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Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation
用于强阳离子交换分馏的浆膜蛋白质的分离、消化和处理

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Abstract

Plasma membrane (PM) proteins play crucial roles in diverse biological processes. But their low abundance, alkalinity and hydrophobicity make their isolation a difficult task. This protocol describes an efficient method for PM proteins isolation, digestion and fractionation so that they can be well prepared for mass spectrometry analysis.

Keywords: Plasma membrane proteins(浆膜蛋白), RapiGest SF(RapiGest SF), Trypsin(胰蛋白酶), Strong cation exchange(强阳离子交换), Fractionation(分馏)

Background

Plasma membrane (PM) proteins participate in diverse biological processes including signal transduction, ion transport and membrane trafficking, and are the first responders in cell-environment communication. They have a complicated composition varying from species, cell types and developmental stages (Alexandersson et al., 2008). Revealing their components and the expression features comprehensively with mass spectrometry (MS) is of great importance for developmental biology. However, their hydrophobic nature and the low abundance are a big challenge for the proteomic analysis (Wu and Yates, 2003). Additives like normal surfactants, organic solvents and urea are often used to improve PM proteins’ solubility, but they will reduce the proteases’ activities and create ion suppression during MS analysis (Zhang, 2015). RapiGest SF is a novel acid-labile anionic surfactant, which is structurally and functionally similar to SDS but does not inhibit the common endopeptidases activities at low concentration (0.1% w/v). Thus RapiGest SF used in solubilizing PM proteins can not only facilitate their digestion by exposing cleavage sites but is also easily quenched by strong acid and removed through centrifugation so that the surfactant does not affect the MS identification (Yu et al., 2003). Peptides yield by the RapiGest SF-assisted digestion can directly undergo the strong cation exchange (SCX) fractionation (Yang and Wang, 2017), so that the low abundance peptides can be detected by the MS.

Materials and Reagents

  1. 1.5 ml tubes (Corning, Axygen®, catalog number: MCT-150-C )
  2. ZipTip® pipette tip (Merck Millipore, catalog number: ZTC18S096 )
  3. PierceTM Spin columns, screw cap (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 69705 )
  4. C18 (FUJIGEL HANBAI, catalog number: MB 100 - 40/75 )
  5. Tris (2-carboxyethyl) phosphine hydrochloride solution, 0.5 M, pH 7.0 (TCEP) (Sigma-Aldrich, catalog number: 646547 )
  6. Formic acid (Sigma-Aldrich, catalog number: 94318 )
  7. Acetonitrile (Sigma-Aldrich, catalog number: 34851 )
  8. RapiGest SF surfactant (WATERS, catalog number: 186001861 )
  9. Iodoacetamide (Sigma-Aldrich, catalog number: I1149 )
  10. Trypsin (Roche Diagnositics, catalog number: 11418025001 )
  11. Potassium phosphate dibasic trihydrate (K2HPO4·3H2O)
  12. Potassium phosphate monobasic (KH2PO4)
  13. Phosphoric acid (H3PO4)
  14. Ammonium chloride (Sigma-Aldrich, catalog number: A9434 )
  15. Hydrochloric acid (HCl)
  16. 0.5 M iodoacetamide stock (see Recipes)
  17. 0.1 μg/μl trypsin stock (see Recipes)
  18. 50 mM potassium phosphate buffer, pH 7.8 (see Recipes)
  19. 100 ml solvent A (5 mM NH4Cl, 25% [v/v] acetonitrile, pH 3.0) (see Recipes)
  20. 100 ml solvent B (500 mM NH4Cl, 25% [v/v] acetonitrile, pH 3.0) (see Recipes)

Equipment

  1. 2 μl, 10 μl, 100 μl, 1,000 μl Pipetman (Gilson, France)
  2. Optimal MAX-XP ultracentrifuge (Beckman Coulter, model: Optimal MAX-XP )
  3. AKTA purifier-10 (GE Healthcare, model: AKTApurifier 10 )
  4. ISS 110 SpeedVac System (Thermo Fisher Scientific, Thermo ScientificTM, model: ISS 110 )
  5. PolySULFOETHYL ATM columns, 2.1 x 200 mm, 5 μm particles, 300 Å poresize (PolyLC, catalog number: 202SE05 )

