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Mar 2015

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Inositol Phosphates Purification Using Titanium Dioxide Beads
利用二氧化钛珠纯化磷酸肌醇   

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Abstract

Inositol phosphates (IPs) comprise a family of ubiquitous eukaryotic signaling molecules. They have been linked to the regulation of a pleiotropy of important cellular activities, but low abundance and detection difficulties have hampered our understanding. Here we present a method to purify and enrich IPs or other phosphate-rich metabolites from mammalian cells or other sample types. Acid-extracted IPs from cells bind selectively via their phosphate groups to titanium dioxide beads. After washing, the IPs are easily eluted from the beads by increasing the pH. This technique, in combination with downstream analytical methods such as PAGE or SAX-HPLC, opens unprecedented investigative possibilities, allowing appropriate analysis of IPs from virtually any biological or non-biological source.

Keywords: Inositol polyphosphate (肌醇多磷酸盐), Inositol pyrophosphate (肌醇焦磷酸盐), IP6 (IP6), IP7 (IP7), Metabolism (代谢), Metabolites (代谢产物), Signaling (信号转导)

Background

Inositol phosphates (IPs) are a family of conserved signaling molecules, ubiquitous in eukaryotes (Irvine and Schell, 2001; Tsui and York, 2010). They have been implicated in the regulation of a broad range of cellular activities, including calcium signaling, trafficking, and phosphate homeostasis (Wilson et al., 2013; Thota and Bhandari, 2015; Azevedo and Saiardi, 2017). However, our understanding of IPs signaling has been hampered by the fact that they can be difficult to study.

Unlike other phosphate-rich molecules such as nucleotides, IPs do not absorb in the UV/Vis range, and are often present at relatively low abundance in cells. The traditional methodology for IP detection and analysis is to radioactively metabolically label cells with 3H-inositol that is taken up and incorporated into IPs over 1-5 days (Wilson and Saiardi, 2017). After labeling, IPs are extracted with perchloric acid; these extracts are neutralised before separation by strong anion exchange (SAX) HPLC and scintillation counting of each fraction (Azevedo and Saiardi, 2006). The use of 3H-labelled IPs and chromatography has also been required for in vitro biochemical studies. The requirement for radioactive IPs or metabolic labeling limits the possible lines of investigation. These are time-consuming, technically demanding and expensive experiments.

We previously developed a polyacrylamide gel electrophoresis (PAGE) method for resolving and visualizing IPs (Losito et al., 2009; Loss et al., 2011). This technique was immediately useful in following in vitro reactions, as well as analyzing in vivo high abundance IPs such as IP6, IP7 and IP8 from Dictyostelium discoideum (Pisani et al., 2014), or IP6 from plant seeds (Desai et al., 2014; Kolozsvari et al., 2014). However, in the majority of cell types or model organisms, low IP concentrations make it impossible to run a PAGE gel of enough neutralized extract to visualize IPs while maintaining correct gel migration. In mammalian cells, the most abundant IP is IP6 at 40-100 µM (measured in cell lines such as HL60, C1866 and BAF3; French et al., 1991; Bunce et al., 1993), while the inositol pyrophosphate IP7 is thought to be present at sub-µM levels. We were therefore inspired to develop the present method that uses titanium dioxide beads to purify cold or radioactive IPs regardless of their abundance (Wilson et al., 2015). Titanium dioxide binds the phosphate groups of the IPs. The concentrated IPs can be analyzed by PAGE, SAX-HPLC, or other techniques.

The use of titanium dioxide beads now enables analysis of total unlabeled IPs from any cell type (Pavlovic et al., 2015; Wilson et al., 2015; Gu et al., 2016; Pavlovic et al., 2016). It also allows the study of IPs extracted from previously impossible sample types, including large volumes of liquid media, biofluids, or animal tissues. For biochemical work, the method can be used to remove salt and proteins from IPs preparations. Here we present the method as used for purifying IPs from cultured adherent mammalian cells.

