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Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity
非洲爪蟾卵母细胞联合液相色谱-质谱法的吸收实验检测转运活性   

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Abstract

Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by spectrofluorometric quantification. These techniques are, however, limited to substrates that are available as radiolabeled isotopes or to when the substrate is fluorescent. This prompted us to develop a transport assay where we could in principle detect transport activity for any organic metabolite regardless of its availability as radiolabeled isotope or fluorescence properties.

In this protocol we describe the use of X. laevis oocytes as a heterologous host for expression of secondary active transporters and how to perform uptake assays followed by detection and quantification of transported metabolites by liquid chromatography-mass spectrometry (LC-MS). We have successfully used this method for identification and characterization of transporters of the plant defense metabolites called glucosinolates and cyanogenic glucosides (Jørgensen et al., 2017), however the method is usable for the characterization of any transporter whose substrate can be detected by LC-MS.

Keywords: Xenopus laevis oocytes(非洲爪蟾卵母细胞), Uptake assays(吸收实验), Transporter characterization(转运蛋白功能研究), Liquid chromatography-mass spectrometry(液相色谱 - 质谱)

Background

Oocytes from the African clawed frog (Xenopus laevis) is a well-established expression system for heterologous expression and characterization of membrane proteins (i.e., transporters and channels). The X. laevis oocyte express few endogenous membrane proteins and has a low background transport activity. Furthermore, secondary active transporters from plants (Boorer et al., 1992; Theodoulou and Miller, 1995; Nour-Eldin et al., 2006), animals (Sumikawa et al., 1981; Sigel, 1990) and microbes (Calamita et al., 1995; Wahl et al., 2010) have been successfully expressed in X. laevis oocytes, showing that this system is widely applicable to characterize transporters from any organism.

A transport assay requires the expression of the transport protein in a system capable of folding the protein correctly and localizing it to a membrane across which movement of substrate can be detected. Due to the minute amounts moved, researchers have typically used radiolabeled substrates for transport assays. By washing oocytes after incubation and scintillation counting of the oocytes interior accumulation of substrate inside the oocyte could be detected. We have previously utilized this method to identify and characterize sucrose and glucose transporters from Arabidopsis thaliana using the Xenopus oocytes system (Nour-Eldin et al., 2006; Norholm et al., 2006). However, for identification and characterization of plant specialized metabolite transporters, it can be very challenging to generate radiolabeled isotopes of a target substrate. To overcome this challenge we developed a protocol for detecting and quantifying transport of specialized metabolites into X. laevis oocytes by use of LC-MS. Use of this method has allowed us to expand the inventory of assayable substrates to anything that can be detected and quantified by the LC-MS system applied.

Materials and Reagents

  1. Pipette filter tips (e.g., Biotix, catalog numbers: M-0012-9FC , M0020-9FC , M-0300-9FC , M-1250-9FC96 )
  2. Petri dishes for oocyte washing (e.g., 90 mm diameter, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 263991 )
  3. 24-well NuncTM cell-culture dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
  4. Pasteur pipette for oocyte handling
  5. 1.5 ml tubes (e.g., 1.5 ml microfuge tubes, ‘Easy Fit’, Almeco, catalog number: 02.023.01001 )
  6. 0.22 μm PVDF-based filter plate (EMD Millipore, catalog number: MSGVN2250 )
  7. LC-MS vials
  8. Oocytes expressing transporter(s) of interest and water-injected control oocytes (see Jørgensen et al., 2016 for protocols on making and injecting cRNA and handling oocytes)
    Notes:
    1. A high-affinity glucosinolate transporter from Arabidopsis thaliana, ARABIDOPSIS THALIANA GLUCOSINOLATE TRANSPORTER-1 (AtGTR1) (UniProt, catalog number: Q944G5), is used as an example here.
    2. Oocytes can be purchased from Ecocyte-biosciences (http://ecocyte-us.com/).
  9. Substrate
    Note: We use 4-methylthiobutyl glucosinolate (4MTB) and 3-indolylmethylglucosinolate (I3M) obtained from C2 Bioengineering (http://www.glucosinolates.com/) and CFM Oskar Tropitzsch GmbH, Marktredwitz (http://www.cfmot.de/), respectively. Cyanogenic glucoside linamarin can be purchased from Santa Cruz Biotechnology.
  10. Methanol for HPLC ≥99.9% (Sigma-Aldrich, catalog number: 34860 )
  11. Formic acid, reagent grade (Sigma-Aldrich, catalog number: F0507 )
  12. Acetonitrile for HPLC (Sigma-Aldrich, catalog number: 34851 )
  13. Sodium chloride (NaCl) (Duchefa Biochemie, catalog number: S0520.5000 )
  14. Potassium chloride (KCl) (Merck, catalog number: 1.04936.1000 )
  15. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  16. Calcium chloride dihydrate (CaCl2·2H2O) (EMD Millipore, catalog number: 1.02382 )
  17. HEPES (Sigma-Aldrich, catalog number: H4034 )
  18. Tris-HCl solutions (Trisma hydrochloride) (Sigma-Aldrich, catalog number: T3253 )
  19. MES (Sigma-Aldrich, catalog number: M8250 )
  20. Kulori media (pH 7.4) (see Recipes)
  21. Kulori media (pH 5.6) (see Recipes)

