Name: proteomic analysis of human macrophage polarization under a low oxygen environment
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment at JoVE.com

am 由青年小组领导人方案 (ATIP/Avenir inserm-cnrs)、"癌症国家联盟" 和 "癌症研究基金会" 资助。我们感谢来自生物质谱的玛丽特·马松多 (巴黎巴斯德研究所, utecs msbio)。我们感谢劳伦·安德森对手稿的阅读。

作为授权协议 (codeceh dc-3114年) 的一部分, 从法国国家血液服务机构 (efs) 获得了健康、被取消鉴定的捐献者的人体血液样本 (lrsc)。捐献者签署了使用血液的同意。
1. 介质和缓冲液准备
<ol><li>制备巨噬细胞培养基 [rpmi 谷氨酰亚胺 + 10 mm hepes + 1x 非必需氨基酸 (neaa)], 并将其加热至37°c。</li>
	<li>从 ab 血浆 (sab) 制备巨噬细胞培养基 + 10% 的人血清, 过滤 (0.22μm 过滤器), 然后将其加?...

<ol>
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由于蛋白质组学是研究来自整个细胞或亚细胞隔间的不同蛋白质表达的有力工具, 因此细胞裂解方案的优化和蛋白质的消化已被许多研究所解决。主要有三类方法, 包括凝胶内消化 (聚丙烯酰胺凝胶基质中蛋白质的消化)17、溶液18中的消化和过滤辅助样品制备19。最后一种方法, 最初被描述为通用的, 已被报告表现出低重现性和可能的蛋白质损失?...

巨噬细胞是一种先天免疫细胞, 涉及许多生理功能, 从反应到传染性病原体, 再到组织稳态, 包括切除凋亡细胞和重塑细胞外基质 1。这些细胞的特点是具有强大的表型可塑性2 , 转化为许多可能的激活状态, 这也被称为极化。这些不同极化的精确分子描述是巨噬细胞生物学领域的一个优先事项3。有人建议使用所谓的 mém2 二分法对这些极化进行分类, 其中 m1 代表促炎, m2 代表抗炎巨噬细胞。这种模型很适合各种病理情况, 如急性感染, 过敏, 和肥胖4。然而, 在长期发炎的组织和癌症中, 已经证明, 这种分类无法掌握巨噬细胞存在于某些细胞环境5,6,7. 目前的共识是, 使用多层面模型更好地描述巨噬细胞极化, 以整合具体的微环境信号8。通过对人类巨噬细胞的转录分析证实了这一结论, 表明 m好久亚模型在描述所获得的极化方面效率低下9。
该研究旨在提供一种协议, 以获得人类巨噬细胞中各种极化的蛋白质组特征。我们描述了如何区分人类巨噬细胞在不同的氧水平的环境中, 并从整个巨噬细胞蛋白质组中获得肽, 以执行无标签的定量。这种量化允许比较各种蛋白质的表达水平。由于对干细胞的研究揭示了氧作为环境关键参数10的重要性, 我们试图了解这种组织参数如何影响人类巨噬细胞的极化。研究发现, 人体的氧分压在 3% 至 2 0% (大气总压力中) 之间, 其中 2 0% 大致相当于细胞培养孵化器中常用的压力 (在取水的同时, 确切值在 1 8. 6% 左右)考虑到)。
先前的研究表明, 肺泡不同于间质巨噬细胞的功能和形态观点 11 , 这些差异的一部分可能是由于不同的氧气水平, 他们暴露 12.此外, 骨髓衍生的巨噬细胞在接触低氧环境时显示出更强的吞噬细菌能力。相反的影响已经发现 thp1 分化人类巨噬细胞 13, 但这些结果支持的观点, 氧气是一个调节巨噬细胞生物学, 有必要澄清这一作用, 在分子水平上的人类巨噬细胞。在之前的一项研究中, 我们应用了蛋白质组学方法来解决这些问题。通过同时测量数千种蛋白质的表达水平, 我们强调了氧气对极化的影响, 并提供了一个新的分子标记列表。我们还能够将这些发现与一些巨噬细胞功能联系起来。值得注意的是, 我们发现 ilni-il13 极化巨噬细胞的凋亡率提高, 这与 alox15 的上升有关, 结果通过蛋白质组分析14显示.在本研究中, 我们描述了如何进行这样的分析。

