概要

从人体二尖瓣提取蛋白质的优化方案

Published: June 14, 2017
doi:

概要

人二尖瓣的蛋白质组成仍然是部分未知的,因为其分析由于细胞数量低,因此通过低蛋白质生物合成而复杂化。这项工作提供了有效提取蛋白质用于二尖瓣蛋白质组分析的方案。

Abstract

细胞蛋白质组学的分析可以帮助阐明基于疾病的分子机制,这是由于技术的发展,允许大规模鉴定和定量复制生物系统中存在的蛋白质。从蛋白质组学方法获得的知识有可能导致更好地了解基础疾病的致病机制,允许鉴定新的诊断和预后疾病标志物,以及希望的治疗靶点。然而,心脏二尖瓣代表蛋白质组学分析的非常具有挑战性的样品,因为蛋白多糖和富含胶原蛋白的细胞外基质的细胞数量较少。这使得挑选提取蛋白质进行全球蛋白质组学分析。该工作描述了与随后的蛋白质分析相容的方案,如定量蛋白质组学和免疫印迹。这可以允许数据关联的相关性g蛋白表达与定量mRNA表达和非定量免疫组化分析数据。实际上,这些方法在一起执行时,可以更全面地了解基因疾病的基因分子机制,从mRNA到翻译后蛋白质的修饰。因此,这种方法可以与对心脏瓣膜病理学研究感兴趣的研究者有关。

Introduction

最近的证据改变了对mRNA合成后发生的许多调节机制的作用的理解。实际上,翻译,转录后和蛋白水解过程可以调节蛋白丰度和功能。假设转录本水平是蛋白质丰度的主要决定因素,mRNA的浓度代表相应蛋白质的代谢 – 已被部分修订。目前,转录本水平仅部分预测蛋白质丰度,这表明转录后事件发生调节细胞内的蛋白质1,2

此外,蛋白质最终决定细胞的功能,因此决定其表型,其可以经受自分泌,旁分泌和内分泌因子的响应的动态变化;血液介体;温度;药物治疗和疾病发展换货。因此,以蛋白质水平为重点的表达分析有助于表征蛋白质组,并且揭示其作为疾病发病机理的一部分发生的关键变化3

因此,尽管现有的技术挑战,蛋白质组学所提出的澄清健康和疾病状况的机会也是艰巨的。蛋白质组学可以贡献的特别有希望的研究领域包括:在任何水平( 全细胞或组织,亚细胞区室和生物液体)上鉴定改变的蛋白质表达;用于疾病诊断和预后的新型生物标志物的鉴定,验证和验证;并希望能够鉴定可用于治疗目的的新蛋白质靶标,以及药物功效和毒性的评估。

捕捉复杂性蛋白质组是技术挑战。目前的蛋白质组学工具提供了进行大规模,高通量分析来鉴定,定量和验证改变的蛋白质水平的机会。此外,引入分馏和富集技术,旨在避免由最丰富的蛋白质引起的干扰,还通过包括最少丰富的蛋白质来改善蛋白质鉴定。最后,蛋白质组学被补充了翻译后修饰的分析,其逐渐成为蛋白质功能的重要调节剂。

然而,分析中的生物标本中的样品制备和蛋白质回收仍然是蛋白质组学工作流程中的限制步骤,并增加可能陷阱的潜力5 。事实上,在大多数必须优化的分子生物学技术中,第一步是组织匀浆离子和细胞裂解,特别是在不存在扩增方法的低丰度蛋白质的分析过程中。此外,蛋白质的化学性质可以影响其自身的恢复。例如,高疏水性蛋白质的分析是非常具有挑战性的,因为它们在等电聚焦过程中容易沉淀,而跨膜蛋白几乎不溶(参见参考文献5)。此外,组织组成变异性为开发通用提取方法创造了显着的障碍。最后,由于几乎所有临床标本的数量都是有限的,所以必须以最小的样品量使蛋白质制备具有最大的恢复和重现性。

这项工作描述了从正常人心脏二尖瓣进行蛋白质提取的优化方案,这代表了蛋白质组学分析的非常具有挑战性的样品。正常二尖瓣是一种lex结构位于左心房和心脏左心室之间( 图1 )。它在控制从心房到脑室的血液流量方面起着重要的作用,防止回流,并确保适当的氧气供应给全身,从而保持足够的心输出量。然而,它通常被认为是一种“无活性”组织,细胞率低,组成少,主要是细胞外基质。这是因为在正常情况下,驻留的瓣膜间质细胞(VIC)具有低蛋白质生物合成速率7的静止表型。

然而,已经证明,在病理状态下,海绵体中的VIC的数量增加并且其蛋白质合成被激活,以及其他功能和表型变化8 。因此,最少的数据可用并不奇怪文献集中于病理性二尖瓣9,10的分析,其中活化的VIC的增加的数量可能解释相对较高数量的鉴定的蛋白质。

总之,本议定书可能通过研究二尖瓣蛋白组分来发展对负责二尖瓣疾病的致病机制的理解。事实上,更深入地了解潜在的病理过程可能有助于改善瓣膜疾病的临床管理,其目前的干预指标主要取决于血液动力学考虑因素。