Equipment

  1. 17 °C incubator for oocyte storage (BINDER, model: Model KB 23 )
  2. Pipettes (10 µl, 50 µl, 200 µl and 1,000 µl)
  3. Table top centrifuge for 1.5 ml microfuge tubes (e.g., Ole Dich Instrumentmakers, model: Ole Dich 157 , max speed 20,000 x g, refrigerated)
  4. Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 μm, 100 Å) (Phenomenex, catalog number: 00D-4499-AN )
  5. LC-MS Triple Quadrupole system for sample analysis/data acquisition (Advance UHPLC coupled to an EVOQ Elite Triple Quadrupole mass spectrometer) (Bruker, model: EVOQ EliteTM Triple Quadrupole )

Software

  1. Microsoft Excel for data analysis and presentation
  2. Bruker MS Workstation software (Version 8.2, Bruker, Bremen, Germany)

Procedure

In the following, we will provide an example of a typical assay where we test the uptake activity of a given transport protein towards glucosinolates. We test seven oocytes that have been injected with cRNA for our transporter of interest and seven oocytes which have been injected with water to be used as negative control. A set of negative control oocytes should be included for every compound to be tested.

  1. Assay preparation
    1. Prepare seven oocytes expressing the transporter of interest (injected with Complimentary RNA (cRNA) [25-50 ng] and incubated at 17 °C for 72 h prior) and seven oocytes that have been injected with water to be used as control oocytes (and incubated at 17 °C for 72 h prior). Injection volume is typically 50 nl.
      Note: See Jørgensen et al., 2016 for a detailed protocol for cRNA generation and injection and for oocyte handling from injection to assay.
    2. 24-well NuncTM cell-culture dish plates are prepared as shown in Figure 1. The pre-incubation well (Figure 1B) is filled with 2 ml Kulori media (pH 5.6, see Recipes). The assay well (Figure 1C) is filled with 1 ml Kulori media (pH 5.6) with substrate (for AtGTRs typically between 100 µM and 1 mM glucosinolate to measure uptake in the high-affinity range).
      Note: A mastermix of Kulori pH 5.6 media and substrate is prepared so exactly that the same concentration of substrate is found in each well.


      Figure 1. 24-well assay plates suitable for 12 assays at a time

    3. Three Petri dishes are filled with cold (4 °C) Kulori media (pH 7.4, see Recipes) and stored in fridge.
    4. Number two sets of 12 microfuge tubes from 1-12. Tubes numbered 1-6 will be used for samples from oocytes expressing the transporter. Tubes numbered 7-12 will be used for the samples from the control oocytes. 
      Note: Use different colours for the two sets of tubes (e.g., the first set of 12 tubes are labelled with a black marker and the second set is marked with a red marker).
    5. To keep track of the assay during the experiment and for laboratory book reporting, we recommend using an assay schematic as shown in Table 1.

      Table 1. Assay schematic


  2. Assay
    1. To start the first transport assay, preincubate 6-7 oocytes expressing AtGTR1 in the preincubation well containing Kulori media (pH 5.6) for 5 min (Figure 1B). Subsequently, use a Pasteur pipette to transfer oocytes to the assay well containing Kulori media (pH 5.6) with substrate (Figure 1C) and incubate for 2-180 min at room temperature. Make sure to transfer oocytes in only one drop from the pipette (see Figure 2). Wait three minutes and start the next transport assay. Continue this until all the assays you want to run are running. The duration of the assay is determined by the transport activity and should be determined empirically.
      Notes:
      1. We start the assay with 1-2 oocytes more than we want to analyze on the LC-MS as sometimes 1-2 oocytes are lost during the washing steps.
      2. Transfer the oocytes in only one drop of the Pasteur pipette.
    Note: The length of the assay depends on the activity of the transporter and how well the substrate is ionized and thereby detected by the LC-MS system. Consequently it needs to be determined empirically and minimized as much as possible (i.e., you need to start by running a long assay and then gradually reduce the incubation time). This is especially important if transport kinetics are to be performed (and electrophysiology is not an option) as kinetic measurements need to be performed in the linear range of transport.


    Figure 2. Process of transferring oocytes from one well to another using a Pasteur pipette. Please note how the oocytes are allowed to settle in the tip and are expelled in a single drop.

    1. With a < 2 µl pipette, take out 1 µl of each assay media (Figure 1C) and add it to the Eppendorf tube for the media sample (in this example tube 1).
    2. Stop the assay by adding 1 ml cold (4 °C) Kulori media (pH 7.4) to the assay well and immediately transfer the oocytes to the first Petri dish using a Pasteur pipette. Make sure to transfer oocytes in only one drop from the pipette and empty the rest of the solution in the pipette into the waste. Subsequently, and in the same way move the oocytes to Petri dish two and then Petri dish three to wash away any external substrate. This washing procedure effectively dilutes the substrate in the external uptake media to below detection levels.
      Note: Each time make sure to only transfer the oocytes with one drop of Kulori media.
    3. Transfer one oocyte to each of the 1.5 ml Eppendorf tubes numbered 2-6 and carefully remove excess wash media with a 100 µl pipette from each tube.
      Note: Removal of excess wash media is a key step. Complete removal ensures low variability between replicates.
    4. Add 50 μl of 50% MeOH (with an appropriate internal standard. For glucosinolate transport assays, we use 1,250 nM of the glucosinolate sinigrin as it is commercially available) to the five oocyte samples and the media sample. Immediately homogenate the oocytes using a 100 µl pipette.
      Note: Adding MeOH and waiting will result in oocytes that cannot be homogenated due to the dehydration by MeOH. It is therefore important that the homogenization is carried out immediately.
    5. Leave the homogenate for two hours at -20 °C and then centrifuge the samples at 20,000 x g for 15 min at 4 °C to precipitate proteins. Transfer 40 µl of the supernatant to the corresponding tube in the second set of numbered tubes and dilute with 60 μl H2O.
    6. Filter the diluted samples through a 0.22 μm PVDF based filter plate (EMD Millipore) and subsequently analyze by analytical LC-MS.