Materials and Reagents

  1. Pipette tips (Starlab, catalog number: S1112-1830 )
  2. 1.5 ml Eppendorf-style microcentrifuge tubes (Starlab, catalog number: S1615-5500 )
  3. 50 ml Falcon centrifuge tubes (Corning, catalog number: 352070 )
  4. 15 cm tissue culture dishes (Thermo Fisher Scientific, catalog number: 168381 )
  5. pH test strips (Sigma-Aldrich, catalog number: P4536-100EA )
  6. Titansphere TiO2 beads, 5 µm (Hichrom, catalog number: 5020-75000 )
  7. PBS (Thermo Fisher Scientific, catalog number: 20012019 )
  8. 0.25% Trypsin-EDTA (Thermo Fisher Scientific, catalog number: 25200056 )
  9. Double distilled water (ddH2O) or Milli-Q water (Millipore)
  10. Perchloric acid, 70% (Sigma-Aldrich, catalog number: 244252-1L )
  11. Ammonium hydroxide, 28-30% (Sigma-Aldrich, catalog number: 221228-1L )
  12. 1 M perchloric acid (see Recipes)
  13. ~2.8% ammonium hydroxide (see Recipes)

Equipment

  1. Pipettes (Gilson, models: P1000 and P200, catalog numbers: F123602 , F123601 )
  2. Ice box
  3. Balance (Acculab, model: ALC-80.4 )
  4. Humidified incubator (Eppendorf, model: Galaxy® 170 R , catalog number: CO17311002)
  5. Benchtop centrifuge (Eppendorf, catalog number: 5702000365 )
  6. Benchtop centrifuge with cooling (LaboGene, model: ScanSpeed 1730R )
  7. Rotator (Cole-Parmer, Stuart, model: SB3 )
    Note: This should be placed in a fridge or cold room.
  8. Vortex mixer (Scientific Industries, model: Vortex Genie 2 , catalog number: SI-0266)
  9. Centrifugal evaporator (Martin Christ Gefriertrocknungsanlagen, catalog number: RVC 2-18 )
  10. Tilt table (Cole-Parmer, Stuart, catalog number: SSM4 )
  11. Cell scrapers (Greiner Bio One International, catalog number: 541070 )

Procedure

  1. Before starting the extraction
    1. Switch on the cooled centrifuge and centrifugal evaporator.
    2. Prepare 2.5% ammonium hydroxide and 1 M perchloric acid. Cool to 4 °C before use.
      Note: The dilute ammonium hydroxide solution can be made in advance and stored at room temperature or 4 °C indefinitely. Check that the pH is > 10 and vortex mix before using.
    3. Weigh titanium dioxide beads, 4 mg/sample, into an Eppendorf. Suspend all the beads together in 1 ml ddH2O by pipetting or vortexing, then centrifuge at 3,500 x g for 1 min at 4 °C. Remove supernatant and wash again using 1 ml 1 M perchloric acid. Remove supernatant and suspend beads in 50 µl 1 M perchloric acid per sample, then aliquot into the correct number of Eppendorfs.
      Note: Beads can be mixed by pipetting or vortexing. They will not stay in suspension so mix frequently when aliquotting. The beads are not damaged by shear stress; standard pipette tips can be used for this protocol.

  2. Preparing the cells
    1. Culture the cell line of interest under standard conditions, for example in a 37 °C humidified incubator with 5% CO2.
      Note: For PAGE analysis after titanium dioxide purification, prepare enough cells for 10 mg equivalent total protein per condition. Titanium dioxide purification with PAGE analysis has been validated in many cell types (Wilson et al., 2015). Depending on IPs of interest, cell type, or analysis method, more or fewer cells may be required.
    2. Wash once with warm PBS then incubate in trypsin-EDTA until the cells have detached. Collect them into a Falcon tube and centrifuge at 200 x g for 3 min. Remove supernatant.
    3. Resuspend cells in 1 ml cold PBS and transfer to Eppendorfs on ice. Adjust the volumes so all samples have approximately the same volume, then remove 40 µl into a separate Eppendorf. These cells are for normalization; they can be counted now or, for example, extracted to quantify protein concentration.
    4. Centrifuge the cells at 200 x g for 3 min and remove supernatant. Samples can be processed immediately or frozen at -80 °C.

  3. Perchloric acid extraction
    1. Resuspend the pelleted cells in 1 ml cold 1 M perchloric acid. Mix by pipetting until fully suspended. The samples will immediately become white and cloudy as proteins precipitate.
      Note: When using frozen cells, there is no need to defrost the samples before adding perchloric acid.
    2. Incubate the samples on ice for 10-15 min, with frequent 2-5 sec vortex intervals.
      Note: Many inositol phosphate species, especially inositol pyrophosphates, are labile under acidic conditions. Therefore all steps before addition of ammonium hydroxide should be performed at 4 °C to minimize degradation, and the acid incubation time should be kept to a minimum. Extraction from certain sample types may require longer incubation.
    3. Centrifuge at 18,000 x g for 5 min at 4 °C. The pellet will contain membranes and proteins. Small polar molecules such as IPs will be in the supernatant.