<table>
	<tbody>
		<tr>
			<td>Hypoxia Working Station</td>
			<td>Oxford Optronix</td>
			<td>Hypoxylab</td>
			<td></td>
		</tr>
		<tr>
			<td>C6 Flow cytometer</td>
			<td>BD</td>
			<td>Accuri C6</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Agilent Technologies</td>
			<td>5188-6435</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (FA)</td>
			<td>ARISTAR</td>
			<td>450122M</td>
			<td></td>
		</tr>
		<tr>
			<td>R-250 Coomassie blue</td>
			<td>Biorad</td>
			<td>1,610,436</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide, E.Coli (LPS)</td>
			<td>Calbiochem</td>
			<td>437627</td>
			<td></td>
		</tr>
		<tr>
			<td>2D clean-up kit</td>
			<td>GE Healthcare</td>
			<td>80-6484-51</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium, glutamax supplement</td>
			<td>Gibco</td>
			<td>61870044</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids (NEAA) Solution 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS) 1X</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Harvard Apparatus column Reverse C18 micro spin column</td>
			<td>Harvard Apparatus</td>
			<td>74-4601</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5 M, pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Bis-Tris 4-12%</td>
			<td>Life Technologies SAS</td>
			<td>NP0321 BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14 Microbeads human</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS separation column LS</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage colony-stimulating factor (M-CSF)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-485</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin 4 (IL4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-917</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin 13 (IL13)</td>
			<td>Miltenyi Biotec</td>
			<td>130-112-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Interferon gamma (INF&#947;)</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-482</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-FITC (clone T&#220;K4)</td>
			<td>Miltenyi Biotec</td>
			<td>130-080-701</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSmix Tube Rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Pierce</td>
			<td>28904</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C Mix</td>
			<td>PROMEGA</td>
			<td>V5073</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete Mini, EDTA-free Protease Inhibitor cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Density Gradient Solution (Histopaque 1077)</td>
			<td>Sigma Aldrich</td>
			<td>10771-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Sigma Aldrich</td>
			<td>A7089-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum from human male AB plasma (SAB)</td>
			<td>Sigma Aldrich</td>
			<td>H4522-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) solution 30%</td>
			<td>Sigma Aldrich</td>
			<td>A9576-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisma-base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma Aldrich</td>
			<td>114405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS) 20%</td>
			<td>Sigma Aldrich</td>
			<td>5030</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34888</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma Aldrich</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma Aldrich</td>
			<td>57670</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiourea</td>
			<td>Sigma Aldrich</td>
			<td>T8656</td>
			<td></td>
		</tr>
		<tr>
			<td>CHAPS</td>
			<td>Sigma Aldrich</td>
			<td>C9426</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL sterile plastic pipette</td>
			<td>VWR</td>
			<td>612-1685</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer C Eppendorf</td>
			<td>VWR</td>
			<td>460-0223</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak tC18 reverse phase cartridges, 100 mg</td>
			<td>Waters</td>
			<td>WAT036820</td>
			<td></td>
		</tr>
	</tbody>
</table>

proteomic analysis of human macrophage polarization under a low oxygen environment

巨噬细胞是先天免疫细胞, 涉及许多生理功能, 从反应到传染性病原体, 再到组织稳态。这些细胞的各种功能与它们的激活状态有关, 这也被称为极化。这些不同极化的精确分子描述是巨噬细胞生物学领域的一个优先事项。目前人们认识到, 有必要采取多层面的办法来描述环境信号如何控制两极分化。在本报告中, 我们描述了一个协议, 旨在获得各种极化的蛋白质组学签名的人类巨噬细胞。该协议是基于无标签定量的巨噬细胞蛋白表达获得在凝胶分馏和赖西 cl 消化细胞裂解的内容。我们还提供了一个基于溶液中消化和等电聚焦分馏的协议, 作为一种替代方案。由于氧浓度是组织中的一个相关环境参数, 我们使用该协议来探讨大气成分或低氧环境如何影响巨噬细胞极化的分类。

该协议从差离心获得的外周血单个核细胞 (pbmc) 开始, 允许通过流式细胞术获得评估纯度超过98% 的 cd14+单核细胞种群 (图 1)。这些单核细胞被二次分化为各种极化 (图 2)。当选择凝胶上的分馏时, sds 页凝胶上的迁移被调整以获得所需的带数, 并对带进行切割 (图 3)。消化是在凝胶的切除带进行二次, ?...

Watch this Scientific Journal Video about Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment at JoVE.com