  3. LC-MS analysis of glucosinolates and cyanogenic glucosides
    1. Analysis can be performed by any type of UHPLC coupled to a Triple Quadrupole mass spectrometer. Separation of analytes is routinely done by reverse phase liquid chromatography using a C18-type column using MilliQ-grade water with 0.05% formic acid and acetonitrile with 0.05% formic acid as gradient solvents. Electrospray ionization (ESI) is then followed by detection by the MS using Multi Reaction Monitoring (MRM) which allows for detection of analytes to very low concentrations depending on ionization efficiency and other instrument parameters. Typically, analytes such as glucosinolates have a lower limit of detection (LLOD) around 5-10 nM (approx. 5-10 fmol on column) (Crocoll et al., 2016) while cyanogenic glucosides have a LLOD of around 200-250 nM (approx. 200-250 fmol on column). The lower limit of quantification (LLOQ) is around 20-50 nM and 400-500 nM for glucosinolates and cyanogenic glycosides, respectively.
    2. Here, chromatography was performed on an Advance UHPLC system (Bruker, Bremen, Germany). Separation was achieved on a Kinetex 1.7u XB-C18 column (100 x 2.1 mm, 1.7 μm, 100 Å, Phenomenex, Torrance, CA, USA). Formic acid (0.05%) in water and acetonitrile (supplied with 0.05% formic acid) were employed as mobile phases A and B, respectively. The elution profile was: 0-0.2 min, 2% B; 0.2-1.8 min, 2-30% B; 1.8-2.5 min 30-100% B, 2.5-2.8 min 100% B; 2.8-2.9 min 100-2% B and 2.9-4.0 min 2% B. The mobile phase flow rate was 400 μl/min. The column temperature was maintained at 40 °C. The liquid chromatography was coupled to an EVOQ Elite Triple Quadrupole mass spectrometer (Bruker, Bremen, Germany) equipped with an electrospray ion source (ESI) operated in combined positive and negative ionization mode. The instrument parameters were optimized by infusion experiments with pure standards. The ion spray voltage was maintained at 5,000 V or -4,000 V for cyanogenic glucoside and glucosinolate analysis, respectively. Cone temperature was set to 300 °C and cone gas to 20 psi. Heated probe temperature was set to 180 °C and probe gas flow to 50 psi. Nebulizing gas was set to 60 psi and collision gas to 1.6 mTorr. Nitrogen was used as probe and nebulizing gas and argon as collision gas. Active exhaust was constantly on. Multiple Reaction Monitoring (MRM) was used to monitor analyte parent ion → product ion transitions: MRM transitions were chosen based on direct infusion experiments. Detailed values for mass transitions can be found in supplemental Table S3 of Jørgensen et al. (2017). Both Q1 and Q3 quadrupoles were maintained at unit resolution. Bruker MS Workstation software (Version 8.2, Bruker, Bremen, Germany) was used for data acquisition and processing. Linearity in ionization efficiencies was verified by analyzing dilution series of standard mixtures. Quantification of all compounds was achieved by use of sinigrin as an internal standard.

  4. LC-MS analysis–preparation of standards
    The LC-MS analysis parameters are highly dependent on the type of equipment available, the setup of the LC-MS system and the compound to be analyzed. It is therefore important to consult the person running the LC-MS equipment prior to starting assays.
    We utilize an internal standard (e.g., sinigrin) and an external standard curve to (semi)quantitatively measure the amount of glucosinolates that is taken up into oocytes during the assay. Based on the external standard curve we can calculate a response factor that we can then use to calculate a sample’s substrate concentration. Using an internal standard for analysis has several advantages over only using an external standard curve, e.g., correction for handling during extraction, correction for technical variation during LC-MS acquisition and it does not require running an external standard curve every single time which reduces sample number and running costs (especially when considering triple injection of a standard curve with 10-12 concentrations covering the linear range of detection). The linear range of modern mass spectrometers often covers 4-5 orders of magnitude (e.g., from as low as 1 nM to up 100 µM). The linearity should always be checked as some analytes might show a non-linear behavior or the linear range is reduced.
    1. Prepare your standard dilution series in 20% MeOH (same as samples to be analyzed). The range of your standard dilution series should be determined empirically based on your substrates ionization efficiency and the transporter’s activity. In our case, we prepare a dilution series from 1 nM to 20,000 nM sinigrin in 20% MeOH.
      Note: How to empirically determine the correct dilution series range? Perform an uptake experiment and as default use a dilution series from 1 nM to 20,000 nM internal standard. If your sample concentrations are not within the standards linear range, you should increase the range of your dilution series or dilute your sample appropriately.
    2. Prepare 11 LC-MS vials (one per standard curve concentration) and add 100 µl of the appropriate standard curve solution in each.
    3. Standard curve samples are measured by LC-MS in triplicate and an average is calculated (see Table 2).