  4. Titanium dioxide beads purification
    1. Transfer the supernatants from Step C3 to the Eppendorfs containing titanium dioxide beads prepared in Step A3. Vortex briefly to mix.
    2. Rotate samples at 4 °C for 15-20 min.
    3. Centrifuge samples at 3,500 x g for 1 min at 4 °C. Carefully discard supernatants as IPs will be bound to the titanium dioxide beads.
      Note: Inositol pyrophosphates can be degraded by adsorption onto the beads as well as by acidic conditions. The protocol should take 1.5-2 h depending on sample number, excluding time taken for centrifugal evaporation.
    4. Wash the beads by resuspending in 500 µl cold 1 M perchloric acid, centrifuging at 3,500 x g for 1 min at 4 °C, then removing supernatant. Repeat this step.
    5. Resuspend the beads in 200 µl ~2.8% ammonium hydroxide to elute IPs. Vortex or pipette mix, then rotate samples for 5 min.
    6. Centrifuge at 3,500 x g for 1 min. Transfer the supernatant to a new Eppendorf.
    7. Elute again with another 200 µl ammonium hydroxide. After rotation and centrifugation, combine that supernatant with the first, for 400 µl sample volume.
    8. Use the centrifugal evaporator to reduce the sample volume to 20-60 µl, or until the pH is 7-8. Do not store the samples until they reach neutral pH. The samples can be heated up to 60 °C during this process to speed up evaporation. If samples are accidentally dried, resuspend in ddH2O.
      Note: Test pH of samples by spotting 1-2 µl onto pH test strips.
    9. Store the neutralized samples at 4 °C. They are quite stable, and can be kept for a few weeks before analysis.

Notes

  1. In this protocol, we have described harvesting cells by trypsinization, but other methods can be used depending on experimental goals. In metabolite extraction protocols it is desirable to quench the cellular metabolism as quickly as possible, to obtain the truest picture of intracellular concentrations. For optimum quenching of adherent cells: quickly remove the culture medium before washing twice in 5 ml cold PBS (for 15 cm dish). Add sufficient cold 1 M perchloric acid to cover the plate (3-5 ml). Incubate the dishes on a tilt table at 4 °C for 10-15 min. Carefully remove the perchloric acid into a Falcon tube and centrifuge to remove contaminating debris. Transfer the supernatant to a fresh tube and proceed with titanium dioxide purification. Almost complete recovery of IPs is possible from volumes up to 10-20 ml (Wilson et al., 2015). Alternatively, after washing, scrape the cells in 1 ml cold PBS, transfer to Falcon or Eppendorf, centrifuge, discard supernatant, and resuspend in a more convenient volume of perchloric acid. The cellular yield is reduced when scraping compared to trypsinizing cells. The chief downside of harvesting by immediate extraction or scraping is that there is no potential for saving an aliquot of cells for normalization purposes. If the experiment will need normalization, e.g., by cell number, extra dishes must be prepared in parallel for this.
  2. Elution of IPs from the titanium dioxide beads is very efficient, allowing reuse of the beads. Wash twice in ddH2O, and store in ddH2O at 4 °C. If carry-over is a concern, either elute again from the washed beads and perform the intended downstream assay to confirm lack of IPs, or use fresh beads each time. The beads we recommend are of defined diameter and intended for scientific use. Cheap cosmetic grade titanium dioxide powder is available to buy online; we found it to be poorer at purifying IPs from cell extracts, although perhaps using greater quantities would compensate.
  3. Aside from IPs, other phosphate-containing molecules will co-purify from cells using acid extraction and titanium dioxide beads. These include nucleotides and other phosphate-containing metabolic intermediates. This protocol can therefore be used to enrich many different small molecules of interest for appropriate downstream analysis. Co-purifying nucleotides such as GTP and ATP resolve separately from IP6, IP7 and IP8 when analyzing by PAGE (Figure 1).


    Figure 1. Higher IPs can be separated from co-purifying contaminant molecules by PAGE. This representative PAGE gel, stained with toluidine blue, shows IPs purified from the human colon carcinoma cell line HCT116 using titanium dioxide beads. An amount of cells equivalent to 20 mg of total protein was used. Inorganic polyphosphate (polyP), and IP6, IP7 and IP8 derived from Dictyostelium discoideum are shown as markers. The identity of each band has been previously confirmed by co-migration with standards, and by enzymatic treatment (phytase to degrade IPs, apyrase to degrade nucleotides). The identity of the Unk band is unknown.

