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Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen
成熟番茄花粉膜蛋白质组富集实验   

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Abstract

We established and elaborated on a method to enrich the membrane proteome of mature pollen from economically relevant crop using the example of Solanum lycopersicum (tomato). To isolate the pollen protein fraction enriched in membrane proteins, a high salt concentration (750 mM of sodium chloride) was used. The membrane protein-enriched fraction was then subjected to shotgun proteomics for identification of proteins, followed by in silico analysis to annotate and classify the detected proteins.

Keywords: Membrane proteome(膜蛋白质组), Pollen(花粉), Tomato(番茄), Proteomics(蛋白质组学)

Background

As proper distribution of proteins and solutes between different cellular compartments or the insertion of newly-synthesized proteins into membranes is largely dependent on membrane proteins, the membrane proteome is central for maintenance of cellular and organellar homeostasis (Paul et al., 2013; 2014 and 2016a). Considering the importance of membrane proteins in general, these are also essential for pollen function and development (Paul et al., 2016b). Many global pollen proteomic studies have been performed in the past (Chaturvedi et al., 2013 and 2016); however, information about the intracellular distribution of proteins and the composition of the membrane proteome in pollen is rarely discussed (Pertl et al., 2009). One reason might be the low abundance and solubility of membrane proteins. Here we describe a protocol to isolate and analyze a protein fraction enriched in membrane proteins from mature pollen, which was established for tomato (Figure 1).


Figure 1. Overview of the protocol for isolation of the membrane proteome of mature tomato pollen

Materials and Reagents

  1. 1.7 ml microtubes (Corning, Axygen®, catalog number: MCT-175-C )
  2. Pipette tips
  3. Cheesecloth Miniwipes (Fisher Scientific, catalog number: 06-665-28 )
  4. Sharp blade (Red Devil, catalog number: 3272 )
  5. Bond-Elute C-18 SPEC plate (Agilent Technologies, Santa Clara, CA, USA)
  6. Seeds of the cultivar of choice. For the method development–seeds of Solanum lycopersicum cv. Moneymaker (Accesion: LA2706) and cv. Red setter were used
  7. Liquid nitrogen
  8. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500 )
  9. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
  10. 6x Laemmli buffer (Alfa Aesar, catalog number: J61337 )
  11. Urea (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 29700 )
  12. Acrylamide/bis-acrylamide (Fisher Scientific, catalog number: BP1408-1 )
  13. TEMED (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17919 )
  14. Ammonium persulfate (Acros Organics, catalog number: 327081000 )
  15. Acetonitrile (Fisher Scientific, catalog number: A9561 )
  16. Formic acid (Fisher Scientific, catalog number: A117-50 )
  17. Boric acid (Fisher Scientific, catalog number: A73-500 )
  18. Calcium nitrate tetrahydrate (Fisher Scientific, catalog number: C109-500 )
  19. Magnesium sulphate anhydrous (Fisher Scientific, catalog number: M65-500 )
  20. Potassium nitrate (Fisher Scientific, catalog number: P263-500 )
  21. Potassium chloride (Fisher Scientific, catalog number: P217-500 )
  22. Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
  23. Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA) (Fisher Scientific, catalog number: S311-100 )
  24. Tris base (Fisher Scientific, catalog number: BP152-500 )
  25. Methanol (Fisher Scientific, catalog number: A456-1 )
  26. Acetic acid (Fisher Scientific, catalog number: A507-P500 )
  27. Double distilled water
  28. Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20278 )
  29. Trypsin solution sequencing grade (Roche Diagnostics, catalog number: 11418475001 )
  30. Ammonium bicarbonate (Fisher Scientific, catalog number: A643-500 )
  31. Calcium chloride (Fisher Scientific, catalog number: C70-500 )
  32. Trifluoroacetic acid (TFA) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28904 )
  33. Germination solution (see Recipes)
  34. Homogenization buffer (see Recipes)
  35. Staining solution (see Recipes)
  36. Destaining solution (see Recipes)
  37. Trypsin buffer (see Recipes)