      Table 2. Standard curve measurements


    4. We plot the analyte concentration (sinigrin and 4MTB in this example) as a function of the signal intensity from the LC-MS and calculate the linear equation to describe the relationship between these two values (see Figure 3) (Crocoll et al., 2016).


      Figure 3. Plot of the standard concentrations relative to the peak area measured by the mass spectrometer

      We calculate the response factor (RF) between our internal standard with known concentration and our substrate (4MTB in this example) by dividing the slope of the internal standard with the slope of the substrate.
      Note: RF values can NOT be transferred between instruments as the response of each analyte depends on instrument settings for source temperature, ionization energy, collision gas, collision energy and other settings that also can be specific to instruments from different vendors (Crocoll et al., 2016). 



      The RF value is used to quantify the amount of substrate taken up during our transport assays.

Data analysis

Upon completion of the LC-MS analysis, we calculate the amount of transported substrate into the oocytes. To calculate the number of mol substrate transported per oocyte we multiply the area of the substrate with the amount of internal standard added in the sample and multiplied this with the response factor we calculated in step D4. This value is divided by the area of the internal standard (Equation 2, see Table 3 and Figure 4 for example data).



The 50% MeOH solution in which we bust oocytes contains 1,250 nM sinigrin as an internal standard. Consequently, we have 50 picomole internal standard in the analyte.



Table 3. Example of data analysis

Note: These values can be plotted in a bar graph to visually compare uptake by AtGTR1-expressing oocytes and non-expressing control oocytes.


Figure 4. Transport assay with glucosinolate transporter AtGTR1 and non-injected control oocytes

Recipes

  1. Kulori media (pH 7.4)
    90 mM NaCl
    1 mM KCl
    1 mM MgCl2
    1 mM CaCl2
    10 mM HEPES
    Adjust to pH 7.4 with Tris
  2. Kulori media (pH 5.6)
    90 mM NaCl
    1 mM KCl
    1 mM MgCl2
    1 mM CaCl2
    10 mM MES
    Adjust to pH 5.6 with Tris

Acknowledgments

We thank Meike Burow and Bo Larsen for their help with the initial LCMS method development for glucosinolate detection from oocyte uptake assays. Morten Egevang Jørgensen is supported by a grant from the Danish Council for Independent Research: DFF–6108-00122. BAH and CC were funded by DNRF99 grant from the Danish National Research Foundation. HHN was funded by DNRF99 grant from the Danish National Research Foundation and by Innovation Fund Denmark J.nr.: 76-2014-3.

References

  1. Boorer, K. J., Forde, B. G., Leigh, R. A. and Miller, A. J. (1992). Functional expression of a plant plasma membrane transporter in Xenopus oocytes. FEBS Lett 302(2): 166-168.
  2. Calamita, G., Bishai, W. R., Preston, G. M., Guggino, W. B. and Agre, P. (1995). Molecular cloning and characterization of AqpZ, a water channel from Escherichia coli. J Biol Chem 270(49): 29063-29066.
  3. Crocoll, C., Halkier, B. A. and Burow, M. (2016). Analysis and quantification of glucosinolates. Curr Protoc Plant Biol 1: 385-409.
  4. Jørgensen, M. E., Nour-Eldin, H. H. and Halkier, B. A. (2016). A Western blot protocol for detection of proteins heterologously expressed in Xenopus laevis oocytes. Methods Mol Biol 1405: 99-107.
  5. Jørgensen, M. E. Xu, D., Crocoll, C., Ramírez, D., Motawia, M. S., Olsen, C. E., Nour-Eldin, H. H. and Halkier, B. A. (2017). Origin and evolution of transporter substrate specificity within the NPF family. eLife 6: e19466.
  6. Norholm, M. H., Nour-Eldin, H. H., Brodersen, P., Mundy, J. and Halkier, B. A. (2006). Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death. FEBS Lett 580(9): 2381-2387.
  7. Nour-Eldin, H. H., Norholm, M. H. and Halkier, B. A. (2006). Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes. Plant Methods 2: 17.
  8. Sigel, E. (1990). Use of Xenopus oocytes for the functional expression of plasma membrane proteins. J Membr Biol 117(3): 201-221.
  9. Sumikawa, K., Houghton, M., Emtage, J. S., Richards, B. M. and Barnard, E. A. (1981). Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature 292(5826): 862-864.
  10. Theodoulou, F. L. and Miller, A. J. (1995). Xenopus oocytes as a heterologous expression system for plant proteins. Mol Biotechnol 3(2): 101-115.
  11. Wahl, R., Wippel, K., Goos, S., Kamper, J. and Sauer, N. (2010). A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis. PLoS Biol 8(2): e1000303.