Software

  1. UNICORN 5.2 software (GE Healthcare, USA)

Procedure

  1. Plasma membrane protein preparation
    1. Suspend the plasma membrane (PM) pellets (isolated according to Han et al., 2010) with 100 μl 50 mM potassium phosphate buffer, pH 7.8.
      Note: The recommended PM protein concentration is 1 μg/μl. This protocol set 100 μg PM protein as an example, so use 100 μl buffer to suspend the pellets.
    2. Add 25 μl 1% (w/v) RapiGest SF stock to the working concentration of 0.2% (w/v), mix well and then boil the mixture at ~100 °C for 5 min to dissolve proteins from PM.
      Note: RapiGest SF is an acid labile denaturant (Figure 1A) which could solubilize and unfold proteins to make them more amenable for cleavage (Figure 1B). The recommended RapiGest SF working concentration is 0.1% (w/v). For PM proteins increase the incubation time (at 37 °C, up to 1 h is safe; at 100 °C, 5 min is enough) or use higher RapiGest SF concentration (up to 0.5% [w/v] is safe) could make the following digestion more efficient.


      Figure 1. Working principle of RapiGest SF surfactant. A. RapiGest SF can be easily broken down by acidification and then removed by centrifugation. B. RapiGest SF is a mild denaturant which making proteins more amenable for cleavage without disrupting the endoproteases activity. Pictures were downloaded from the official website of Waters: (http://www.waters.com/waters/en_US/RapiGestSFSurfactant/nav.htm?locale=/&cid=1000941)

    3. Cool down the mixture to room temperature (RT) for the following digestion.

  2. Plasma membrane protein digestion
    1. Reduction: add 2.5 μl 0.5 M TCEP, pH 7.0 to the protein mixture to a working concentration of 10 mM, mix them gently and then incubate at 56 °C for 1 h.
    2. Alkylation: cool down the mixture to RT, and add 14 μl 0.5 M iodoacetamide stock to a working concentration of 50 mM, mix them gently and then incubate at RT, dark, for 45 min.
    3. Digestion: add 20 μl 0.1 μg/μl trypsin stock to the protein mixture to a working concentration of 1:50 (w/w), mix them gently and then incubate at 37 °C for 12 h.
    4. Acidification: add 0.5 μl formic acid to the mixture to a working concentration of 0.2-0.5% (v/v) (pH < 2), incubate at 37 °C for 30-45 min, then centrifuge at 4 °C, 20,000 x g for 20 min to remove RapiGest SF. Discharge the pellets, transfer the supernatant to a new tube.
    5. Desalting: use C18 filled spin column to concentrate and purify the peptides mixture.
      Note: If the peptides mixture is less than 5.0 μg, please use ZipTip® pipette tip to do the desalting.
    6. Put the purified peptides mixture into SpeedVac system to lyophilize for the following fractionation use.

  3. Peptides mixture fractionation
    The following protocol was modified from Zhu et al. (2009). It performs on the AKTA purifier-10 system with the strong cation exchange (SCX) PolySULFOETHYL ATM column.
    1. Resuspend the lyophilized peptides mixture in solvent A.
    2. Run solvent B for 20 min, then solvent A for 30 min through the AKTA purifier-10 system to balance the PolySULFOETHYL ATM column.
    3. Load the suspended peptides and run the following gradient at a flow rate of 200 μl/min, collect the products in every 2 min.
      0-5 min: 100% solvent A
      5-95 min: 0-60% solvent B
      95-115 min: 60%-100% solvent B
      115-145 min: 100% solvent B
      145-155 min: 100% solvent A
    4. Run ddH2O for 20 min, then 100% acetonitrile for 10 min to store the column.
    5. Pool the products according to the 214 nm UV absorption peak (Figure 2).


