN,目录号:69980)
  2. 超声波浴(Cole-Parmer Instrument Company,型号:8545-4)
  3. SpeedVac真空离心机(LabConco)
  4. 使用Agilent Jetstream(AJS)ESI离子源接口的Agilent 6530高分辨率精确质量LC-MS Q-TOF
  5. Agilent Zorbax Eclipse高分辨率XDB-C18 2.1 x 50 mm,1.8μmLC色谱柱

软件

  1. 用于数据采集和数据分析的Agilent MassHunter软件版本B.05.00

程序

  1. 该分析测定可以应用于在平板培养基或土壤上生长的新鲜冷冻根组织。 对于我们的实验,发芽的M. truncatula 幼苗(以前发芽过夜水琼脂在培养皿中)在​​培养皿中的Fåhraeus培养基上生长四天(Ng等人,2015)。随后可以进行若干分析:
    1. 为了研究结瘤的早期阶段,在接种部位周围(不包括尖端)收获4mm的区段。
    2. 为了确定结节中的生长素浓度,收获整根根瘤
    3. 为了测量总根生长素浓度以在植物基因型,物种之间进行比较等,收获全部根组织。
  2. M。切下根剪毛,收集在无菌,非高压灭菌的2ml Eppendorf管中,并立即在液氮中快速冷冻。对于在M中的生长素定量。 truncatula根,50-100mg组织就足够了。在具有预冷却样品架的TissueLyser LT(储存在-20℃下)中用不锈钢球粉碎冷冻的根组织。
    注意:

    1. 在不锈钢球放在2ml Eppendorf管中
    2. 将TissueLyser LT频率设置为不高于40-45Hz,并且1分钟以充分研磨年轻根组织。延长的时间对于更坚韧和/或更老的组织可能是必要的。
    3. 相关地,组织被完全研磨以最大化对于生长素的溶剂提取效率(图1)。


      图1.在TissueLyser LT中通过不锈钢球研磨之前和之后的疣状Medic根根组织的实例。完全研磨组织是至关重要的最佳代谢物提取(组织应具有粉末稠度)
  3. 将20ng的内标(D5-IAA)等分到每个样品中并使其被根样品基质吸收(样品总是保持在干冰上)。
    注意:
    1. 要添加20 ng内标,准确吸取20μl1 mg/L内标原液至每个样品。
    2. 如果没有干冰,请将样品保存在冰上。这同样适用于步骤4.
  4. 接下来,将600μl包含20:79:1甲醇:丙醇:冰醋酸(v/v/v)的提取溶剂等分到每个样品(样品仍保持在干冰或冰上),然后剧烈涡旋, 5秒。    
    注意:提取溶剂通过使用酸洗的100ml量筒测量上述指定有机溶剂的体积来制备。然后将溶液转移到酸洗涤的100ml Schott瓶中以更安全地储存。建议在任何新的批次萃取之前将萃取溶液制成新鲜的,以消除可能的交叉污染。
  5. 生长素代谢物在超声波浴中在4℃下提取30分钟。
  6. 将样品在4℃下以16,100×g离心15分钟。
  7. 将来自每个样品的上清液转移到新的,无菌的,非高压灭菌的1.5ml微管中。
  8. 重复步骤4-6,并将第二次提取的上清液与第一次合并。将合并的提取物在真空离心机(30℃,〜20分钟;图2A)中减少至干。
  9. 向每个样品管中加入100μl甲醇,然后剧烈涡旋,确保甲醇与管的整个内壁接触,使生长素提取液充分重悬,然后将溶液转移到Nanosep MF GHP 0.45μm过滤器离心装置(图2B)用于样品过滤。将另外100μl甲醇加入每个样品管中,涡旋,并将溶液合并在相应的Nanosep MF GHP0.45μm过滤器离心装置中。将样品在室温下以16,100×g离心1分钟以除去否则会堵塞和弄脏LC窄孔样品和样品管的颗粒物质; ESI雾化器和室;和Q-TOF玻璃毛细管传输线和分离器

    图2.用于提取和定量生长素的消耗品和设备 A.真空离心机用于通过蒸发掉多余的溶剂来浓缩样品中的生长素和生长素代谢物。 B.用于在分析前过滤样品的离心装置。 C. Amber自动进样器玻璃小瓶,其具有用于在分析之前最终再悬浮生长素的插入物。 D.安捷伦6530 HPLC设置。 E.安捷伦高分辨率精确质量LC-ESI-QTOF设置
  10. 将滤液(含有可溶性植物生长素)转移到具有250μl插入物的琥珀色(以使光氧化降解最小化,因为植物生长素是光敏感的)玻璃自动进样器小瓶中(图2C)。在分析前,将样品在真空离心机中减少至干燥,并在较小体积(例如,50μl)的甲醇/水混合物(60:40,v/v)中重悬。
    注意:真空离心机设置为30°C。对于200μl混合物,需要约20分钟来干燥样品。   
  11. 立即用优化的LC-ESI-QTOF分析提取的生长素(图2D-E)。或者,可以在分析之前将生长素提取物干燥(冷冻干燥或真空离心)并在惰性气氛(高纯度N 2或Ar气)下在-80℃下储存长达两个星期。
  12. 将浓缩的,重悬浮的生长素提取物注射(7μl)到Agilent Zorbax Eclipse高分辨率XDB-C182.1x50mm,1.8μmLC柱上,连接到Agilent 6530高分辨率精确质量LC-ESI-MS Q-TOF系统图2D-E)。溶剂A由99.9%水:0.1%甲酸组成,溶剂B由90%甲醇:9.9%水:0.1%甲酸组成。使用表1中所述的线性梯度,以200μlmin -1 -1的流速从柱中洗脱出营养素。