简介

非洲爪蟾卵母细胞是用于表征异源表达的第二活性转运蛋白的广泛使用的模型系统。历史上,研究人员依靠通过闪烁计数或荧光标记的底物通过分光荧光定量测量放射性标记底物的积累来检测运输活动。然而,这些技术仅限于可用作放射性标记的同位素的底物或当底物是荧光时。这促使我们开发了一种运输测定法,我们可以原则上检测任何有机代谢物的转运活性,无论其作为放射性标记的同位素或荧光性质如何。

在这个协议中,我们描述了X的使用。卵母细胞作为表达次要活性转运蛋白的异源宿主,以及如何进行摄取测定,然后通过液相色谱 - 质谱(LC-MS)检测和定量运输的代谢物。我们已经成功地使用这种方法来鉴定和表征称为硫代葡萄糖苷和氰基葡糖苷的植物防御代谢物的转运蛋白(Jørgensen等人,2017),然而该方法可用于表征任何转运蛋白底物可以通过LC-MS检测。

【背景】来自非洲爪蟾蛙(Xenopus laevis)的卵母细胞是用于异源表达和表征膜蛋白(即,转运蛋白和通道)的完整表达系统。 X。卵母细胞表达少量内源性膜蛋白,具有低的背景转运活性。此外,来自植物的次要活性转运蛋白(Boorer等人,1992; Theodoulou和Miller,1995; Nour-Eldin等人,2006),动物(Sumikawa 1981); Sigel,1990)和微生物(Calamita等人,1995; Wahl等人,2010)已被成功地表达在 X。 laevis 卵母细胞,表明该系统广泛适用于表征来自任何生物体的转运蛋白。

运输测定法需要在能够正确折叠蛋白质的系统中表达运输蛋白质,并将其定位于可以检测底物运动的膜上。由于移动量很小,研究人员通常使用放射性标记的底物进行运输测定。孵化后洗涤卵母细胞,可以检测卵母细胞内部积累基质内部卵母细胞的闪烁计数。我们之前已经使用这种方法来鉴定和表征来自拟南芥的蔗糖和葡萄糖转运蛋白,使用非洲爪蟾卵母细胞系统(Nour-Eldin等人。 ,2006; Norholm等人,2006)。然而,为了鉴定和表征植物专用代谢物转运蛋白,产生目标底物的放射性标记的同位素是非常具有挑战性的。为了克服这个挑战,我们开发了一种检测和定量将特异性代谢物转运到X中的方案。通过使用LC-MS来测定卵母细胞。使用这种方法使我们能够将可分析底物的库存扩大到所应用的LC-MS系统可以检测和量化的任何物质。

关键字:非洲爪蟾卵母细胞, 吸收实验, 转运蛋白功能研究, 液相色谱 - 质谱

材料和试剂

  1. 移液器过滤嘴(例如,,Biotix,目录号:M-0012-9FC,M0020-9FC,M-0300-9FC,M-1250-9FC96)
  2. 用于卵母细胞洗涤的培养皿(例如,直径90mm,Thermo Fisher Scientific,Thermo Scientific TM,目录号:263991)
  3. 24孔Nunc TM细胞培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:142475)
  4. 用于卵母细胞处理的巴斯德移液器
  5. 1.5ml管(例如,1.5ml微量离心管,“Easy Fit”,Almeco,目录号:02.023.01001)
  6. 0.22μmPVDF基过滤板(EMD Millipore,目录号:MSGVN2250)
  7. LC-MS小瓶
  8. 卵母细胞表达感兴趣的转运蛋白和注射水的对照卵母细胞(参见Jørgensen等人,2016,关于制备和注射cRNA和处理卵母细胞的方案)
    注意:
    1. 使用来自拟南芥的高亲和力硫代葡萄糖苷转运蛋白,ARABIDOPSIS THALIANA GLUCOSINOLATE TRANSPORTER-1(AtGTR1)(UniProt,目录号:Q944G5)作为这里的实例。/
    2. 卵母细胞可以从Ecocyte-biosciences购买( http://ecocyte-us.com /
  9. 基板
    注意:我们使用从C2 Bioengineering( http://www.glucosinolates.com/ )和CFM Oskar Tropitzsch GmbH,Marktredwitz( http://www.cfmot.de/ )。 Cyanogenic glucoside linamarin可购自Santa Cruz Biotechnology。
  10. 甲醇用于HPLC≥99.9%(Sigma-Aldrich,目录号:34860)
  11. 甲酸,试剂级(Sigma-Aldrich,目录号:F0507)
  12. 用于HPLC的乙腈(Sigma-Aldrich,目录号:34851)
  13. 氯化钠(NaCl)(Duchefa Biochemie,目录号:S0520.5000)
  14. 氯化钾(KCl)(Merck,目录号:1.04936.1000)
  15. 氯化镁六水合物(MgCl 2•6H 2 O)(Sigma-Aldrich,目录号:M2670)
  16. 氯化钙二水合物(CaCl 2•2H 2 O)(EMD Millipore,目录号:1.02382)
  17. HEPES(Sigma-Aldrich,目录号:H4034)
  18. Tris-HCl溶液(Trisma盐酸盐)(Sigma-Aldrich,目录号:T3253)
  19. MES(Sigma-Aldrich,目录号:M8250)
  20. Kulori培养基(pH 7.4)(参见食谱)
  21. Kulori培养基(pH 5.6)(参见食谱)