Recipes

  1. 1 M perchloric acid
    1. For 200 ml, mix 17.2 ml stock (70%, 11.6 M) with 182.8 ml ddH2O
    2. Store at room temperature or 4 °C
  2. ~2.8% ammonium hydroxide
    1. For 30 ml, mix 3 ml stock (28-30%) with 27 ml ddH2O
    2. Store at room temperature

Acknowledgments

This work was supported by the UK Medical Research Council (MRC) core support to the MRC/UCL Laboratory for Molecular Cell Biology University Unit (MC UU 1201814). This protocol is an expanded and slightly modified version of that originally described in Wilson et al. (2015). The authors declare that they have no conflicts of interest.

References

  1. Azevedo, C. and Saiardi, A. (2006). Extraction and analysis of soluble inositol polyphosphates from yeast. Nat Protoc 1(5): 2416-2422.
  2. Azevedo, C. and Saiardi, A. (2017). Eukaryotic phosphate homeostasis: The inositol pyrophosphate perspective. Trends Biochem Sci 42(3): 219-231.
  3. Bunce, C. M., French, P. J., Allen, P., Mountford, J. C., Moor, B., Greaves, M. F., Michell, R. H., Brown, G. (1993). Comparison of the levels of inositol metabolites in transformed haemopoietic cells and their normal counterparts. Biochem J 298(3): 667-673.
  4. Desai, M., Rangarajan, P., Donahue, J. L., Williams, S. P., Land, E. S., Mandal, M. K., Phillippy, B. Q., Perera, I. Y., Raboy, V. and Gillaspy, G. E. (2014). Two inositol hexakisphosphate kinases drive inositol pyrophosphate synthesis in plants. Plant J 80(4): 642-653.
  5. French, P. J., Bunce, C. M., Stephens, L. R., Lord, J. M., McConnell, F. M., Brown, G., Creba, J. A., Michell, R. H. (1991). Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes. Proc Biol Sci 245(1314): 193-201.
  6. Gu, C., Wilson, M. S., Jessen, H. J., Saiardi, A. and Shears, S. B. (2016). Inositol pyrophosphate profiling of two HCT116 cell lines uncovers variation in InsP8 levels. PLoS One 11(10): e0165286.
  7. Irvine, R. F. and Schell, M. J. (2001). Back in the water: the return of the inositol phosphates. Nat Rev Mol Cell Biol 2(5): 327-338.
  8. Kolozsvari, B., Parisi, F. and Saiardi, A. (2014). Inositol phosphates induce DAPI fluorescence shift. Biochem J 460(3): 377-385.
  9. Losito, O., Szijgyarto, Z., Resnick, A. C. and Saiardi, A. (2009). Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis. PLoS One 4(5): e5580.
  10. Loss, O., Azevedo, C., Szijgyarto, Z., Bosch, D. and Saiardi, A. (2011). Preparation of quality inositol pyrophosphates. J Vis Exp (55): e3027.
  11. Pavlovic, I., Thakor, D. T., Bigler, L., Wilson, M. S., Laha, D., Schaaf, G., Saiardi, A. and Jessen, H. J. (2015). Prometabolites of 5-diphospho-myo-inositol pentakisphosphate. Angew Chem Int Ed Engl 54(33): 9622-9626.
  12. Pavlovic, I., Thakor, D. T., Vargas, J. R., McKinlay, C. J., Hauke, S., Anstaett, P., Camuna, R. C., Bigler, L., Gasser, G., Schultz, C., Wender, P. A. and Jessen, H. J. (2016). Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo. Nat Commun 7: 10622.
  13. Pisani, F., Livermore, T., Rose, G., Chubb, J. R., Gaspari, M. and Saiardi, A. (2014). Analysis of Dictyostelium discoideum inositol pyrophosphate metabolism by gel electrophoresis. PLoS One 9(1): e85533.
  14. Thota, S. G. and Bhandari, R. (2015). The emerging roles of inositol pyrophosphates in eukaryotic cell physiology. J Biosci 40(3): 593-605.
  15. Tsui, M. M. and York, J. D. (2010). Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear processes. Adv Enzyme Regul 50(1): 324-337.
  16. Wilson, M. S. C. and Saiardi, A. (2017). Importance of radioactive labelling to elucidate inositol polyphosphate signalling. Top Curr Chem (Cham) 375(1): 14.
  17. Wilson, M. S., Bulley, S. J., Pisani, F., Irvine, R. F. and Saiardi, A. (2015). A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine. Open Biol 5(3): 150014.
  18. Wilson, M. S., Livermore, T. M. and Saiardi, A. (2013). Inositol pyrophosphates: between signalling and metabolism. Biochem J 452(3): 369-379.