Equipment

  1. Vortex (e.g., Fisher Scientific, model: Fisher ScientificTM Analog Vortex Mix , catalog number: 02-215-365)
  2. Centrifuge (e.g., Eppendorf, model: 5424 R )
  3. Ultracentrifuge (e.g., Thermo Fisher Scientific, model: Sorvall® DiscoveryTM 90 SE )
  4. Daisy BBs (Daisy, model: 24, catalog number: 980024-001 )
  5. Tissuelyser II (QIAGEN, model: Tissuelyser II, catalog number: 85300 )
  6. Speed-Vac Labogene ApS (LabGeneTM, model: ScanVac CoolSafe 110-4 , catalog number: H01130032)
  7. Pipette
  8. Sonicator (BANDELIN electronic, model: Sonorex RK 100 , catalog number: 301.00044253.024)
  9. Precolumn (Eksigent, Redwood City, CA, USA)
  10. Ascentis column (Sigma-Aldrich, Supelco, model: Ascentis® Express Peptide ES-C18 HPLC Column, catalog number: 53552-U ), dimension 15 x 100 μm, pore size 2.7 μm
  11. Orbitrap LTQ XL mass spectrometer (Thermo Fisher Scientific, model: LTQ XLTM )

Software

  1. Proteome Discoverer 1.3 (Thermo, Germany) or other software like MaxQuant (http://www.maxquant.org/), Protmax (http://www.univie.ac.at/mosys/software.html) and MASCOT (http://www.matrixscience.com/)
  2. PROMEX (http://promex.pph.univie.ac.at/promex/) or other software like ProteomeXchange (http://www.proteomexchange.org/)

Procedure

  1. Harvesting of mature pollen grains
    1. Grow plants (e.g., tomato cv. Moneymaker–MM hereafter and cv. Red setter–RS hereafter) under controlled greenhouse conditions (for tomato: 24 °C/20 °C, day/night; 60% humidity (Fragkostefanakis et al., 2015).
      Note: Growth conditions need to be adapted for the plant species or cultivar in use.
    2. Harvest anthers (> 10 mm length for MM and RS) from flower buds after anthesis as they contain mature pollen grains (preferable time is 0-72 h post anthesis).
    3. Make a horizontal cut on the collected anther and put four anthers in one 1.7 ml microtube containing 500 µl of germination solution.
    4. Squeeze anther tissue with slight mechanical stress using pipette tips and vortex for 15-20 sec to release the mature pollen from anther locules.
    5. Further, pass germination solution containing isolated mature pollen grains through cheesecloth to remove anther debris.
    6. Centrifuge the collected pollen grains at 10,000 x g for 15 min at 4 °C.
    7. Remove the supernatant, wash the pelleted pollen grains with 200 µl of germination solution and centrifuge at 10,000 x g for 1 min at 4 °C.
    8. Remove the supernatant and repeat the washing step A7.
    9. Remove the supernatant, immerse the collected pollen in liquid nitrogen and store at -80 °C till further usage.

  2. Isolation of membrane-enriched fraction
    1. Add metal beads (Daisy BB) that help to homogenize the tissue to microtubes and grind the collected mature pollen grains with TissueLyser II (at 25/sec for 1 min).
    2. Add 10-20 ml homogenisation buffer containing sodium chloride (NaCl, 750 mM) and protease inhibitor cocktail to the ground mature pollen, re-suspend and leave it on ice for 30 min.
    3. Centrifuge the homogenised mixture at 100,000 x g for 60 min at 4 °C.
    4. Re-suspend the pellet in Laemmli buffer and quantify the protein concentration.
      Note: The supernatant is enriched in soluble proteins and can also be subjected to shotgun proteomics protocol, if needed.