设备

  1. 17℃用于卵母细胞储存的培养箱(BINDER,型号:KB23型)
  2. 移液管(10μl,50μl,200μl和1,000μl)
  3. 用于1.5ml微量离心管(例如,Ole Dich Instrumentmakers,型号:Ole Dich 157,最大速度20,000×g,冷藏)的台式离心机
  4. Kinetex 1.7u XB-C18色谱柱(100 x 2.1 mm,1.7μm,100)(Phenomenex,目录号:00D-4499-AN)
  5. 用于样品分析/数据采集的LC-MS三重四极杆系统(与EVOQ Elite Triple Quadrupole质谱仪耦合的Advance UHPLC)(Bruker,型号:EVOQ Elite TM三重四极杆)

软件

  1. 用于数据分析和演示的Microsoft Excel
  2. Bruker MS Workstation软件(版本8.2,Bruker,Bremen,Germany)

程序

在下文中,我们将提供一个典型测定的例子,其中我们测试给定的转运蛋白对硫代葡萄糖苷的摄取活性。我们测试已经注射了cRNA的七个卵母细胞,用于我们的感兴趣的转运蛋白和已经注入水的七个卵母细胞用作阴性对照。每种待测化合物应包括一组阴性对照卵母细胞。

  1. 测定准备
    1. 准备表达感兴趣的转运蛋白的七个卵母细胞(注射免疫RNA(cRNA)[25-50ng],并在17℃孵育72小时)和已注入水的七个卵母细胞用作对照卵母细胞(和在17℃下孵育72小时)。注射体积通常为50 nl。
      注意:请参阅Jørgensen等,2016,了解cRNA生成和注射的详细方案,以及从注射到测定的卵母细胞处理。
    2. 如图1所示制备24孔Nunc TM细胞培养皿。预培养井(图1B)用2ml Kulori培养基(pH5.6,参见食谱)填充。测定孔(图1C)用底物填充1ml Kulori培养基(pH 5.6)(对于AtGTR通常在100μM和1mM硫代葡萄糖苷之间以测量在高亲和力范围内的摄取)。
      注意:准备好Kulori pH 5.6培养基和底物的主剂,以确定每个孔中都有相同浓度的底物。


      图1.一次适合12次测定的24孔测定板

    3. 三个培养皿充满冷(4℃)Kulori培养基(pH 7.4,参见食谱)并储存在冰箱中。
    4. 来自1-12的两套12个微量离心管。编号1-6的管将用于表达转运蛋白的卵母细胞的样品。编号为7-12的管将用于对照卵母细胞的样品。&nbsp;
      注意:对于两组管子使用不同的颜色(例如,第一组12个管子用黑色标记标记,第二组用红色标记标记)。
    5. 为了跟踪实验期间的实验和实验室书籍报告,我们建议使用如表1所示的分析原理图。

      表1.分析原理图


  2. 化验
    1. 为了开始第一次运输测定,在含有Kulori培养基(pH 5.6)的预温育5分钟内将6-7个表达AtGTR1的卵母细胞预孵育5分钟(图1B)。随后,使用巴斯德吸管将卵母细胞转移到含底物的含有Kulori培养基(pH 5.6)的测定中(图1C),并在室温下孵育2-180分钟。确保从移液器只传输一滴液体中的卵母细胞(见图2)。等待三分钟,开始下一次运输测定。继续这样,直到您要运行的所有测定正在运行。测定的持续时间由运输活动决定,应根据经验确定 注意:
      1. 我们开始使用1-2个卵母细胞进行测定,超过我们想在LC-MS上分析,有时1-2个卵母细胞在洗涤步骤期间丢失。
      2. 只有一滴巴斯德移液器才能转移卵母细胞
    注意:测定的长度取决于转运蛋白的活性以及底物如何被离子化,从而由LC-MS系统检测。因此,需要尽可能经验地确定和最小化(即,您需要首先运行长测定法,然后逐渐减少孵育时间)。如果需要在线性运输范围内进行动力学测量,则要进行运输动力学(电生理学不是一个选择),这一点尤为重要。


    图2.使用巴斯德吸管将卵母细胞从一口转移到另一口的过程。 请注意,卵母细胞如何被允许沉淀在提示中,并在一滴中排出。

    1. 使用&lt;取2μl移液管,取出1μl各种测定培养基(图1C),并将其加入到用于培养基样品(本例中为管1)的Eppendorf管中。
    2. 通过加入1 ml冷(4°C)Kulori培养基(pH 7.4),测定孔,并使用巴斯德吸管立即将卵母细胞转移到第一培养皿中。确保将卵母细胞从移液管中移出一滴,并将其余的移液管中的溶液清空到废物中。随后,以相同的方式将卵母细胞移动到培养皿中,然后将培养皿三次洗去任何外部底物。这种洗涤过程有效地将外部吸收介质中的底物稀释到低于检测水平 注意:每次确保只用一滴Kulori培养基转移卵母细胞。
    3. 将一个卵母细胞转移到编号为2-6的1.5ml Eppendorf管中,并从每个管中小心地用100μl移液管除去多余的洗涤介质。
      注意:去除多余的洗涤介质是关键的一步。完全移除可确保复制之间的低变异性。
    4. 加入50μl50%的MeOH(具有适当的内标。对于硫代葡萄糖苷转运测定,我们使用1250nM的硫代葡萄糖苷sinigrin,如可商购的)至五个卵母细胞样品和培养基样品。立即用100μl移液管匀浆卵母细胞。
      注意:添加MeOH和等待将导致由于MeOH脱水而不能匀浆的卵母细胞。因此,必须立即进行均质化。
    5. 将匀浆在-20℃下放置2小时,然后在4℃下以20,000×g离心样品15分钟以沉淀蛋白质。将40μl上清转移到第二组编号管中的相应管中,并用60μlH 2 O稀释。
    6. 通过0.22μmPVDF滤膜(EMD Millipore)过滤稀释的样品,随后通过分析LC-MS分析。