简介

肌醇磷酸(IP)包含普遍存在的真核信号分子家族。 它们与重要细胞活动的多效性的调节有关,但低丰度和检测困难阻碍了我们的理解。 在这里,我们提出了一种从哺乳动物细胞或其他样本类型中纯化和富集IP或其他富含磷酸盐的代谢物的方法。 来自细胞的酸提取的IP通过其磷酸基团选择性地结合到二氧化钛珠子上。 洗涤后,通过增加pH容易从珠中洗脱IP。 该技术与下游分析方法(如PAGE或SAX-HPLC)相结合,开启了前所未有的研究可能性,允许从几乎任何生物或非生物来源对IP进行适当分析。

【背景】肌醇磷酸(IP)是保守信号分子家族,在真核生物中普遍存在(Irvine和Schell,2001; Tsui和York,2010)。它们涉及广泛的细胞活动的调节,包括钙信号传导,运输和磷酸盐稳态(Wilson et al。,2013; Thota和Bhandari,2015; Azevedo和Saiardi,2017; )。然而,我们对IP信号的理解受到了它们难以研究的事实的阻碍。

与其他富含磷酸盐的分子(例如核苷酸)不同,IP在UV / Vis范围内不吸收,并且通常以相对低的丰度存在于细胞中。用于IP检测和分析的传统方法是用 3 H-肌醇对细胞进行放射性代谢标记,所述H-肌醇在1-5天内被摄取并掺入IP中(Wilson和Saiardi,2017)。标记后,用高氯酸提取IP;在通过强阴离子交换(SAX)HPLC分离之前中和这些提取物并对每个级分进行闪烁计数(Azevedo和Saiardi,2006)。 体外生化研究也需要使用 3 H标记的IP和色谱法。对放射性IP或代谢标签的要求限制了可能的调查线。这些是耗时的,技术要求高且昂贵的实验。

我们之前开发了一种用于解析和可视化IP的聚丙烯酰胺凝胶电泳(PAGE)方法(Losito et al。,2009; Loss et al。,2011)。该技术可立即用于跟踪体外反应,以及分析体内高丰度IP,如IP 6 ,IP 7 和来自 Dictyostelium discoideum 的IP 8 (Pisani et al。,2014),或IP 6 来自植物种子(Desai et al。,2014; Kolozsvari et al。,2014)。然而,在大多数细胞类型或模式生物中,低IP浓度使得不可能运行足够中和的提取物的PAGE凝胶来可视化IP,同时保持正确的凝胶迁移。在哺乳动物细胞中,最丰富的IP是40-100μM的IP 6 (在细胞系如HL60,C1866和BAF3中测量;法国等人,1991; Bunce et al。,1993),而肌醇焦磷酸酯IP 7 被认为以亚μM水平存在。因此,我们受到启发,开发了使用二氧化钛珠子净化冷或放射性IP而不论其丰度如何的本方法(Wilson et al。,2015)。二氧化钛结合IP的磷酸基团。可以通过PAGE,SAX-HPLC或其他技术分析浓缩的IP。

二氧化钛珠子的使用现在可以分析来自任何细胞类型的总未标记IP(Pavlovic et al。,2015; Wilson et al。,2015; Gu 等人,,2016; Pavlovic et al。,2016)。它还允许研究从先前不可能的样品类型中提取的IP,包括大量液体培养基,生物流体或动物组织。对于生物化学工作,该方法可用于从IP制剂中除去盐和蛋白质。在这里,我们提出了用于从培养的贴壁哺乳动物细胞中纯化IP的方法。

关键字:肌醇多磷酸盐, 肌醇焦磷酸盐, IP6, IP7, 代谢, 代谢产物, 信号转导

材料和试剂

  1. 移液器吸头(Starlab,目录号:S1112-1830)
  2. 1.5毫升Eppendorf式微量离心管(Starlab,目录号:S1615-5500)
  3. 50毫升Falcon离心管(Corning,目录号:352070)
  4. 15厘米组织培养皿(Thermo Fisher Scientific,目录号:168381)
  5. pH试纸(Sigma-Aldrich,目录号:P4536-100EA)
  6. Titansphere TiO 2 珠,5μm(Hichrom,目录号:5020-75000)
  7. PBS(赛默飞世尔科技,目录号:20012019)
  8. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,目录号:25200056)
  9. 双蒸水(ddH 2 O)或Milli-Q水(Millipore)
  10. 高氯酸,70%(Sigma-Aldrich,目录号:244252-1L)
  11. 氢氧化铵,28-30%(Sigma-Aldrich,目录号:221228-1L)
  12. 1 M高氯酸(见食谱)
  13. ~2.8%的氢氧化铵(见食谱)