  3. Shotgun proteomics
    1. Load 40 µg of total proteins onto an SDS-PAGE (12.5% with maximum thickness 1 mm) and run the gel at 80 V constant for 1.5 cm in resolving gel (Valledor and Weckwerth, 2014). Laemmli buffer is used as loading buffer.
    2. Stain the gel with methanol:acetic acid:water:Coomassie Brilliant Blue R-250 (40:10:50:0.001) for 30 min and de-stain it overnight in methanol:water (40:60). Afterwards, slice the thick stacked protein band from the remaining gel and chop it into smaller pieces with a sharp blade. Immerse the chopped gel pieces into double distilled water until all samples are processed.
    3. Afterwards, remove water and add 1 ml of sodium bicarbonate (25 mM) and incubate at 37 °C for 15 min. Discard supernatant and add 1 ml of 25 mM sodium bicarbonate and 50% acetonitrile. Incubate the samples at 37 °C for 15 min. Repeat this step.
    4. Remove the supernatant and dehydrate gel pieces in 300 μl 100% acetonitrile for 5 min at room temperature. Discard supernatant and dry out in a Speed-Vac for 5 min.
      Note: Gel pieces should have a ‘transparent-plastic’ look. If the gel-pieces are still translucent, repeat the step.
    5. Digest the chopped gel pieces with 50 µl trypsin solution (10 ng/μl) and incubate for 15 min at 37 °C. If required, add more trypsin solution until the gel pieces are completely rehydrated. Incubate for 14-16 h at 37 °C and avoid shaking during incubation. Afterwards, stop the reaction by adding 5 μl of 1.0% trifluoroacetic acid (pH should be < 4 after addition of TFA).
      Note: Add 1 ml of trypsin buffer to dilute trypsin to 10 ng/μl. Incubate on ice for 10 min and then pipette up and down to completely re-suspend the pellet. After dilution, trypsin can be stored at -20 °C for one month. It is highly recommended to store it in aliquots (Valledor and Weckwerth, 2014).
    6. For peptide extraction, add 150 µl of 50% acetonitrile (v/v) and 1% formic acid (v/v) into the microtube containing the gel pieces and incubate for 5 min at room temperature followed by sonication for 3 min (low intensity ultrasound bath).
    7. Spin microtubes (1,000 x g for 1 min) and collect the supernatant in a new microtube ‘A’. Repeat step C6 and transfer the supernatant to microtube ‘A’.
    8. Add 100 µl of 90% acetonitrile (v/v) and 1% formic acid (v/v) to the microtube containing the gel pieces and incubate for 5 min at room temperature. Transfer the supernatant to microtube ‘A’.
    9. Dry the samples in Speed-Vac (microtube A). For long-term storage of extracted peptides -80 °C is preferred.
    10. Afterwards, proceed with peptide desalting using spec 96 well C18, Agilent (Valledor and Weckwerth, 2014).
    11. Dissolve peptides in 4% (v/v) acetonitrile, 0.1% (v/v) formic acid.
    12. Inject digested peptides into a one-dimensional nanoflow LC-MS/MS system that has a precolumn.
    13. Elute the peptides with an Ascentis column during 80 min and a gradient ranging from 5% to 50% (v/v) acetonitrile, 0.1% (v/v) formic acid and perform mass spectral analysis on an Orbitrap LTQ XL mass spectrometer with a controlled flow rate of 500 nl per minute and specific tune settings for MS as follows: spray voltage set to 1.8 kV; temperature of the heated transfer capillary set to 180 °C.
    14. After each full MS scan, perform ten MS/MS scans, and dynamically select ten most abundant peptide molecular ions, with a dynamic exclusion window set to 90 sec.
    15. Omit ions with a +1 or unidentified charge state in the full MS from MS/MS analysis.

Data analysis

  1. Shotgun proteome analysis
    1. Search the obtained raw data (in form of mass spectra) with SEQUEST algorithm present in Proteome Discoverer version 1.3. For this, set identification confidence to 5% FDR (false discovery rate) and the variable modifications to acetylation of N-terminus and oxidation of methionine, with a mass tolerance of 10 ppm for the parent ion and 0.8 Da for the fragment ion.
    2. Match peptides to the genome sequence of the respective plant species, tomato in this case (Sol Genomics Network), and consider a significant hit when the peptide confidence is at medium or high and an Xcorr threshold is established at 1 per charge (2 for +2 ions, 3 for +3 ions, etc.).
      Note: Choose high thresholds to minimize false detection of proteins.

  2. Prediction of membrane proteins
    First, use a database like ARAMEMNON for prediction of membrane proteins (Schwacke et al., 2003). ARAMEMNON considers 20 and 11 different databases (or algorithms) to predict alpha-helical and beta-barrel proteins, respectively.
    1. If the plant species of your interest is not listed in ARAMEMNON, you can predict Arabidopsis co-orthologs of the detected protein by well-established approaches (Simm et al., 2015).
    2. The detected proteins should be verified by a second program like THMMM (http://www.cbs.dtu.dk/services/TMHMM/), which predicts transmembrane alpha helices based on hidden Markov models (Krogh et al., 2001).

