JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생화학

通过亲和色谱与西布洛特或质谱结合的亲致色谱,识别磷酸磷酸磷酸磷酸醇或磷酸蛋白

Published: July 26, 2019
doi:
10.3791/59865
1Institute of Parasitology,McGill University

Summary

该协议侧重于识别与磷酸肌醇或磷酸磷酸剂结合的蛋白质。它使用亲和色谱与生物异化异酸醇磷酸盐或磷酸磷酸酯,通过链球菌固定到胶质或磁珠。磷酸磷酸磷酸磷酸磷酸磷酸二醇结合蛋白通过西方印迹或质谱法鉴定。

Abstract

肌醇磷酸盐和磷酸磷化物调节真核细胞中的多个细胞过程,包括基因表达、囊泡贩运、信号转导、代谢和发育。这些代谢物通过与蛋白质结合来执行此调节活性,从而改变蛋白质构象、催化活性和/或相互作用。此处描述的方法使用与质谱或西方印迹耦合的亲和色谱法来识别与磷酸肌醇或磷酸磷酸醇相互作用的蛋白质。肌醇磷酸盐或磷酸磷酸剂与生物锡在化学上标记,然后通过链球菌与甘蔗或磁珠结合捕获。蛋白质通过与代谢物结合的亲和力进行分离,然后通过质谱或西方印迹进行洗脱和识别。该方法具有一个简单的工作流程,是敏感的,非放射性的,无脂质体,可定制的,支持蛋白质和代谢物相互作用的精确分析。这种方法可用于无标签或氨基酸标签的定量质谱方法,以识别复杂生物样品中的蛋白质-代谢物相互作用或使用纯化蛋白质。该协议针对分析来自锥虫病的布鲁氏蛋白进行了优化,但可以适应相关的原生动物寄生虫、酵母或哺乳动物细胞。

Introduction

肌醇磷酸盐 (IP) 和磷酸磷酸(PIs) 通过调节细胞过程(如控制基因表达1、2、3、囊泡贩运)在真核生物中起着核心作用4,信号转导5,6,代谢7,8,9,和发育8,10。这些代谢物的调节功能源于它们与蛋白质相互作用的能力,从而调节蛋白质功能。当蛋白质结合后,IP和P可能改变蛋白质构象11,催化活性12,或相互作用13,从而影响细胞功能。IP 和 P 分布在多个亚细胞室中,如核2、3、14、15、内质神经质16、17、等离子体膜1和细胞醇18,要么与蛋白质3,19或与RNA20相关。

磷脂酶C的膜相关PI(4,5)P2的裂解导致Ins(1,4,5)P3的释放,该P3可分别通过IP激酶和磷酸酶进行磷酸化或脱磷。IP 是可溶性分子,可与蛋白质结合并发挥调节功能。例如,元群中的Ins(1,4,5)P3可以通过与IP3受体结合,作为第二信使,诱导受体构象变化,从而从细胞内存储11释放Ca2+。Ins(1,3,4,5)P4与组蛋白脱乙酰酶复合物结合,并调节蛋白质复合组装和活性13。IP调节功能的其他例子包括染色质组织控制21、RNA传输22、23、RNA编辑24和转录1、2、3.相反,PIs通常与血浆膜或细胞膜25中蛋白质的招募有关。然而,PIs的一个新兴特性是在非膜环境中与蛋白质关联的能力3,15,19,26。这是核受体类固醇因子的情况,其转录控制功能受PI(3,4,5)P319和聚A聚合酶的酶,酶活性由核PI(4,5)P226调节。IP和P的调节作用已在许多生物体中显现出来,包括酵母22,27,哺乳动物细胞19,23,果蝇10和蠕虫28。重要的是这些代谢物在锥体中的作用,它很早就偏离了真核系。这些代谢物在锥虫细胞瘤布鲁氏转录控制1,3,发育8,细胞器生物发生和蛋白质流量29,30中起重要作用,31,32,还参与控制病原体T.cruzi33、34、35、弓形虫36和疟原虫的发育和感染5,37.因此,了解IP和P在锥体中的作用可能有助于阐明这些分子的新生物功能,并确定新的药物靶点。