设备

  1. 移液器(Gilson,型号:P1000和P200,目录号:F123602,F123601)
  2. 冰盒
  3. 平衡(Acculab,型号:ALC-80.4)
  4. 加湿培养箱(Eppendorf,型号:Galaxy ® 170 R,目录号:CO17311002)
  5. 台式离心机(Eppendorf,目录号:5702000365)
  6. 带冷却的台式离心机(LaboGene,型号:ScanSpeed 1730R)
  7. Rotator(Cole-Parmer,Stuart,型号:SB3)
    注意:这应该放在冰箱或冷藏室里。
  8. 涡旋混合器(科学工业,型号:Vortex Genie 2,目录号:SI-0266)
  9. 离心蒸发器(Martin Christ Gefriertrocknungsanlagen,目录号:RVC 2-18)
  10. 倾斜台(Cole-Parmer,Stuart,目录号:SSM4)
  11. 细胞刮刀(Greiner Bio One International,目录号:541070)

程序

  1. 在开始提取之前
  1. 打开冷却的离心机和离心蒸发器。
  2. 准备2.5%的氢氧化铵和1M的高氯酸。使用前冷却至4°C。
    注意:稀氢氧化铵溶液可以预先制备,并在室温或4°C下无限期保存。检查pH是否> 10,使用前涡旋混合。
  3. 将4mg /样品的二氧化钛珠粒称入Eppendorf。通过移液或涡旋将所有珠子悬浮在1ml ddH 2 O中,然后在4℃下以3,500 x g 离心1分钟。除去上清液,再用1 ml 1M高氯酸洗涤。去除上清液并将珠子悬浮于每个样品50μl1M高氯酸中,然后等分到正确数量的Eppendorf中。
    注意:可以通过移液或涡旋混合珠子。它们不会保持悬浮状态,因此在等分时会频繁混合。剪切应力不会损坏珠子;标准移液器吸头可用于此协议。

  1. 准备细胞
  1. 在标准条件下培养感兴趣的细胞系,例如在具有5%CO 2 的37℃加湿培养箱中培养。
    注意:对于二氧化钛纯化后的PAGE分析,准备足够的细胞,每种条件下10 mg当量的总蛋白质。使用PAGE分析的二氧化钛纯化已经在许多细胞类型中得到验证(Wilson等人,2015)。根据感兴趣的IP,细胞类型或分析方法,可能需要更多或更少的细胞。
  2. 用温PBS洗涤一次,然后在胰蛋白酶-EDTA中孵育直至细胞分离。将它们收集到Falcon管中并以200 x g 离心3分钟。去除上清液。
  3. 将细胞重悬于1ml冷PBS中,并在冰上转移至Eppendorfs。调整体积使所有样品的体积大致相同,然后将40μl移入单独的Eppendorf中。这些细胞用于标准化;它们现在可以计数,或者例如,提取以量化蛋白质浓度。
  4. 将细胞在200 x g 离心3分钟并除去上清液。样品可立即加工或在-80°C冷冻。

  1. 高氯酸提取
  1. 将沉淀的细胞重悬于1ml冷的1M高氯酸中。通过移液混合直至完全悬浮。当蛋白质沉淀时,样品将立即变白并变浑浊。
    注意:使用冷冻细胞时,在添加高氯酸之前无需对样品进行除霜。
  2. 将样品在冰上孵育10-15分钟,经常以2-5秒的涡旋间隔孵育。
    注意:许多肌醇磷酸盐物种,特别是肌醇焦磷酸盐,在酸性条件下是不稳定的。因此,在加入氢氧化铵之前的所有步骤应在4℃下进行以使降解最小化,并且酸孵育时间应保持最小。从某些样品类型中提取可能需要更长时间的培养。
  3. 在4℃下以18,000 x g 离心5分钟。颗粒含有膜和蛋白质。诸如IP的小极性分子将在上清液中。

  1. 二氧化钛珠净化
  1. 将来自步骤C3的上清液转移至步骤A3中制备的含有二氧化钛珠粒的Eppendorfs。涡旋短暂混合。
  2. 将样品在4°C下旋转15-20分钟。
  3. 在4℃下以3,500 x g 离心1分钟。小心地丢弃上清液,因为IP将与二氧化钛珠粒结合。
    注意:肌醇焦磷酸盐可以通过吸附到珠子上以及酸性条件下降解。该方案需要1.5-2小时,具体取决于样品编号,不包括离心蒸发所需的时间。
  4. 通过重悬于500μl冷的1M高氯酸中,在4,500℃下以3,500×g离心1分钟洗涤珠子,然后除去上清液。重复此步骤。
  5. 将珠子重悬于200μl~2.8%氢氧化铵中以洗脱IP。涡旋或移液管混合,然后旋转样品5分钟。
  6. 在3,500 x g 离心1分钟。将上清液转移到新的Eppendorf。
  7. 再用另外200μl氢氧化铵洗脱。旋转和离心后,将上清液与第一个上清液合并,加入400μl样品。
  8. 使用离心蒸发器将样品体积减少至20-60μl,或直至pH值为7-8。在样品达到中性pH值之前,请勿存放样品。在此过程中,样品可加热至60°C,以加速蒸发。如果样品被意外干燥,则重新悬浮在ddH 2 O中。
    注意:通过在pH试纸上点样1-2μl来测试样品的pH值。
  9. 将中和的样品保存在4°C。它们非常稳定,可以在分析前保存几周。