Recipes

  1. Germination solution
    2 mM boric acid
    2 mM calcium nitrate tetrahydrate
    2 mM magnesium sulphate anhydrous
    1 mM potassium nitrate
    For preparing 100 ml mix:
    0.012 g of boric acid
    0.047 g of calcium nitrate tetrahydrate
    0.024 g of magnesium sulphate anhydrous
    0.010 g of potassium nitrate
  2. Homogenization buffer
    100 mM potassium chloride
    5 mM DTT
    1 mM EDTA
    50 mM Tris pH 7.2
    750 mM sodium chloride
    For preparing 100 ml mix:
    0.745 g of potassium chloride
    0.077 g of DTT
    0.029 g of EDTA
    0.605 g of Tris pH 7.2
    Note: Always add sodium chloride in the last place as per the homogenization buffer; for 20 ml homogenization buffer, add 0.876 g of sodium chloride.
  3. Staining solution
    methanol:acetic acid:water:Coomassie Brilliant Blue R-250 (40:10:50:0.001)
  4. Destaining solution
    Methanol and water (40:60)
  5. Trypsin buffer
    25 mM sodium bicarbonate
    10 % (v/v) acetonitrile
    5 mM calcium chloride
    For preparing 100 ml mix:
    0.21 g of sodium bicarbonate
    100 μl of pure acetonitrile
    645 μl of water
    Note: Always add calcium in the last place as per the trypsin buffer; for 10 ml of trypsin buffer, add 0.005 g of calcium chloride.

Acknowledgments

This protocol is adapted from (Paul et al., 2016a). This is an article of the SPOT-ITN consortium funded by Marie-Curie (EU Grant Agreement Number 289220) to E.S.

References

  1. Chaturvedi, P., Ghatak, A., and Weckwerth, W. (2016). Pollen proteomics: from stress physiology to developmental priming. Plant Reprod 29(1-2): 119-32.
  2. Chaturvedi, P., Ischebeck, T., Egelhofer, V., Lichtscheidl, I. and Weckwerth, W. (2013). Cell-specific analysis of the tomato pollen proteome from pollen mother cell to mature pollen provides evidence for developmental priming. J Proteome Res 12(11): 4892-4903.
  3. Fragkostefanakis, S., Simm, S., Paul, P., Bublak, D., Scharf, K. D. and Schleiff, E. (2015). Chaperone network composition in Solanum lycopersicum explored by transcriptome profiling and microarray meta-analysis. Plant Cell Environ 38(4): 693-709.
  4. Krogh, A., Larsson, B., von Heijne, G. and Sonnhammer, E. L. (2001). Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305(3): 567-580.
  5. Paul, P., Chaturvedi, P., Selymesi, M., Ghatak, A., Mesihovic, A., Scharf, K. D., Weckwerth, W., Simm, S. and Schleiff, E. (2016a). The membrane proteome of male gametophyte in Solanum lycopersicum. J Proteomics 131: 48-60.
  6. Paul, P., Roth, S. and Schleiff, E. (2016b). Importance of organellar proteins, protein translocation and vesicle transport routes for pollen development and function. Plant Reprod 29(1-2): 53-65.
  7. Paul, P., Simm, S., Blaumeiser, A., Scharf, K. D., Fragkostefanakis, S., Mirus, O. and Schleiff, E. (2013). The protein translocation systems in plants - composition and variability on the example of Solanum lycopersicum. BMC Genomics 14: 189.
  8. Paul, P., Simm, S., Mirus, O., Scharf, K. D., Fragkostefanakis, S. and Schleiff, E. (2014). The complexity of vesicle transport factors in plants examined by orthology search. PLoS One 9(5): e97745.
  9. Pertl, H., Schulze, W. X. and Obermeyer, G. (2009). The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen. J Proteome Res 8(11): 5142-5152.
  10. Schwacke, R., Schneider, A., van der Graaff, E., Fischer, K., Catoni, E., Desimone, M., Frommer, W. B., Flugge, U. I. and Kunze, R. (2003). ARAMEMNON, a novel database for Arabidopsis integral membrane proteins. Plant Physiol 131(1): 16-26.
  11. Simm, S., Fragkostefanakis, S., Paul, P., Keller, M., Einloft, J., Scharf, K. D. and Schleiff, E. (2015). Identification and expression analysis of ribosome biogenesis factor co-orthologs in Solanum lycopersicum. Bioinform Biol Insights 9: 1-17.
  12. Valledor, L. and Weckwerth, W. (2014). An improved detergent-compatible gel-fractionation LC-LTQ-Orbitrap-MS workflow for plant and microbial proteomics. Methods Mol Biol 1072: 347-358.