      Figure 2. SCX fractionation of peptides mixture from rice mature pollen grain. According to the 214 nm UV absorption peak, the peptides mixture can be pooled into 14 fractions for LC-MS/MS analysis.

    6. Use C18 spin column or ZipTip® pipette tip to purify the combined products.
    7. Lyophilize the purified products for mass spectrometry identification.

Data analysis

The SCX separated peptides were monitored by their 214 nm UV absorption peak through the UNICORN 5.2 software (GE Healthcare, USA).

Notes

After SCX fractionation, the repooled peptides contains a high concentration of salts. Don’t forget to do the desalting procedure through C18 spin column or ZipTip® pipette tip, or the following mass spectrometry identification will be affected.

Recipes

  1. 1% (w/v) RapiGest SF stock
    Dissolve 1 mg of lyophilized RapiGest SF powder in 100 μl ddH2O, store at -20 °C
  2. 0.5 M iodoacetamide stock
    Dissolve 46 mg of iodoacetamide powder in 500 μl ddH2O, use immediately
  3. 0.1 μg/μl trypsin stock
    Dissolve 25 μg of trypsin powder in 250 μl ddH2O, store at -20 °C
  4. 200 ml 50 mM potassium phosphate buffer, pH 7.8
    2.072 g K2HPO4·3H2O
    0.125 g KH2PO4
    Fill up to 200 ml with ddH2O
    Adjust pH to 7.8 with H3PO4
  5. 100 ml solvent A (5 mM NH4Cl, 25% [v/v] acetonitrile, pH 3.0)
    0.027 g NH4Cl
    25 ml acetonitrile
    Fill up to 100 ml with ddH2O
    Adjust pH to 3.0 with HCl
  6. 100 ml solvent B (500 mM NH4Cl, 25% [v/v] acetonitrile, pH 3.0)
    2.675 g NH4Cl
    25 ml acetonitrile
    Fill up to 100 ml with ddH2O
    Adjust pH to 3.0 with HCl

Acknowledgments

This protocol was mainly modified from Han et al. (2010) and Yang and Wang (2017). This work was supported by the Chinese Ministry of Science and Technology (grant No. 2013CB945101) and the China Postdoctoral Science Foundation (grant No. 2016M591284).

References

  1. Alexandersson, E., Gustavsson, N., Bernfur, K., Karlsson, A., Kjellbom, P. and Larsson, C. (2008). Purification and proteomic analysis of plant plasma membranes. Methods Mol Biol 432: 161-173.
  2. Han, B., Chen, S., Dai, S., Yang, N. and Wang, T. (2010). Isobaric tags for relative and absolute quantification- based comparative proteomics reveals the features of plasma membrane-associated proteomes of pollen grains and pollen tubes from Lilium davidii. J Integr Plant Biol 52(12): 1043-1058.
  3. Wu, C. C. and Yates, J. R., 3rd (2003). The application of mass spectrometry to membrane proteomics. Nat Biotechnol 21(3): 262-267.
  4. Yang, N. and Wang, T. (2017). Comparative proteomic analysis reveals a dynamic pollen plasma membrane protein map and the membrane landscape of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils in rice. BMC Plant Biol 17(1): 2.
  5. Yu, Y. Q., Gilar, M., Lee, P. J., Bouvier, E. S. and Gebler, J. C. (2003). Enzyme-friendly, mass spectrometry-compatible surfactant for in-solution enzymatic digestion of proteins. Anal Chem 75(21): 6023-6028.
  6. Zhang, X. (2015). Less is more: Membrane protein digestion beyond urea-trypsin solution for next-level proteomics. Mol Cell Proteomics 14(9): 2441-2453.
  7. Zhu, M., Dai, S., McClung, S., Yan, X. and Chen, S. (2009). Functional differentiation of Brassica napus guard cells and mesophyll cells revealed by comparative proteomics. Mol Cell Proteomics 8(4): 752-766.