蛋白质和IP或PI结合的特异性取决于蛋白质相互作用域和肌醇13、38的磷酸化状态,尽管与PI脂质部分的相互作用也发生19。I 和 P 的种类及其修饰的激酶和磷酸酶为控制蛋白质功能提供了灵活的细胞机制,该机制受代谢物可用性和丰度、肌醇磷酸化状态和蛋白质的影响交互的亲和力 1,3,13,38。虽然某些蛋白质域具有很好的特征39、40、41, 例如, 普列克斯特林同源域42和 SPX (S YG1/Pho81/XPR1) 域43 ,44,45,一些蛋白质与IP或P通过机制仍然未知相互作用。例如,T.Brucei的抑制剂激活蛋白1(RAP1)缺乏规范的PI结合域,但与PI(3,4,5)P3相互作用,并控制与抗原变异3有关的基因的转录。对来自锥体、酵母或哺乳动物细胞的IP或PI相互作用蛋白的亲和色谱和质谱分析,确定了几种没有已知IP或PI结合域的几种蛋白质8,46。47.数据表明,与这些代谢物结合的其他未定性蛋白质域。因此,识别与IP或P相互作用的蛋白质可能会揭示蛋白质-代谢物相互作用的新机制,以及这些小分子的新细胞调节功能。

此处描述的方法采用亲和色谱与西方印迹或质谱相结合,以识别与 IP 或 P 结合的蛋白质。它使用生物回位化 IP 或 P,它们要么与链球菌珠联结成交结,要么通过链球蛋白结合的磁珠捕获(图 1)。该方法提供了一个简单的工作流程,是敏感的,非放射性的,无脂素体,适合检测来自细胞泄漏或纯化蛋白质3的蛋白质结合(图2)。该方法可用于无标签8、46或与氨基酸标签定量质谱47耦合,以识别复杂生物样品中的IP或PI结合蛋白。因此,这种方法是研究IP或P与细胞蛋白相互作用的少数方法的替代方法,将有助于了解这些代谢物在锥体中,也许在其他真核生物中的调节功能。

Protocol

1. 通过亲和色谱和西方印迹分析IP或PI结合蛋白 细胞生长、致莱和亲和色谱 将T.布鲁氏细胞生长到中日志阶段,并监测细胞的存活性和密度。总共 5.0 x 107个细胞足以进行一次结合测定。 对于血液形式,在HMI-9培养基中生长细胞,在37°C和5%CO2下辅以10%的胎儿牛血清(FBS)。使细胞密度保持在 8.0 x 105至 1.6 x 106细胞/mL 之间。注:密度?…

Representative Results

亲和力色谱与西方印迹对RAP1和PI(3,4,5)P3相互作用的分析此示例说明了此方法的应用,通过T. 布鲁氏酸酶或重组T. 布鲁比RAP1 蛋白分析 RAP1 对 PI 的结合。T. 布鲁西亚血流形式的流合物形成表达血凝素 (HA) 标记 RAP1 用于结合测定.RAP1是一种参与转录控制变异表面糖蛋白(VSG)基因3,48的蛋白质,它编码了抗原变异49中参与寄生虫?…

Discussion

识别与IP或P结合的蛋白质对于理解这些代谢物的细胞功能至关重要。与西印体或质谱结合的亲和色谱提供了识别IP或PI相互作用蛋白的机会,从而获得对其调节功能的见解。IP 或 P 化学标记 [例如,Ins(1,4,5)P3 化学上与生物蛋白相关]并通过链球蛋白与腺糖珠交联,或由链球蛋白磁珠捕获,从而可以分离相互作用的蛋白质,然后通过质量识别这些蛋白光谱学或西方污点。这里描述的修饰已经被用来识别来?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了加拿大自然科学和工程研究理事会(NSERC,RGPIN-2019-04658)的支持;NSERC 发现发布早期职业研究人员补充 (DGECR-2019-00081) 和由麦吉尔大学。