简介

我们建立并阐述了利用番茄茄子(番茄)的例子丰富经济相关作物的成熟花粉膜蛋白质组的方法。为了分离富含膜蛋白质的花粉蛋白质级分,使用高盐浓度(750mM氯化钠)。然后将富含膜蛋白的级分进行霰弹枪蛋白质组学鉴定蛋白质,然后进行计算机分析,以对所检测的蛋白质进行注释和分类。

背景 由于蛋白质和溶质在不同细胞区室之间的适当分布或将新合成的蛋白质插入膜中很大程度上取决于膜蛋白质,所以膜蛋白质组是维持细胞和细胞器内稳态的核心(Paul等人 2013,2014和2016a)。考虑到膜蛋白的重要性,这些对于花粉功能和发育也是至关重要的(Paul等人,2016b)。许多全球花粉蛋白质组学研究已经在过去进行(Chaturvedi等人,2013和2016);然而,很少讨论关于蛋白质的胞内分布和膜蛋白质组学在花粉中的组成的信息(Pertl et al。,2009)。一个原因可能是膜蛋白的低丰度和溶解度。在这里,我们描述了一种方法,用于分离和分析富含成熟花粉的膜蛋白的蛋白质级分,这是为番茄建立的(图1)。


图1.成熟番茄花粉膜蛋白质组分离方案概述

关键字:膜蛋白质组, 花粉, 番茄, 蛋白质组学

材料和试剂

  1. 1.7ml微管(Corning,Axygen ,目录号:MCT-175-C)
  2. 移液器提示
  3. Cheesecloth Miniwipes(Fisher Scientific,目录号:06-665-28)
  4. 锋利刀片(红魔,目录号:3272)
  5. Bond-Elute C-18 SPEC板(Agilent Technologies,Santa Clara,CA,USA)
  6. 种子种子的选择。对于Solanum lycopersicum的方法开发种子cv。 Moneymaker(Accesion:LA2706)和cv。使用红色设置器
  7. 液氮
  8. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-500)
  9. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P9599)
  10. 6x Laemmli缓冲液(Alfa Aesar,目录号:J61337)
  11. 尿素(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:29700)
  12. 丙烯酰胺/双丙烯酰胺(Fisher Scientific,目录号:BP1408-1)
  13. TEMED(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17919)
  14. 过硫酸铵(Acros Organics,目录号:327081000)
  15. 乙腈(Fisher Scientific,目录号:A9561)
  16. 甲酸(Fisher Scientific,目录号:A117-50)
  17. 硼酸(Fisher Scientific,目录号:A73-500)
  18. 硝酸钙四水合物(Fisher Scientific,目录号:C109-500)
  19. 无水硫酸镁(Fisher Scientific,目录号:M65-500)
  20. 硝酸钾(Fisher Scientific,目录号:P263-500)
  21. 氯化钾(Fisher Scientific,目录号:P217-500)
  22. 二硫苏糖醇(DTT)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0861)
  23. 乙二胺四乙酸,脱水二钠盐(EDTA)(Fisher Scientific,目录号:S311-100)
  24. Tris基(Fisher Scientific,目录号:BP152-500)
  25. 甲醇(Fisher Scientific,目录号:A456-1)
  26. 乙酸(Fisher Scientific,目录号:A507-P500)
  27. 双蒸水
  28. 考马斯亮蓝R-250(Thermo Fisher Scientific,Thermo Scientific TM,目录号:20278)
  29. 胰蛋白酶溶液测序级(Roche Diagnostics,目录号:11418475001)
  30. 碳酸氢铵(Fisher Scientific,目录号:A643-500)
  31. 氯化钙(Fisher Scientific,目录号:C70-500)
  32. 三氟乙酸(TFA)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:28904)
  33. 萌发溶液(见配方)
  34. 均质缓冲液(见配方)
  35. 染色溶液(参见食谱)
  36. 解决方案(见配方)
  37. 胰蛋白酶缓冲液(参见食谱)