简介

血浆膜(PM)蛋白在不同生物过程中起关键作用。但是,他们的低丰度,碱度和疏水性使其隔离成为一项艰巨的任务。该方案描述了PM蛋白分离,消化和分级的有效方法,以便它们可以很好地准备用于质谱分析。

背景 血浆膜(PM)蛋白参与不同的生物学过程,包括信号转导,离子运输和膜运输,是细胞与环境交流中的第一反应者。它们具有从物种,细胞类型和发育阶段变化的复杂组成(Alexandersson等人,2008)。用质谱(MS)全面揭示其组分和表达特征对发育生物学非常重要。然而,它们的疏水性和低丰度对于蛋白质组学分析是一个很大的挑战(Wu和Yates,2003)。通常使用像正常表面活性剂,有机溶剂和尿素这样的添加剂来改善PM蛋白的溶解度,但它们会降低蛋白酶的活性并在MS分析过程中产生离子抑制(Zhang,2015)。 Rapi Gest SF是一种新型酸不稳定的阴离子表面活性剂,其结构和功能上与SDS类似,但不抑制低浓度(0.1%w / v)下的常见内肽酶活性。因此,Rapi 用于增溶PM蛋白的Gest SF不仅可以通过暴露裂解位点促进它们的消化,而且还容易被强酸淬灭并通过离心去除,使表面活性剂不影响MS鉴定(Yu 等人,2003)。通过Rapi Gest SF辅助消化的肽产量可以直接进行强阳离子交换(SCX)分离(Yang和Wang,2017),从而可以通过MS检测低丰度肽。

关键字:浆膜蛋白, RapiGest SF, 胰蛋白酶, 强阳离子交换, 分馏

材料和试剂

  1. 1.5ml管(Corning,Axygen ,目录号:MCT-150-C)
  2. ZipTip ®移液器吸头(Merck Millipore,目录号:ZTC18S096)
  3. Pierce TM旋转柱,螺帽(Thermo Fisher Scientific,Thermo Scientific TM,目录号:69705)
  4. C18(FUJIGEL HANBAI,目录号:MB 100 - 40/75)
  5. 0.5M,pH 7.0(TCEP)(Sigma-Aldrich,目录号:646547)的三(2-羧乙基)膦盐酸盐溶液
  6. 甲酸(Sigma-Aldrich,目录号:94318)
  7. 乙腈(Sigma-Aldrich,目录号:34851)
  8. Rapi Gest SF表面活性剂(WATERS,目录号:186001861)
  9. 碘乙酰胺(Sigma-Aldrich,目录号:I1149)
  10. 胰蛋白酶(Roche Diagnositics,目录号:11418025001)
  11. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)
  12. 磷酸二氢钾(KH 2 PO 4)
  13. 磷酸(H 3 3 PO 4)
  14. 氯化铵(Sigma-Aldrich,目录号:A9434)
  15. 盐酸(HCl)
  16. 0.5 M碘乙酰胺原料(见配方)
  17. 0.1μg/μl胰蛋白酶原料(见食谱)
  18. 50mM磷酸钾缓冲液,pH 7.8(参见食谱)
  19. 100ml溶剂A(5mM NH 4 Cl,25%[v/v]乙腈,pH3.0)(参见食谱)
  20. 100ml溶剂B(500mM NH 4 Cl,25%[v/v]乙腈,pH 3.0)(参见食谱)

设备

  1. 2μl,10μl,100μl,1000μlPipetman(Gilson,France)
  2. 最佳MAX-XP超速离心机(Beckman Coulter,型号:Optimal MAX-XP)
  3. AKTA净化器-10(GE Healthcare,型号:AKTApurifier 10)
  4. ISS 110 SpeedVac系统(Thermo Fisher Scientific,Thermo Scientific TM ,型号:ISS 110)
  5. PolySULFOETHYL A TM柱,2.1×200mm,5μm颗粒,300A孔(PolyLC,目录号:202SE05)