  3. 硫代葡萄糖苷和氰基葡萄糖苷的LC-MS分析
    1. 分析可以通过耦合到三重四极杆质谱仪的任何类型的UHPLC进行。通过使用C18型柱的反相液相色谱法分离分析物,使用具有0.05%甲酸的MilliQ级水和具有0.05%甲酸的乙腈作为梯度溶剂。然后通过使用多反应监测(MRM)的MS检测电喷雾离子化(ESI),其允许根据电离效率和其它仪器参数将分析物检测到非常低的浓度。通常,诸如硫代葡萄糖苷的分析物具有约5-10nM的检测下限(LLOD)(柱上约5-10fmol)(Crocoll等人,2016),而氰基葡糖苷具有约200-250nM的LLOD(在柱上约200-250fmol)。定量下限(LLOQ)分别为20-50nM,对于硫代葡萄糖苷和氰基糖苷为400-500nM。
    2. 这里,色谱在Advance UHPLC系统(Bruker,Bremen,Germany)上进行。在Kinetex 1.7u XB-C18柱(100×2.1mm,1.7μm,100,Phenomenex,Torrance,CA,USA)上进行分离。分别用水和乙腈(0.05%甲酸提供)中的甲酸(0.05%)作为流动相A和B。洗脱曲线为:0-0.2分钟,2%B; 0.2-1.8分钟,2-30%B; 1.8-2.5分钟30-100%B,2.5-2.8分钟100%B; 2.8-2.9min 100-2%B和2.9-4.0min 2%B.流动相流速为400μl/ min。柱温保持在40℃。将液相色谱与装有电喷雾离子源(ESI)的EVOQ Elite Triple Quadrupole质谱仪(Bruker,Bremen,Germany)相结合,其以组合的正离子和负离子模式进行操作。仪器参数通过纯标准品的输注实验进行优化。离子喷雾电压分别维持在5000V或-4,000V,用于产氰葡萄糖苷和硫代葡萄糖苷分析。锥体温度设定为300℃,锥形气体设定为20psi。加热探针温度设定为180℃,探测气体流量为50psi。雾化气体设定为60psi,碰撞气体为1.6mTorr。使用氮气作为探针,雾化气体和氩气作为碰撞气体。主动排气不断开启。多重反应监测(MRM)用于监测分析物亲本离子→产物离子转换:基于直接输注实验选择MRM转换。质量转换的详细值可以在Jørgensen等人的补充表S3中找到(2017)。 Q1和Q3四极都保持单位分辨率。 Bruker MS工作站软件(8.2版,Bruker,Bremen,Germany)用于数据采集和处理。通过分析标准混合物的稀释系列来验证电离效率的线性。所有化合物的定量是通过使用sinigrin作为内标来实现的
  4. LC-MS分析 - 准备标准
    LC-MS分析参数高度依赖于可用设备的类型,LC-MS系统的设置和要分析的化合物。因此,在开始测定之前,请咨询运行LC-MS设备的人员。
    我们利用内部标准(例如,sinigrin)和外部标准曲线((半))定量测量测定期间摄入卵母细胞的硫代葡萄糖苷的量。基于外部标准曲线,我们可以计算一个响应因子,然后我们可以用它来计算样品的底物浓度。使用内部标准进行分析具有优于仅采用外部标准曲线,例如,提取期间的处理更正,LC-MS采集期间技术变化的校正,并且不需要运行外部标准曲线,每一次减少样品数量和运行成本(特别是考虑三次注入标准曲线,覆盖线性检测范围的10-12浓度时)。现代质谱仪的线性范围通常覆盖4-5个数量级(例如,从低至1nM至高达100μM)。始终检查线性度,因为某些分析物可能显示非线性行为或线性范围减少。
    1. 在20%MeOH(与待分析样品相同)中准备标准稀释系列。您的标准稀释系列的范围应根据您的底物离子化效率和转运蛋白的活性凭经验确定。在我们的例子中,我们在20%的MeOH中制备1 nM到20,000 nM的sinigrin稀释系列 注意:如何经验确定正确的稀释系列范围?进行摄取实验,默认使用1 nM至20,000 nM内标的稀释系列。如果您的样品浓度不在标准线性范围内,您应该增加稀释系列的范围或适当稀释样品。
    2. 准备11个LC-MS小瓶(每个标准曲线浓度一个),并在每个样品瓶中加入100μl适当的标准曲线溶液。
    3. 标准曲线样品通过LC-MS一式三份测量,计算平均值(见表2)
      表2.标准曲线测量


    4. 我们将分析物浓度(本示例中的sinigrin和4MTB)作为来自LC-MS的信号强度的函数绘制,并计算线性方程来描述这两个值之间的关系(见图3)(Crocoll等人。,2016)。