设备

  1. 涡旋(例如,Fisher Scientific,型号:Fisher Scientific TM Analog Vortex Mix,目录号:02-215-365)
  2. 离心机(例如,Eppendorf,型号:5424R)
  3. 超速离心机(例如,Thermo Fisher Scientific,型号:Sorvall发现 TM 90 / SE>
  4. 雏菊BB(雏菊,型号:24,目录号:980024-001)
  5. Tissuelyser II(QIAGEN,型号:Tissuelyser II,目录号:85300)
  6. Speed-Vac Labogene ApS(LabGene TM,型号:ScanVac CoolSafe 110-4,目录号:H01130032)
  7. 移液器
  8. 超声波发生器(BANDELIN electronic,型号:Sonorex RK 100,目录号:301.00044253.024)
  9. Precolumn(Eksigent,Redwood City,CA,USA)
  10. Ascentis色谱柱(Sigma-Aldrich,Supelco,型号:Ascentis Express Peptide ES-C18 HPLC柱,目录号:53552-U),尺寸15×100μm,孔径2.7μm
  11. Orbitrap LTQ XL质谱仪(Thermo Fisher Scientific,型号:LTQ XL TM

软件

  1. Proteome Discoverer 1.3(Thermo,德国)或其他软件,如MaxQuant( http://www.maxquant .org / ),Protmax( http:/ /www.univie.ac.at/mosys/software.html )和MASCOT( http://www.matrixscience.com/
  2. PROMEX( http://promex.pph.univie.ac.at / promex / )或其他软件,如ProteomeXchange( http://www.proteomexchange .ORG /

程序

  1. 成熟花粉收获
    1. 在控制的温室条件下(番茄:24℃/ 20℃,白天/晚上; 60日龄)生长植物(例如,以下为番茄cv。Moneymaker-MM,以及cv。Red setter-RS) %湿度(Fragkostefanakis等人,2015)。
      注意:生长条件需要适应正在使用的植物物种或品种。
    2. 在开花后从花芽收获花药(MM和RS的长度> 10 mm),因为它们含有成熟的花粉粒(优选时间是开花后0-72小时)。
    3. 对收集的花药进行水平切割,并将四个花药放入含有500μl发芽液的1.7ml微量管中。
    4. 使用移液管吸头用轻微的机械应力挤压花药组织并涡旋15-20秒,以从花药室释放成熟花粉。
    5. 此外,通过干酪布通过含有分离的成熟花粉粒的发芽溶液以除去另外的碎片
    6. 在4℃下将收集的花粉颗粒以10,000xg离心15分钟。
    7. 取出上清液,用200微升萌发溶液洗涤颗粒状花粉颗粒,并在4℃下以10,000×g离心1分钟。
    8. 取出上清液,重复洗涤步骤A7。
    9. 取出上清液,将收集的花粉浸入液氮中,储存于-80°C直至进一步使用
  2. 隔膜富集级分
    1. 添加金属珠(雏菊BB),帮助将组织均匀化成微管,并用组织培养器II研磨收集的成熟花粉粒(以25 /秒1分钟)。
    2. 加入含有氯化钠(NaCl,750mM)和蛋白酶抑制剂混合物的10-20ml均质缓冲液至研磨的成熟花粉中,重新悬浮并在冰上放置30分钟。
    3. 将匀浆的混合物在10℃下离心,在4℃下离心60分钟
    4. 将沉淀重新悬浮在Laemmli缓冲液中,并定量测定蛋白质浓度。
      注意:上清液富含可溶性蛋白质,如果需要,也可以进行霰弹枪蛋白质组学方案。