    表1.用于洗脱生长素代谢物的优化线性梯度</strong>


  13. 样品在正离子和负离子极性下进行电喷雾电离(在阳离子模式下获得更高的IAA,IBA和IAA-Ala的仪器灵敏度。在负离子模式中更好地检测到其他生长素代谢物)。优化的电喷雾电离条件在表2中描述
    表2.正离子和负离子极性下的优化电喷雾电离条件


  14. 以碰撞诱导解离(在18psi下供应的N 2>碰撞气体)和1.3m/z的目标MS/MS模式(专门为了定量目的开发)运行Q-TOF。 (质荷比)隔离窗口。 Q-TOF在扩展的动态范围(2Hz)中运行,MS模式设置为100-1,000m/z,采集速率为3个光谱s -1 ,2015)。对于单个生长素分析物优化的碰撞能量和特征产物离子列于表3中。注意,这些生长素代谢物在我们的实验中是靶向的,因为它们被假定存在于M中。为了定量其他植物组织,物种中的植物生长素或回答其他生物学问题,还可以使用此MS/MS采集方法靶向额外的生长素或生长素样化合物。

    表3.在正离子模式(上图)和负离子模式(下图)中针对单个分析物优化的碰撞能量和特征产物离子。从Ng em等提取2015)。


  15. 使用Agilent MassHunter软件版本B.05.00分析数据。对于每次运行,使用内部标准(D5-IAA)的可靠校准和质量控制(QC)参考标准用于实际样品中生长素代谢物的无偏的鉴定和定量。保留时间,可靠标准的前体和特征产物离子用于确认实际样品中的假定阳性命中。
    下面在图2中给出了一个实例(使用MassHunter软件中的提取离子色谱图(EIC))。真实标准总是与真实样品一起运行,作为系统性能和质量控制检查说明由于可能的HPLC柱批次变化,脏LC-MS和/或流动相制备而导致的实验之间可能发生的参数(例如,,保留时间偏移)的任何微小变化。
    注意:生长素QC标准混合物是用所有生长素化合物以1mg/L和内标为0.4mg/L制备的。  </em&
  16. 生长素和生长素代谢物将理想地针对其自身的稳定同位素(例如,13 C或2 H标记的内标)定量。然而,这些通常不可用或过于昂贵。尽管不产生理想的绝对定量值,但是使用相关内标(在这种情况下为H 2 H 5 -IAA)将容易地实现不同样品之间的定量比较。
  17. 生长素和生长素代谢物在内标固定在已知浓度的校准范围内验证(Ng等人,2015)。为每个分析物绘制校准曲线以得到用于定量的线性回归方程和相关系数,并确定它们各自的检测限(LOD)和下限定量(LLOQ)。
  18. 为了计算每种生长素代谢物(例如ng/g组织)的相对浓度,使用以下等式:

    提取离子对应于每种分析物的最丰富的产物离子(定量离子)(参见图3)。将峰面积除以内标的提取的离子峰面积以获得面积比。 20ng是加入每个根组织样品的内标的浓度。浓度以鲜重为基础报告,但如果必要,也可以表示为干重

    图3.在苜蓿根瘤样本中鉴定IAA 。使用真实的IAA标准(A,C)作为参考,与真实的IAA命中比较样品(B,D)。样品(B)中鉴定的IAA化合物的保留时间接近于标准物(A)的保留时间。样品(D)中推定的IAA化合物的前体和产物离子与在真实IAA标准(C)中发现的那些紧密匹配。碎片产物离子m/z 130.06用作定量(定量)离子,因为它是最丰富的,而其他标记产物离子是限定(确认)离子。

致谢

该方案改编自Müller和Munné-Bosch(2011)和Buer等人(2013),并由Ng等人(2015)进行。这项工作是由澳大利亚研究委员会未来奖学金授予Ulrike Mathesius(FT100100669)支持的。

参考文献

  1. Buer,CS,Kordbacheh,F.,Truong,TT,Hocart,CHand Djordjevic,MA(2013)。  透明睾丸突变体中黄酮类积累模式的改变会干扰生长素传输,重力反应,并对根和枝条结构产生长期影响。 (1):171-189。
  2. Muller,M。和Munné-Bosch,S.(2011)。  通过与电喷雾电离串联质谱联用的液相色谱法对复杂植物样品进行快速且敏感的激素谱分析。 植物方法 7:37.
  3. Ng,JL,Hassan,S.,Truong,TT,Hocart,CH,Laffont,C.,Frugier,F。和Mathesius,U。(2015)。  黄酮和植物生长素转运抑制剂挽救Medic属truncatula细胞分裂素感知突变em1的共生结瘤/em>。植物细胞 27(8):2210-2226。
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引用:Ng, J. L., Truong, T. T., Hocart, C. H. and Mathesius, U. (2016). Quantifying Auxin Metabolites in Young Root Tissue of Medicago truncatula by Liquid Chromatography Electrospray-ionisation Quadrupole Time-of-flight (LC-ESI-QTOF) Tandem Mass Spectrometry. Bio-protocol 6(12): e1843. DOI: 10.21769/BioProtoc.1843.
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