软件

  1. UNICORN 5.2软件(GE Healthcare,USA)

程序

  1. 血浆膜蛋白制备
    1. 用100μl50mM磷酸钾缓冲液(pH7.8)悬浮质膜(PM)颗粒(根据Han等人,2010)分离。
      注意:推荐的PM蛋白浓度为1μg/μl。该方案设定100μgPM蛋白为例,因此使用100μl缓冲液悬浮颗粒。
    2. 加入25μl1%(w/v)的Rapi Gest SF储备液至0.2%(w/v)的工作浓度,充分混合,然后在〜100℃下将混合物煮沸5分钟从PM溶解蛋白质。
      注意:RapiGest SF是一种酸不稳定变性剂(图1A),可以溶解和展开蛋白质,使其更易于切割(图1B)。推荐的RapiGest SF工作浓度为0.1%(w/v)。对于PM蛋白质,增加孵育时间(在37℃,至少1小时是安全的;在100℃,5分钟就足够了)或使用更高的RapiGest SF浓度(高达0.5%[w/v]是安全的)使以下消化更有效率。


      图1. Rapi的工作原理 Gest SF表面活性剂。 A. Rapi Gest SF可以通过酸化容易地分解,然后通过离心除去。 B. Rapi Gest SF是一种温和的变性剂,其使蛋白质更适合于切割而不破坏内切蛋白酶活性。图片是从Waters官方网站下载的:( http://www.waters.com/waters/en_US/RapiGestSFSurfactant/nav.htm?locale=/&cid=1000941

    3. 将混合物冷却至室温(RT)以进行以下消化。

  2. 血浆膜蛋白消化
    1. 减少:向蛋白质混合物中加入2.5μl0.5M TCEP,pH7.0至10mM的工作浓度,轻轻混合,然后在56℃温育1小时。
    2. 烷基化:将混合物冷却至室温,并加入14μl0.5M碘乙酰胺原液至50mM的工作浓度,轻轻混合,然后在室温,暗处孵育45分钟。
    3. 消化:向蛋白质混合物中加入20μl0.1μg/μl胰蛋白酶原料,工作浓度为1:50(w/w),轻轻混合,然后在37℃下孵育12小时。
    4. 酸化:向混合物中加入0.5μl甲酸至工作浓度为0.2-0.5%(v/v)(pH <2),37℃孵育30-45分钟,然后在4℃离心20,000 xg 20分钟,以移除 Gest SF。排出小丸,将上清液转移到新管上
    5. 脱盐:使用C18填充的旋转柱浓缩和纯化肽混合物 注意:如果肽混合物小于5.0μg,请使用ZipTip ®移液管进行脱盐。
    6. 将纯化的肽混合物放入SpeedVac系统中冻干以进行以下分级分离。

  3. 肽混合物分馏
    以下协议由Zhu等人修改。 (2009年)。它在具有强阳离子交换(SCX)PolySULFOETHYL A TM 列的AKTA净化器-10系统上进行。
    1. 将冻干的肽混合物重新悬浮在溶剂A中
    2. 运行溶剂B 20分钟,然后通过AKTA净化器-10系统溶剂A 30分钟,以平衡PolySULFOETHYL A TM 柱。
    3. 加载悬浮肽并以200μl/min的流速运行以下梯度,每2分钟收集一次产品。
      0-5分钟:100%溶剂A
      5-95分钟:0-60%溶剂B
      95-115分钟:60%-100%溶剂B
      115-145分钟:100%溶剂B
      145-155分钟:100%溶剂A
    4. 运行ddH 2 O 20分钟,然后运行100%乙腈10分钟以存储色谱柱。
    5. 根据214 nm紫外吸收峰积分产品(图2)

      图2.来自水稻成熟花粉颗粒的肽混合物的SCX分级。根据214nm UV吸收峰,可以将肽混合物合并成14个级分用于LC-MS/MS分析。 >
    6. 使用C18旋转柱或ZipTip ®移液器尖端净化组合的产品。
    7. 对纯化产物进行冻干以进行质谱鉴定。

