基于性光谱的眼微血管蛋白质组学样品制备
Summary
眼微血管床的蛋白质组特征是深入了解人类许多眼部病理的关键。本研究展示了一种有效、快速、稳健的方法, 用于利用猪短的后睫毛动脉作为基于质谱的蛋白质组学分析的模型血管, 从小血管中提取蛋白质和制备样品。
Abstract
利用体外孤立的眼血管, 用先进的技术方法破译眼睛的病理生理状态, 极大地扩大了我们对某些疾病的认识。质谱 (ms) 的蛋白质组学已成为一个强有力的工具, 以解开改变分子机制和蛋白质信号通路的血管床在健康和疾病。然而, 在 ms 分析之前的样品制备步骤对于获得可重现的结果和深入阐明复杂蛋白质组至关重要。这对于眼科微血管的制备尤其重要, 因为在眼科微血管中, 可用于分析的样品数量往往有限, 因此对最佳蛋白质提取构成了挑战。本文试图提供一个有效的, 快速和可靠的方案, 样品制备从一个示范后腰椎眼血管床使用猪短后睫毛动脉。本方法的重点是从样品的上清液和颗粒的蛋白质提取程序后, 均质化, 样品清洗前的离心过滤装置和肽纯化在液相色谱-电喷雾电离-线性离子陷阱-轨道记录 ms 系统中的无标签量化步骤。虽然这种方法是专门为眼科微血管的蛋白质组学分析开发的, 但我们也提供了令人信服的证据, 证明它也可以很容易地用于其他组织基样品。
Introduction
蛋白质组学领域的进步, 使综合和无与伦比的数据收集能力, 极大地改变了我们对某些疾病条件背后的分子机制的理解, 以及在反映特定细胞群或组织的生理状态1、2、3、4。蛋白质组学也被证明是眼科研究的一个重要平台, 因为对不同的眼睛样本进行了敏感性和无偏见的分析, 有助于确定潜在的疾病标志物, 以便最终诊断和预后。近年来的许多研究都很有证明, 包括我们的一些研究 1、5、6、7、8、9、10.然而, 由于伦理原因, 通常很难获得用于蛋白质组学分析的人体样本, 特别是考虑到需要健康的个人提供控制材料进行可靠的比较分析。另一方面, 获得足够数量的样品以进行最佳和可靠的质谱分析也具有挑战性。这对于数量有限的生物材料, 如眼睛的微血管尤其重要。其中一个主要的反流血管, 发挥调节眼血流的关键作用是短后睫毛动脉 (scca)。这个维管床上的任何扰动或异常都可能导致严重的临床影响, 从而导致青光眼和非动脉前缺血性视神经病变 (naion)等几种有视觉症状的疾病的发病机制11,12. 然而, 由于上述缺点, 缺乏阐明该动脉床蛋白质组变化的研究。因此, 近年来, 由于人类和猪13之间的形态和系统发育相似性很高, 家猪 (家养林奈, 1758) 已成为眼科研究中的一个很好的动物模型 。 14,15。猪眼样本很容易获得, 最重要的是, 更准确地表示人体组织。
考虑到这些血管在眼睛中的重要作用, 以及缺乏有效提取蛋白质的方法和从这些微血管中分析的方法, 我们以前已经用内部的方法对猪 pca 的蛋白质组进行了表征。协议, 导致识别了大量的蛋白质16。在这项研究的基础上, 我们在本文中进一步优化和深入地描述了我们的方法, 允许使用猪 scca 作为模型组织从微小数量的样本中进行蛋白质组分析。尽管这项研究的主要目的是建立一个 ms 兼容的方法, 质量有限的眼血管, 我们提供了大量的实验证据, 所述的工作流程也可以广泛地适用于各种组织为基础的样本。
根据设想, 这一工作流程将有助于从少量材料中制备高质量的 ms 兼容样品, 以便进行全面的蛋白质组分析。
Protocol
所有使用动物样本的实验程序都是严格按照视力和眼科研究协会 (arvo) 《关于在眼科和视力研究中使用动物的声明》和机构指南进行的。这项研究是在美因茨大学医疗中心眼科进行和批准的。 请注意:猪的眼睛与视神经和眼外组织是新鲜的从当地屠宰场立即死亡。用冰凉磷酸盐缓冲盐水 (pbs) 将有钙化的眼睛输送到实验室, 并立即使用。所使用的工作流的示意图概述…
Representative Results
样本有限是眼科研究的主要缺陷之一。相应地, 从少量样本 (如眼睛血管) 中提取最佳蛋白质产量的方法也常常值得商榷。到目前为止, 特别是从反疣血管中提取蛋白质的方法很少。因此, 作为方法优化的第一步, 并作为一项原则证明, 将几种常用的蛋白质提取剂的疗效和鲁棒性与相对较新的试剂 t-per 进行了试点研究, 并利用心脏组织进行了研究小鼠的情况 (由于优化步骤的样…
Discussion
对各种眼样本进行全面的蛋白质组分析是阐明健康和疾病中涉及的分子机制和信号通路的重要和不可或缺的第一步。为了获得高质量的数据并确保从这些分析中获得的结果的重现性, 前面的样品制备步骤至关重要, manal 等人在深入讨论样品处理程序的审查中强调了这一点。对于眼睛的不同部位采用二维凝胶电泳和质谱策略 1。在这些研究中, 我们目前的研究提供了一个优化的逐步?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
manicam 博士得到了美因法约翰内斯古腾堡大学医学中心的内部大学研究资助 (stufe 1) 和德国 mforschungsgemeinschaft 的资助 (ma 800 6/1-1) 的资助。
Materials
| A. Chemicals | |||
| 1, 4-Dithiothreitol (DTT) | Sigma-Aldrich | 1.11474 | |