Protocol

在该方案中,尽管正常的超声心动图参数,在多器官移植(冷缺血时间4-12小时,平均6±2小时)内,从器官移植排除器官移植的技术或功能原因收集人心脏。他们被送到米兰的心血管组织银行Monzino心脏中心(意大利米兰),为主动脉瓣和肺动脉瓣的银行服务。二尖瓣后叶不用于临床目的,因此在从供体亲属获得知情同意后,在主动脉瓣和肺动脉瓣隔离期间收集二尖瓣后叶。移植和研究的组织仅在?…

Representative Results

蛋白质在尿素缓冲液中的提取和溶解与基于等电聚焦(二维电泳(2-DE) 11和液相等电聚焦(IEF) 12 )的蛋白质组学方法直接相容,并且在Laemmli缓冲液13中稀释后进行免疫印迹含有蛋白酶抑制剂混合物14 。 对于基于无凝胶质谱法( 即与数据无关质?…

Discussion

该方案的一个关键步骤是使用液氮冷冻样品并冷却研磨机系统。液氮的使用可防止生物降解并有效地进行粉末化,但需要对培训进行安全处理。

在该协议中,具有用于样品研磨的研磨机系统,因为小样品难以从标准的砂浆和杵中回收。在这种情况下,小样品作为细粉扩散到砂浆表面,使收集困难。另一个优点是研磨机是电动的,这允许以可再现的方式处理更多数量的样品并?…

開示

The authors have nothing to disclose.

Acknowledgements

意大利卫生部支持这项研究(RC 2013-BIO 15)。我们非常感谢Barbara Micheli的优秀技术援助。

Materials

Saline solution 0.9 % NaCl
Eurocollins A SALF 30874046 Balanced organ's transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins
Eurocollins B SALF 30874022 Balanced organ's transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins
Wisconsin Bridge life RM/N 4081 Balanced organ's transport medium
Biohazard vertical flow air Burdinola Class A GMP classification
Dewar Flask Thermo Scientific Nalgene 4150-1000
Cryogrinder system OPS diagnostics CG 08-01 Grinder system containing mortars, pestles and screwdriver
Stainless steel forceps
Stainless steel spatula
Disposable sterile scalpel Medisafe MS-10
Stainless steel scissors Autoclavable
Stainless steel picks Autoclavable
Disposable sterile drap Mon&Tex 3.307.08
Sterilizing solution with isopropyl alcohol 70% isopropyl alcohol
Sterilizing solution with hydrogen peroxide 6% hydrogen peroxide
Micropipette, 1 mL, with tips
15 mL centrifuge tubes VWR international 9278
1.7 mL centrifuge tubes VWR international PIER90410
Urea buffer 8 M urea, 2 M thiourea, 4 % w/v CHAPS, 20 mM Trizma, 55 mM Dithiotreitol
Urea Sigma aldrich U6504-1KG To be used for Urea buffer
Thiourea Sigma aldrich T8656 To be used for Urea buffer
CHAPS Sigma aldrich C3023-5GR To be used for Urea buffer
Dithiotreitol Sigma aldrich D0632-5G To be used for Urea buffer
Syringe 50 mL PIC To be used to filter Urea buffer
0.22 µm filter Millipore SLGP033RB To be used to filter Urea buffer
PFTE Pestle, 2 mL Kartell 6302 Part of Potter-Elvehjem homogenizer
Borosilicate glass mortar Kartell 6102 Part of Potter-Elvehjem homogenizer
Stirrer VELP scientifica Stirrer DLH To be used for homogenization by Potter-Elvehjem
Bradford Protein assay Bio-Rad laboratories 5000006
Tube rotator Pbi International F205
Liquid nitrogen
Aluminum foil
Ice
Polystyrene box
Dry ice
Centrifuge For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g
Freezer -80°C
Precision balance
Autoclave For sterilization
Cryogenic gloves for liquid nitrogen
Gloves
Professional forced ventilation and natural air convection oven For sterilization
Protease inhibitor cocktail Sigma aldrich P8340-5ML 100X solution
ProteoExtract Protein Precipitation Kit Calbiochem 539180
RapiGest Waters 186001861
Cytoscape www.cytoscape.org version 2.7 Software platform for Gene Ontology analysis
BiNGO http://apps.cytoscape.org/apps/bingo version 3.0.3 Plugin for Gene ontology analysis
AlphaB Crystallin/CRYAB Antibody Novus Biologicals NBP1-97494 Mouse monoclonal antibody against CryAB
Septin-11 Antibody Novus Biologicals NBP1-83824 Rabbit polyclonal antibody against septin-11
FHL1 Antibody Novus Biologicals NBP-188745 Rabbit polyclonal antibody against FHL-1
Dermatopontin Antibody Novus Biologicals NB110-68135 Rabbit polyclonal antibody against dermatopontin
Goat Anti mouse IgG HRP Sigma aldrich A4416-0.5ML Secondary antibody for immunoblotting
Goat Anti rabbit IgG HRP Bio-Rad laboratories 170-5046 Secondary antibody for immunoblotting

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記事を引用
Banfi, C., Guarino, A., Brioschi, M., Ghilardi, S., Mastrullo, V., Tremoli, E., Polvani, G. Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve. J. Vis. Exp. (124), e55762, doi:10.3791/55762 (2017).

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