笔记

  1. 在该方案中,我们已经描述了通过胰蛋白酶消化收获细胞,但是可以根据实验目标使用其他方法。在代谢物提取方案中,希望尽可能快地淬灭细胞代谢,以获得细胞内浓度的最真实图像。对于贴壁细胞的最佳淬灭:快速除去培养基,然后在5ml冷PBS(15cm培养皿)中洗涤两次。加入足够的冷的1M高氯酸覆盖板(3-5ml)。将培养皿在4°C的倾斜台上孵育10-15分钟。小心地将高氯酸去除到Falcon管中并离心以去除污染的碎屑。将上清液转移到新管中并进行二氧化钛纯化。从高达10-20毫升的体积中可以几乎完全回收IP(Wilson et al。,2015)。或者,洗涤后,将细胞在1ml冷PBS中刮擦,转移至Falcon或Eppendorf,离心,弃去上清液,并重悬于更方便体积的高氯酸中。与胰蛋白酶消化细胞相比,刮擦时细胞产量降低。通过立即提取或刮擦收获的主要缺点是没有可能为了标准化目的而保存等份的细胞。如果实验需要标准化,例如,按细胞数量,必须为此平行准备额外的菜肴。
  2. 从二氧化钛珠粒中洗脱IP非常有效,允许重复使用珠粒。在ddH 2 O中洗涤两次,并在4℃下储存在ddH 2 O中。如果遗留物是一个问题,要么从洗过的珠子中再次洗脱并进行预期的下游分析以确认缺乏IP,或每次使用新鲜的珠子。我们推荐的珠子具有确定的直径并且用于科学用途。廉价的化妆品级二氧化钛粉末可在网上购买;我们发现它从细胞提取物中纯化IP更差,尽管可能使用更多的量会补偿。
  3. 除了IP之外,其他含磷酸盐的分子将使用酸提取和二氧化钛珠子从细胞中共纯化。这些包括核苷酸和其他含磷酸盐的代谢中间体。因此,该方案可用于富集许多不同的目标小分子以进行适当的下游分析。当通过PAGE分析时,共纯化核苷酸如GTP和ATP与IP 6 ,IP 7 和IP 8 分开解析(图1)。


    图1.通过PAGE可以将较高的IP与共纯化污染物分子分开。 用甲苯胺蓝染色的该代表性PAGE凝胶显示使用二氧化钛珠从人结肠癌细胞系HCT116纯化的IP。使用相当于20mg总蛋白质的细胞量。来自 Dictyostelium discoideum 的无机多磷酸盐(polyP)和IP 6 ,IP 7 和IP 8 显示为标记。先前通过与标准物的共迁移和酶处理(植酸酶降解IP,腺苷三磷酸双磷酸酶降解核苷酸)证实了每个条带的特性。 Unk乐队的身份不明。

食谱

  1. 1 M高氯酸
    1. 对于200毫升,将17.2毫升原液(70%,11.6毫升)与182.8毫升ddH 2 O混合
    2. 储存在室温或4°C
  2. ~2.8%的氢氧化铵
    1. 对于30ml,将3ml原液(28-30%)与27ml ddH 2 O混合
    2. 在室温下储存

致谢

这项工作得到了英国医学研究委员会(MRC)对MRC / UCL分子细胞生物学大学实验室(MC UU 1201814)核心支持的支持。该协议是最初在(Wilson et al。,2015)中描述的扩展和略微修改的版本。作者声明他们没有利益冲突。