Materials

Acetone Sigma-Aldrich 650501 Ketone
Acetonitrile Sigma-Aldrich 271004 Solvent 
Ammonium bicarbonate Sigma-Aldrich A6141 Inorganic salt
Centrifuge Avanti J6-MI Beckman Coulter Avanti J6-MI Centrifuge for large volumes (e.g., 1L)
Centrifuge botles Sigma-Aldrich B1408 Bottles for centrifugation of 1L of culture
Control Beads Echelon P-B000-1ml Affinity chromatography reagent – control
D-(+)-Glucose Sigma-Aldrich G8270 Sugar, Added in PBS to keep cells viable
Dithiothreitol (DTT)  Bio-Rad 1610610 Reducing agent
Dynabeads M-270 Streptavidin ThermoFisher Scientific 65305 Streptavidin beads for binding to biotin ligands
EDTA-free Protease Inhibitor Cocktail Roche 11836170001 Protease inhibitors
Electrophoresis running buffer Bio-Rad 1610732 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3
Falcon 15 mL Conical Centrifuge Tubes Corning Life Sciences 430052 To centrifuge 10 mL cultures
Formic acid Sigma-Aldrich 106526 Acid
Glycine Sigma-Aldrich G7126 Amino acid
HMI-9 cell culture medium ThermoFisher Scientific ME110145P1 Cell culture medium for T. brucei bloodstream forms
Imperial Protein Stain ThermoFisher Scientific 24615 Coomassie staining for protein detection in SDS/PAGE
Ins(1,4,5)P3 Beads Echelon Q-B0145-1ml Affinity chromatography reagent 
Instant Nonfat Dry Milk Thomas Scientific C837M64 Blocking reagent for Western blotting
Iodoacetamide Sigma-Aldrich I6125 Alkylating reagent for cysteine proteins or peptides
Lab Rotator Thomas Scientific 1159Z92 For binding assays
LoBind Microcentrifuge Tubes ThermoFisher Scientific 13-698-793 Low protein binding tubes for mass spectrometry
Nonidet P-40 (Igepal CA-630) Sigma-Aldrich 21-3277 Detergent
PBS, pH 7.4 ThermoFisher Scientific 10010031 Physiological buffer
Peroxidase substrate for chemiluminescence ThermoFisher Scientific 32106 Substrate for Western bloting detection of proteins
PhosSTOP Phosphatase Inhibitor Cocktail Tablets Roche 4906845001 Phosphatase inhibitors
PI(3)P PIP Beads Echelon P-B003a-1ml Affinity chromatography reagent 
PI(3,4)P2 PIP Beads Echelon P-B034a-1ml Affinity chromatography reagent 
PI(3,4,5)P3 diC8 Echelon P-3908-1mg Affinity chromatography reagent 
PI(3,4,5)P3 PIP Beads Echelon P-B345a-1ml Affinity chromatography reagent 
PI(3,5)P2 PIP Beads Echelon P-B035a-1ml Affinity chromatography reagent 
PI(4)P PIP Beads Echelon P-B004a-1ml Affinity chromatography reagent 
PI(4,5)P2 diC8 Echelon P-4508-1mg Affinity chromatography reagent 
PI(4,5)P2 PIP Beads Echelon P-B045a-1ml Affinity chromatography reagent 
PI(5)P PIP Beads Echelon P-B005a-1ml Affinity chromatography reagent 
Ponceau S solution Sigma-Aldrich P7170 Protein staining (0.1% [w/v] in 5% acetic acid)
Potassium hexacyanoferrate(III) Sigma-Aldrich 702587 Potassium salt 
PtdIns PIP Beads Echelon P-B001-1ml Affinity chromatography reagent 
PVDF Membrane Bio-Rad 1620177 For Western blotting 
Refrigerated centrifuge Eppendorf 5910 R Microcentrifuge for small volumes (e.g., 1.5 mL)
Sodium dodecyl sulfate Sigma-Aldrich 862010 Detergent
Sodium thiosulfate Sigma-Aldrich 72049 Chemical 
SpeedVac Vacuum Concentrators ThermoFisher Scientific SPD120-115 Sample concentration (e.g., for mass spectrometry)
T175 flasks for cell culture  ThermoFisher Scientific 159910 To grow 50 mL T. brucei culture
Trypsin, Mass Spectrometry Grade Promega V5280 Trypsin for protein digestion
Urea Sigma-Aldrich U5128 Denaturing reagent
Vortex Fisher Scientific 02-215-418 For mixing reactions
Western blotting transfer buffer Bio-Rad 1610734 25 mM Tris, 192 mM glycine, pH 8.3 with 20% methanol
Whatman 3 mm paper Sigma-Aldrich WHA3030861 Paper for Wester transfer
2-mercaptoethanol (14.2 M) Bio-Rad 1610710 Reducing agent
2x Laemmli Sample Buffer Bio-Rad 161-0737 Protein loading buffer
4–20% Mini-PROTEAN TGX Precast Protein Gels Bio-Rad 4561094 Gel for protein electrophoresis
4x Laemmli Sample Buffer Bio-Rad 161-0747 Protein loading buffer
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Inositol PhosphatePhosphoinositideProtein BindingAffinity ChromatographyWestern BlotMass SpectrometryRegulatory FunctionSignal TransductionCell DevelopmentBiotinylatedLyposome FreeT. BruceiPBS-GLysis BufferProtease InhibitorPhosphatase InhibitorBinding Assay
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Cestari, I. Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry. J. Vis. Exp. (149), e59865, doi:10.3791/59865 (2019).
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