      图3.标准浓度相对于通过质谱仪测量的峰面积的曲线

      我们通过将内标的斜率除以衬底的斜率,计算已知浓度的内标与本底(本例中为4MTB)之间的响应因子(RF)。
      注意:RF值不能在仪器之间传输,因为每个分析物的响应取决于仪器设置的源温度,电离能,碰撞气体,碰撞能量和其他可能来自不同供应商的仪器特定的设置(Crocoll等等,2016)。&nbsp;



      RF值用于量化我们运输测定过程中吸收的底物量。

数据分析

在完成LC-MS分析后,我们计算出转移到卵母细胞中的底物量。为了计算每个卵母细胞转移的基质的摩尔数,我们将底物的面积乘以样品中加入的内标量,并将其与步骤D4中计算的响应因子相乘。该值除以内部标准的面积(等式2,参见表3和图4的示例数据)。



我们胸围卵母细胞的50%MeOH溶液含有1,250 nM的sinigrin作为内标。因此,我们在分析物中有50皮摩尔内标。



表3.数据分析示例

注意:这些值可以在条形图中进行绘制,目的是比较表达AtGTR1的卵母细胞和非表达对照卵母细胞的摄取。

图4.使用硫代葡萄糖苷转运蛋白AtGTR1和非注射对照卵母细胞的运输测定

食谱

  1. Kulori培养基(pH 7.4)
    90 mM NaCl
    1 mM KCl
    1mM MgCl 2
    1mM CaCl 2
    10 mM HEPES
    用Tris
    调节至pH 7.4
  2. Kulori培养基(pH 5.6)
    90 mM NaCl
    1 mM KCl
    1mM MgCl 2
    1mM CaCl 2
    10 mM MES
    用Tris
    调节至pH 5.6

致谢

我们感谢Meike Burow和Bo Larsen对从卵母细胞摄取测定中的硫代葡萄糖苷检测的初步LCMS方法开发的帮助。 Morten EgevangJørgensen得到丹麦独立研究委员会的资助:DFF-6108-00122。 BAH和CC由丹麦国家研究基金会的DNRF99拨款资助。 HHN由丹麦国家研究基金会和丹麦创新基金DNRF99拨款资助:76-2014-3。

参考

  1. Boorer,K.J.,Forde,B.G.,Leigh,R.A。和Miller,A.J。(1992)。 非洲爪蟾卵母细胞中植物质膜转运蛋白的功能表达。 a> FEBS Lett 302(2):166-168。
  2. Calamita,G.Bishai,W.R.,Preston,G.M.,Guggino,W.B。和Agre,P。(1995)。 AqpZ,大肠杆菌水通道的分子克隆和表征。 生物化学 270(49):29063-29066。
  3. Crocoll,C.,Halkier,B.A。和Burow,M。(2016)。 硫代葡萄糖苷的分析和定量。 Curr Protoc Plant Biol 1:385-409。
  4. Jørgensen,M.E.,Nour-Eldin,H.H.和Halkier,B.A。(2016)。 用于检测在非洲爪蟾卵母细胞中异源表达的蛋白质的蛋白质印迹方案。 Methods Mol Biol 1405:99-107。
  5. Jørgensen,M. E. Xu,D.,Crocoll,C.,Ramírez,D.,Motawia,M. S.,Olsen,C.E.,Nour-Eldin,H.H。和Halkier,B.A。(2017)。 NPF家族内转运体底物特异性的起源和进化 eilife < 6:e19466。
  6. Norholm,M.H.,Nour-Eldin,H.H.,Brodersen,P.,Mundy,J.and Halkier,B.A。(2006)。 表达拟南芥高亲和力己糖转运蛋白STP13与编程细胞相关死亡。 FEBS Lett 580(9):2381-2387。
  7. Nour-Eldin,H.H.,Norholm,M.H。和Halkier,B.A。(2006)。 通过表达正常拟南芥全长cDNA筛选植物转运蛋白功能非洲爪蟾卵母细胞中的图书馆 植物我 thods 2:17.
  8. Sigel,E。(1990)。 使用非洲爪蟾卵母细胞进行质膜蛋白的功能表达。 / a> J Membr Biol 117(3):201-221。
  9. Sumikawa,K.,Houghton,M.,Emtage,J.S.,Richards,B.M。和Barnard,E.A。(1981)。 通过在非洲爪蟾中翻译异源mRNA组装的活性多亚基ACh受体卵母细胞。
    自然 292(5826):862-864。
  10. Theodoulou,F.L。和Miller,A.J。(1995)。作为植物蛋白质的异源表达系统的卵母细胞的 。 > Mol Biotechnol 3(2):101-115。
  11. Wahl,R.,Wippel,K.,Goos,S.,Kamper,J.and Sauer,N。(2010)。 需要一种新颖的高亲和力蔗糖转运蛋白,用于植物病原体的毒力 Ustilago maydis < 。 8(2):e1000303。
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Copyright Jørgensen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jørgensen, M. E., Crocoll, C., Halkier, B. A. and Nour-Eldin, H. H. (2017). Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity. Bio-protocol 7(20): e2581. DOI: 10.21769/BioProtoc.2581.
  2. Jørgensen, M. E. Xu, D., Crocoll, C., Ramírez, D., Motawia, M. S., Olsen, C. E., Nour-Eldin, H. H. and Halkier, B. A. (2017). Origin and evolution of transporter substrate specificity within the NPF family. eLife 6: e19466.
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