  3. 猎枪蛋白质组学
    1. 将40μg总蛋白加载到SDS-PAGE(12.5%,最大厚度为1mm)上,并以80V恒定的凝胶运行1.5cm的解析凝胶(Valledor and Weckwerth,2014)。 Laemmli缓冲区用作加载缓冲区。
    2. 用甲醇:乙酸:水:考马斯亮蓝R-250(40:10:50:0.001)染色凝胶30分钟,并在甲醇:水(40:60)中脱色一夜。然后,从剩余的凝胶中切下厚的堆叠蛋白质条带,并用锋利的刀片将其切成较小的碎片。将切碎的凝胶片浸入双蒸水中,直到所有样品被处理
    3. 然后取出水,加入1ml碳酸氢钠(25mM),37℃孵育15分钟。弃上清,加入1ml 25mM碳酸氢钠和50%乙腈。将样品在37℃孵育15分钟。重复此步骤。
    4. 去除上清液,并在室温下将300μl100%乙腈中的凝胶片脱水5分钟。弃上清液,并在Speed-Vac中干燥5分钟 注意:凝胶片应该具有“透明塑料”的外观。如果凝胶片仍然是半透明的,请重复步骤。
    5. 用50μl胰蛋白酶溶液(10ng /μl)对切碎的凝胶片进行消化,并在37℃下孵育15分钟。如果需要,加入更多的胰蛋白酶溶液,直到凝胶片完全再水合。在37°C孵育14-16 h,避免孵化期间发生振荡。然后,加入5μl的1.0%三氟乙酸(加入TFA后,pH <4)停止反应。
      注意:加入1 ml胰蛋白酶缓冲液,将胰蛋白酶稀释至10 ng /μl。在冰上孵育10分钟,然后上下移液以完全重新悬浮沉淀。稀释后,胰蛋白酶可以在-20°C保存一个月。强烈建议将其存放在等分试样中(Valledor and Weckwerth,2014)。
    6. 对于肽提取,将150μl50%乙腈(v / v)和1%甲酸(v / v)加入到含有凝胶片的微管中,并在室温下孵育5分钟,然后超声处理3分钟(低强度超声波浴)。
    7. 旋转微管(1000×g 1分钟),并将上清液收集在新的微管“A”中。重复步骤C6并将上清液转移到微管“A”
    8. 加入100μl90%乙腈(v / v)和1%甲酸(v / v)到含有凝胶片的微管中,并在室温下孵育5分钟。将上清液转移到微管“A”
    9. 在Speed-Vac(微管A)中干燥样品。长期保存提取的肽-80℃是优选的
    10. 然后,使用规格96孔C18,Agilent(Valledor和Weckwerth,2014)进行肽脱盐。
    11. 将肽溶解于4%(v / v)乙腈,0.1%(v / v)甲酸中。
    12. 将消化的肽注入具有预柱的一维纳流LC-MS / MS系统。
    13. 用Ascentis柱在80分钟内洗脱肽,梯度为5%至50%(v / v)乙腈,0.1%(v / v)甲酸,并在Orbitrap LTQ XL质谱仪上进行质谱分析,控制流量为500 nl每分钟,MS的特定调谐设置如下:喷射电压设置为1.8 kV;加热的转移毛细管的温度设置为180°C。
    14. 在每次完整的MS扫描后,执行10次MS / MS扫描,并动态选择十个最丰富的肽分子离子,动态排除窗口设置为90秒。
    15. 在MS / MS分析的完整MS中,省略+1或未识别电荷状态的离子。

数据分析

  1. 霰弹枪蛋白质组学分析
    1. 使用Proteome Discoverer 1.3版本中的SEQUEST算法搜索获得的原始数据(以质谱的形式)。为此,将识别置信度设置为5%FDR(假发现率)和对N-末端的乙酰化和甲硫氨酸的可变修饰,母体离子的质量公差为10ppm,碎片离子为0.8Da。 br />
    2. 将肽与相应植物物种的基因组序列匹配,在这种情况下为番茄(Sol Genomics Network),并且当肽置信度处于中等或高时考虑到显着的命中,并且以每次充电1为1建立Xcorr阈值(2为+ 2离子,3个用于+3离子,等等)。
      注意:选择高阈值以最小化蛋白质的错误检测。

  2. 膜蛋白的预测
    首先,使用像ARAMEMNON这样的数据库来预测膜蛋白(Schwacke等人,2003)。 ARAMEMNON分别考虑20和11个不同的数据库(或算法)来分别预测α-螺旋和β-桶蛋白。
    1. 如果您感兴趣的植物种类没有在ARAMEMNON中列出,您可以通过成熟的方法预测检测到的蛋白质的共同直系同源物(Simm等人,2015年) )。
    2. 检测到的蛋白质应通过第二个程序(如THMMM)进行验证( http: //www.cbs.dtu.dk/services/TMHMM/ ),其预测基于隐马尔可夫模型的跨膜α螺旋(Krogh等人,2001)。