参考

  1. Azevedo,C。和Saiardi,A。(2006)。 从酵母中提取和分析可溶性肌醇多磷酸盐。 Nat Protoc 1(5):2416-2422。
  2. Azevedo,C。和Saiardi,A。(2017)。 真核生物磷酸盐稳态:焦磷酸肌醇的观点。 趋势Biochem Sci 42(3):219-231。
  3. Bunce,C.M.,French,P.J.,Allen,P.,Mountford,J.C.,Moor,B.,Greaves,M.F.,Michell,R.H.,Brown,G。(1993)。 转化的造血细胞及其正常对应物中肌醇代谢物水平的比较。 < em> Biochem J 298(3):667-673。
  4. Desai,M.,Rangarajan,P.,Donahue,J.L.,Williams,S.P.,Land,E.S.,Mandal,M.K.,Phillippy,B.Q.,Perera,I.Y.,Raboy,V。和Gillaspy,G.E。(2014)。 两种肌醇六磷酸激酶在植物中驱动肌醇焦磷酸合成。 植物J < / em> 80(4):642-653。
  5. French,P.J。,Bunce,C.M.,Stephens,L.R.,Lord,J.M.,McConnell,F.M.,Brown,G.,Creba,J.A.,Michell,R.H。(1991)。 HL60早幼粒细胞向中性粒细胞或单核细胞分化过程中肌醇脂和磷酸盐水平的变化。 Proc Biol Sci 245(1314):193-201。
  6. Gu,C.,Wilson,M。S.,Jessen,H.J。,Saiardi,A。和Shears,S.B。(2016)。 两种HCT116细胞系的肌醇焦磷酸盐谱分析揭示了InsP8水平的变化。 PLoS One 11(10):e0165286。
  7. Irvine,R。F.和Schell,M。J.(2001)。 回到水中:肌醇磷酸盐的回归。 Nat Rev Mol Cell Biol 2(5):327-338。
  8. Kolozsvari,B.,Parisi,F。和Saiardi,A。(2014)。 肌醇磷酸盐诱导DAPI荧光移位。 Biochem J 460 (3):377-385。
  9. Losito,O.,Szijgyarto,Z.,Resnick,A。C.和Saiardi,A。(2009)。 肌醇焦磷酸盐及其独特的代谢复杂性:凝胶电泳分析。 PLoS一个 4(5):e5580。
  10. 损失,O.,Azevedo,C.,Szijgyarto,Z.,Bosch,D。和Saiardi,A。(2011)。 优质肌醇焦磷酸盐的制备。 J Vis Exp ( 55):e3027。
  11. Pavlovic,I.,Thakor,D.T.,Bigler,L.,Wilson,M。S.,Laha,D.,Schaaf,G.,Saiardi,A。和Jessen,H.J。(2015)。 5-diphospho-myo-inositol pentakisphosphate的代谢物。 Angew Chem Int Ed Engl 54(33):9622-9626。
  12. Pavlovic,I.,Thakor,DT,Vargas,JR,McKinlay,CJ,Hauke,S.,Anstaett,P.,Camuna,RC,Bigler,L.,Gasser,G.,Schultz,C.,Wender,PA and Jessen,HJ(2016)。 笼中肌醇焦磷酸的细胞传递和光化学释放诱导纤维素中的PH结构域易位。 Nat Commun 7:10622。
  13. Pisani,F.,Livermore,T.,Rose,G.,Chubb,J.R.,Gaspari,M。和Saiardi,A。(2014)。 通过凝胶电泳分析 Dictyostelium discoideum 肌醇焦磷酸代谢。 PLoS One 9(1):e85533。
  14. Thota,S。G.和Bhandari,R。(2015)。 肌醇焦磷酸盐在真核细胞生理学中的新兴作用。 J Biosci < / em> 40(3):593-605。
  15. Tsui,M。M.和York,J。D.(2010)。 肌醇磷酸盐和肌醇焦磷酸盐在发育,细胞信号传导和核过程中的作用。 < em> Adv Enzyme Regul 50(1):324-337。
  16. Wilson,M。S. C.和Saiardi,A。(2017)。 放射性标记的重要性,以阐明肌醇多磷酸盐信号。 Top Curr Chem( (Cham) 375(1):14。
  17. Wilson,M。S.,Bulley,S.J。,Pisani,F.,Irvine,R。F.和Saiardi,A。(2015)。 从生物样品中纯化肌醇磷酸酯的新方法显示,人血浆中不存在肌醇六磷酸或者尿液。 Open Biol 5(3):150014。
  18. Wilson,M。S.,Livermore,T。M.和Saiardi,A。(2013)。 肌醇焦磷酸盐:信号与新陈代谢之间的关系。 Biochem J 452(3):369-379。
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引用:Wilson, M. S. C. and Saiardi, A. (2018). Inositol Phosphates Purification Using Titanium Dioxide Beads. Bio-protocol 8(15): e2959. DOI: 10.21769/BioProtoc.2959.
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