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
发布: 2017年06月05日第7卷第11期 DOI: 10.21769/BioProtoc.2315 浏览次数: 8073
评审: Samik BhattacharyaNing LiuAnonymous reviewer(s)
PDF 
Q&A
参见作者原研究论文
The authors used this protocol in:
Jan 2016
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
- 1.7 ml microtubes (Corning, Axygen®, catalog number: MCT-175-C )
- Pipette tips
- Cheesecloth Miniwipes (Fisher Scientific, catalog number: 06-665-28 )
- Sharp blade (Red Devil, catalog number: 3272 )
- Bond-Elute C-18 SPEC plate (Agilent Technologies, Santa Clara, CA, USA)
- Seeds of the cultivar of choice. For the method development–seeds of Solanum lycopersicum cv. Moneymaker (Accesion: LA2706) and cv. Red setter were used
- Liquid nitrogen
- Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500 )
- Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P9599 )
- 6x Laemmli buffer (Alfa Aesar, catalog number: J61337 )
- Urea (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 29700 )
- Acrylamide/bis-acrylamide (Fisher Scientific, catalog number: BP1408-1 )
- TEMED (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17919 )
- Ammonium persulfate (Acros Organics, catalog number: 327081000 )
- Acetonitrile (Fisher Scientific, catalog number: A9561 )
- Formic acid (Fisher Scientific, catalog number: A117-50 )
- Boric acid (Fisher Scientific, catalog number: A73-500 )
- Calcium nitrate tetrahydrate (Fisher Scientific, catalog number: C109-500 )
- Magnesium sulphate anhydrous (Fisher Scientific, catalog number: M65-500 )
- Potassium nitrate (Fisher Scientific, catalog number: P263-500 )
- Potassium chloride (Fisher Scientific, catalog number: P217-500 )
- Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
- Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA) (Fisher Scientific, catalog number: S311-100 )
- Tris base (Fisher Scientific, catalog number: BP152-500 )
- Methanol (Fisher Scientific, catalog number: A456-1 )
- Acetic acid (Fisher Scientific, catalog number: A507-P500 )
- Double distilled water
- Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20278 )
- Trypsin solution sequencing grade (Roche Diagnostics, catalog number: 11418475001 )
- Ammonium bicarbonate (Fisher Scientific, catalog number: A643-500 )
- Calcium chloride (Fisher Scientific, catalog number: C70-500 )
- Trifluoroacetic acid (TFA) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28904 )
- Germination solution (see Recipes)
- Homogenization buffer (see Recipes)
- Staining solution (see Recipes)
- Destaining solution (see Recipes)
- Trypsin buffer (see Recipes)
Equipment
- Vortex (e.g., Fisher Scientific, model: Fisher ScientificTM Analog Vortex Mix , catalog number: 02-215-365)
- Centrifuge (e.g., Eppendorf, model: 5424 R )
- Ultracentrifuge (e.g., Thermo Fisher Scientific, model: Sorvall® DiscoveryTM 90 SE )
- Daisy BBs (Daisy, model: 24, catalog number: 980024-001 )
- Tissuelyser II (QIAGEN, model: Tissuelyser II, catalog number: 85300 )
- Speed-Vac Labogene ApS (LabGeneTM, model: ScanVac CoolSafe 110-4 , catalog number: H01130032)
- Pipette
- Sonicator (BANDELIN electronic, model: Sonorex RK 100 , catalog number: 301.00044253.024)
- Precolumn (Eksigent, Redwood City, CA, USA)
- 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
- Orbitrap LTQ XL mass spectrometer (Thermo Fisher Scientific, model: LTQ XLTM )
Software
- 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/)
- PROMEX (http://promex.pph.univie.ac.at/promex/) or other software like ProteomeXchange (http://www.proteomexchange.org/)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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.
Download Citation in RIS Format
分类
植物科学 > 植物生物化学 > 蛋白质
生物化学 > 蛋白质 > 分离和纯化
您对这篇实验方法有问题吗？
在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。
提问指南
+ 问题描述
写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。
发布问题