| Ammonium bicarbonate (ABC, CH₅NO₃) | Sigma-Aldrich | 5.33005 | |
| Calcium chloride dihydrate (CaCl2) | Carl Roth | 5239.1 | 2.5 mM |
| Dulbecco's phosphate-buffered saline (PBS) | Thermo Fisher Scientific | 14190169 | |
| Formic acid (CH2O2) | AppliChem | A0748 | |
| HPLC-grade acetonitrile (ACN, C2H3N) | AppliChem | A1605 | |
| HPLC-grade methanol (CH3OH) | Fisher Scientific | M/4056/17 | |
| HPLC-grade water | AppliChem | A1589 | |
| Iodoacetamide (IAA) | Sigma-Aldrich | I6125 | |
| Kalium chloride (KCl) | Carl Roth | 6781.1 | 4.7 mM |
| Kalium dihydrogen phosphate (KH2PO4) | Carl Roth | 3904.2 | 1.2 mM |
| LC-MS-grade acetic acid | Carl Roth | AE69.1 | |
| Magnesium sulphate (MgSO4) | Carl Roth | 261.2 | 1.2 mM |
| NuPAGE Antioxidant | Thermo Fisher Scientific (Invitrogen) | NP0005 | |
| NuPAGE LDS Sample buffer | Thermo Fisher Scientific (Invitrogen) | NP0007 | 4x |
| NuPAGE MES SDS Running Buffer | Thermo Fisher Scientific (Invitrogen) | NP0002 | 20x |
| NuPAGE Sample reducing agent | Thermo Fisher Scientific (Invitrogen) | NP0004 | 10x |
| SeeBlue Plus2 pre-stained protein standard | Thermo Fisher Scientific (Invitrogen) | LC5925 | |
| Sequencing grade modified trypsin | Promega | V5111 | |
| Sodium chloride (NaCl) | Carl Roth | 9265.2 | 118.3 mM |
| Sodium hydrogen carbonate (NaHCO3) | Carl Roth | 965.3 | 25 mM |
| Trifluoroacetic acid (TFA, C2HF3O2) | Merck Millipore | 108178 | |
| α-(D)-(+)- Glucose monohydrate | Carl Roth | 6780.1 | 11 mM |
| B. Reagents and Kits | |||
| 0.5mm zirconium oxide beads | Next Advance | ZROB05 | |
| 1.0mm zirconium oxide beads | Next Advance | ZROB10 | |
| Colloidal Blue Staining Kit | Thermo Fisher Scientific (Invitrogen) | LC6025 | To stain 25 mini gels per kit |
| NuPAGE 4-12 % Bis-Tri gels | Thermo Fisher Scientific (Invitrogen) | NP0321BOX | 1.0 mm, 10-well |
| Pierce Bicinchoninic Acid (BCA) Protein Assay Kit | Thermo Fisher Scientific | 23227 | |
| ProteoExtract Transmembrane Protein Extraction Kit, TM-PEK | Merck Millipore | 71772-3 | 20 reactions per kit |
| Tissue Protein Extraction Reagent (T-PER) | Thermo Scientific | 78510 | |
| C. Tools | |||
| 96-well V-bottom plates | Greiner Bio-One | 651180 | |
| Corning 96-well flat-bottom plates | Sigma-Aldrich | CLS3595-50EA | |
| Disposable microtome blades | pfm Medical | 207500014 | |