食谱

  1. 萌发方案
    2 mM硼酸
    2mM硝酸钙四水合物
    2毫摩尔无水硫酸镁
    1mM硝酸钾/ / 准备100ml混合物:
    0.012g硼酸
    0.047g硝酸钙四水合物
    0.024克无水硫酸镁 0.010g硝酸钾/ /
  2. 均质缓冲液
    100 mM氯化钾 5 mM DTT
    1 mM EDTA
    50 mM Tris pH 7.2
    750 mM氯化钠
    准备100ml混合物:
    0.745克氯化钾
    0.077g DTT
    0.029g EDTA
    0.605g Tris pH7.2
    注意:根据匀浆缓冲液,最后加入氯化钠;对于20ml均质缓冲液,加入0.876g氯化钠。
  3. 染色溶液
    甲醇:乙酸:水:考马斯亮蓝R-250(40:10:50:0.001)
  4. 解决方案
    甲醇和水(40:60)
  5. 胰蛋白酶缓冲液
    25 mM碳酸氢钠
    10%(v / v)乙腈 5 mM氯化钙 准备100ml混合物:
    0.21克碳酸氢钠
    100μl纯乙腈
    645微升水
    注意:根据胰蛋白酶缓冲液,最后添加钙。对于10ml胰蛋白酶缓冲液,加入0.005克氯化钙。

致谢

该协议来自(Paul等人,2016a)。这是由Marie-Curie(欧盟赠款协议号289220)资助的SPOT-ITN联盟的一篇文章。

参考

  1. Chaturvedi,P.,Ghatak,A.和Weckwerth,W。(2016)。花粉蛋白质组学:从压力生理学到发育启动。植物生殖发育 29(1-2):119-32。
  2. Chaturvedi,P.,Ischebeck,T.,Egelhofer,V.,Lichtscheidl,I。和Weckwerth,W。(2013)。&nbsp; 从花粉母细胞到成熟花粉的番茄花粉蛋白质组的细胞特异性分析为发育启动提供了证据。 em> 12(11):4892-4903。
  3. Fragkostefanakis,S.,Simm,S.,Paul,P.,Bublak,D.,Scharf,KD和Schleiff,E.(2015)。&nbsp; 通过转录组分析和微阵列荟萃分析研究的Solanum lycopersicum中的分子伴侣网络组合植物细胞环境38(4):693-709。
  4. Krogh,A.,Larsson,B.,von Heijne,G.and Sonnhammer,EL(2001)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov / pubmed / 11152613“target =”_ blank“>使用隐马尔可夫模型预测跨膜蛋白拓扑:应用于完整的基因组。 J Mol Biol 305(3):567-580。 br />
  5. Paul,P.,Chaturvedi,P.,Selymesi,M.,Ghatak,A.,Mesihovic,A.,Scharf,KD,Weckwerth,W.,Simm,S。和Schleiff,E.(2016a)一个class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26455813”target =“_ blank”>茄属茄子中雄配子体的膜蛋白质组> 蛋白质组学 131:48-60。
  6. Paul,P.,Roth,S。和Schleiff,E.(2016b)。细胞器蛋白质的重要性,蛋白质易位和囊泡运输途径,用于花粉发育和功能。植物生长 29(1-2):53-65。 >
  7. Paul,P.,Simm,S.,Blaumeiser,A.,Scharf,KD,Fragkostefanakis,S.,Mirus,O. and Schleiff,E。(2013)。&lt; a class =“ke-insertfile”href = “http://www.ncbi.nlm.nih.gov/pubmed/23506162”target =“_ blank”>植物中的蛋白质易位系统 - 茄子茄子的实例的组成和变异性 / a> BMC基因组学 14:189.
  8. Paul,P.,Simm,S.,Mirus,O.,Scharf,KD,Fragkostefanakis,S。和Schleiff,E.(2014)。通过正交搜索检查的植物中囊泡运输因子的复杂性 9(5) :e97745。
  9. Pertl,H.,Schulze,WX和Obermeyer,G。(2009)。花粉细胞膜蛋白质组在百合花粉的发芽和管生长期间显示出高度的空间 - 时间动力学.J蛋白质组Res。8(11):5142-5152。 />
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引用:Paul, P., Chaturvedi, P., Mesihovic, A., Ghatak, A., Weckwerth, W. and Schleiff, E. (2017). Protocol for Enrichment of the Membrane Proteome of Mature Tomato Pollen. Bio-protocol 7(11): e2315. DOI: 10.21769/BioProtoc.2315.
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