数据分析

通过UNICORN 5.2软件(GE Healthcare,USA),通过其214nm UV吸收峰监测SCX分离的肽。

笔记

在SCX分馏后,转化后的肽含有高浓度的盐。不要忘记通过C18旋转柱或ZipTip ®移液器尖端进行脱盐程序,否则将影响以下质谱鉴定。

食谱

  1. 1%(w/v)Rapi Gest SF股票
    将1mg冻干的Rapi/Gest SF粉末溶于100μlddH 2 O中,储存在-20℃下。
  2. 0.5M碘乙酰胺原料
    将46mg碘乙酰胺粉末溶于500μlddH 2 O中,立即使用
  3. 0.1μg/μl胰蛋白酶库存
    将25μg胰蛋白酶粉末溶于250μlddH 2 O中,储存在-20℃下。
  4. 200毫升50毫克磷酸钾缓冲液,pH 7.8。
    2.072g K 2 HPO 4·3H 2 O
    0.125g KH 2 PO 4
    填充高达200毫升与ddH 2 O
    用H 3 PO 4将调节至7.8
  5. 100ml溶剂A(5mM NH 4 Cl,25%[v/v]乙腈,pH3.0)
    0.027g NH 4 Cl
    25ml乙腈
    用ddH 2 O
    填充100 ml 用HCl调节pH至3.0
  6. 100ml溶剂B(500mM NH 4 Cl,25%[v/v]乙腈,pH 3.0)
    2.675g NH 4 Cl
    25ml乙腈
    用ddH 2 O
    填充100 ml 用HCl调节pH至3.0

致谢

这个协议主要是从Han等人修改的。 (2010),杨和王(2017)。这项工作得到了中国科技部(授权号:2013CB945101)和中国博士后科学基金(授权号:2016M591284)的支持。

参考

  1. Alexandersson,E.,Gustavsson,N.,Bernfur,K.,Karlsson,A.,Kjellbom,P.和Larsson,C。(2008)。植物血浆膜的纯化和蛋白质组学分析。方法Mol Biol 432:161-173。
  2. Han,B.,Chen,S.,Dai,S.,Yang,N。和Wang,T。(2010)。用于相对和绝对定量的比较蛋白质组学的等压标签揭示了百合花粉花粉粒和花粉管的质膜相关蛋白质组的特征, em> .J Integr Plant Biol 52(12):1043-1058。
  3. Wu,CC和Yates,JR,3rd(2003)。  质谱法在膜蛋白质组学中的应用 Nat Biotechnol 21(3):262-267。
  4. Yang,N.和Wang,T.(2017)。比较蛋白质组学分析显示动态花粉质膜蛋白图谱和受体样激酶和转运蛋白的膜景观对于花粉管生长和水稻雌蕊相互作用是重要的。 BMC植物生物学 17 (1):2.
  5. Yu,YQ,Gilar,M.,Lee,PJ,Bouvier,ES and Gebler,JC(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/14588046"target ="_ blank">用于溶解酶促消化蛋白质的酶友好的质谱相容表面活性剂。分析化学75(21):6023- 6028.
  6. Zhang,X.(2015)。  Less more more膜蛋白消化超出尿素 - 胰蛋白酶溶液用于下一级蛋白质组学。细胞蛋白质组学 14(9):2441-2453。
  7. Zhu,M.,Dai,S.,McClung,S.,Yan,X.和Chen,S。(2009)。通过比较蛋白质组学揭示的欧洲油菜保护细胞和叶肉细胞的功能分化。细胞蛋白质组学 em> 8(4):752-766。
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引用:Yang, N., Han, B. and Wang, T. (2017). Protein Isolation from Plasma Membrane, Digestion and Processing for Strong Cation Exchange Fractionation. Bio-protocol 7(10): e2298. DOI: 10.21769/BioProtoc.2298.
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