| Disposable scalpels #21 | pfm Medical | 200130021 | |
| Dissection pins | Carl Roth | PK47.1 | |
| Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | |
| Falcon conical centrifuge tubes (50 mL) | Fisher Scientific | 14-432-22 | |
| Mayo scissors, Tough cut | Fine Science Tools | 14130-17 | |
| Precision tweezers | Fine Science Tools | 11251-10 | Type 5 |
| Precision tweezers, straight with extra fine tips | Carl Roth | LH53.1 | Type 5 |
| Self-adhesive sealing films for microplates | Ratiolab (vWR) | RATI6018412 | |
| Standard pattern forceps | Fine Science Tools | 11000-12 | |
| Student Vannas spring scissors | Fine Science Tools | 91501-09 | |
| Vannas capsulotomy scissors | Geuder | 19760 | Straight, 77 mm |
| ZipTipC18 pipette tips | Merck Millipore | ZTC18S096 | |
| D. Equipment and devices | |||
| 150 × 0.5 mm BioBasic C18 column | Thermo Scientific, Rockford, USA | 72105-150565 | |
| 30 × 0.5 mm BioBasic C18 pre-column | Thermo Scientific, Rockford, USA | 72105-030515 | |
| Amicon Ultra-0.5 3K Centrifugal Filter Devices | Merck Millipore | UFC500396 | Pack of 96. |
| Analytical balance | Sartorius | H51 | |
| Autosampler | CTC Analytics AG, Zwingen, Switzerland | HTS Pal | |
| BBY24M Bullet Blender Storm | Next Advance | NA-BB-25 | |
| Eppendorf concentrator, model 5301 | Sigma-Aldrich | Z368172 | |
| Eppendorf microcentrifuge, model 5424 | Fisher Scientific | 05-403-93 | Non-refrigerated |
| Heraeus Primo R Centrifuge | Thermo Scientific | 75005440 | Refrigerated |
| Labsonic M Ultrasonic homogenizer | Sartorius | BBI-8535027 | |
| LC-MS pump, model Rheos Allegro | Thermo Scientific, Rockford, USA | 22080 | |
| LTQ Orbitrap XL mass spectrometer | Thermo Scientific, Bremen, Germany | ||
| Multiskan Ascent plate reader | Thermo Labsystems | v2.6 | |
| Rotator with vortex | neoLab | 7-0045 | |
| Titanium probe (Ø 0.5mm, 80mm long) | Sartorius | BBI-8535612 | |
| Ultrasonic bath, type RK 31 | Bandelin | 329 | |
| Xcell Surelock Mini Cell | Life Technologies | El0001 |
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Cite This Article
Perumal, N., Straßburger, L., Schmelter, C., Gericke, A., Pfeiffer, N., Grus, F. H., Manicam, C. Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels. J. Vis. Exp. (144), e59140, doi:10.3791/59140 (2019).