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

我们提出了一个协议, 以获得蛋白质组的特征的人类巨噬细胞, 并应用于确定低氧环境对巨噬细胞极化的影响。

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

因此，该方法有助于回答巨噬细胞生物学领域的关键问题，例如如何在蛋白质水平上描述不同环境下的活化状态。该技术的主要优点是，它提供了利用无标签定量方法获取人类巨噬细胞中许多蛋白质不同表达的机会。演示程序将是玛丽·马利耶和马加利法院从我的实验室。首先，在两个50毫升离心管中加入15毫升密度梯度细胞分离溶液，使溶液在接收LRSC之前可以加热到室温。这一点至关重要，因为密度取决于温度。将 LRSC 清空到 50 毫升离心管中。加入 1x PBS 至 50 毫升并混合。然后，非常缓慢地，在平衡密度梯度溶液的 15 毫升上添加 25 毫升的混合。注意不要在此步骤中混合阶段。血液必须添加到密度梯度溶液上，而不受此阶段的任何干扰。在700 G下将两个离心管离心25分钟，无中断。在密度梯度离心结束时，从下到上层是红细胞和粒细胞，形成颗粒、密度梯度溶液相、PBMC 层和稀释血浆。使用移液器，通过等离子相，无需吸气。然后，将PBMC层收集到新的50毫升离心管中。将高达 50 毫升 1x PBS 的 PBS 加入 PBMC，作为洗涤装置，并在 300 G 下离心 10 分钟。吸升液，并在 40 毫升巨噬细胞介质中重新悬浮颗粒。在马拉塞兹腔室中将 PBMC 放在后，将进行实验所需的 PBMC 量转移到离心管中。在300G下将细胞离心10分钟，吸制上流液，每1000万PBMC分拣缓冲液的80微升中重新悬浮颗粒。然后，每 1000 万 PBMC 添加 20 微升 CD14 微珠。混合好，在4摄氏度的不断搅拌下孵育15分钟。孵育后，每1000万PBMC添加1毫升分拣缓冲液作为洗涤步骤，并像以前一样离心。然后，吸升液，每1亿PBMC的500微升分拣缓冲液中重新悬浮颗粒。现在，在分离器的磁场中放置一个列。使用三毫升的分拣缓冲器冲洗列来准备列。将单元格悬浮应用到列上。然后收集流通过包含未标记的细胞，以便随后染色（如有必要）。用三毫升的分拣缓冲液清洗柱子三次。小心不要擦干你的列。然后，在列下放置一个收集管，并将其从分隔符中卸下。洗涤后，移液器将五毫升的分拣缓冲液放入柱中。通过将柱塞牢固地推入柱体，立即冲洗出磁性标记的细胞。最后，在电镀单核和执行文本协议中描述的巨噬细胞极化之前，用新列重复这些步骤。在氧气控制的环境中保持单核细胞和巨噬细胞，以进行缺氧状况分析。在实验过程中，使用缺氧工作站将细胞保持在所需的氧气部分压力下。在低氧压力下工作时，在站下准备所有介质和洗涤缓冲液非常重要，并充分等待以获得液体中正确的部分压力。在经过调整的发动机罩下在缓冲器中执行细胞解解。将蛋白质当量为30万个细胞，每个样品在4-12%双三丙烯酰胺凝胶上。控制电泳迁移的持续时间，使每个蛋白质样本分裂成六个凝胶带，如下所示。修复和染色后，如文本协议所述，用干净的手术刀切除蛋白质带。将每个切除带切成500微升管之前，先将每个切入带子切成骰子。现在，在200微升25毫摩尔碳酸氢盐中洗三次凝胶片，在37摄氏度下洗涤20分钟，在每一步之间丢弃碳酸氢铵。遵循这一点与25毫摩尔碳酸氢铵和醋酸酯洗一洗。然后，用200微升100%乙酰三酯脱水凝胶片10分钟。在56摄氏度下，用10毫摩尔DTT和25毫摩尔碳酸氢铵孵育每块45分钟。然后，丢弃DTT，在室温下在黑暗中用55毫摩尔碘多乙酰胺孵育凝胶片，在25毫摩尔碳酸氢铵中孵育35分钟。要停止烷基化，请丢弃用过的溶液，在室温下用200微升10毫摩尔DTT孵育10毫摩尔DTT，在室温下孵化10分钟。用200微升25毫摩尔碳酸氢铵清洗凝胶片。然后，用200微升100%乙酰三酯脱水10分钟。根据制造商的说明，在37摄氏度下用三辛/Lys-C混合体在37摄氏度下隔夜消化蛋白质。在低吸收管中加入50微升50%乙酰三叶酸，从凝胶片中提取产生的肽15分钟。加入50微升5%甲酸15分钟后，加入50微升100%乙酰三叶酸15分钟。在低吸收管中拉干每个分数，以限制多肽的吸收和样品损失。将样品存放在 80 摄氏度，直到进一步分析，如文本协议中详细说明。此处显示的是流式细胞学在选择 CD14 正单核细胞后由流式细胞测量评估的代表性纯度。分化人类巨噬细胞的相位对比成像显示，经过九天的培养，两种不同极化获得的形态的异质性。显示了使用银染色 SDS 页面凝胶的凝胶外消化示例。从细胞解解和溶液内消化后对蛋白质的评估表明，在分解过程中没有降解，消化效率也高。此处显示的是一个碰撞引起的分离光谱，该肽在MS光谱上的质量-电荷比为597.29，电荷为正2。在此频谱中，确定了相应的序列。分析后的最后结果为使用分效应蛋白质表示所有极化状态的分层聚类的热图。分析显示，在3%氧条件下，所有极化中，一组蛋白质过度表达。在尝试此过程时，重要的是要记住，您必须首先决定您将对新样本执行哪种分馏，以便相应地调整协议。使用此工作的蛋白质组学方法是对微噬细胞生物学极化研究领域近年来使用的基因组方法的补充。该技术开发后，为免疫学领域的研究人员探索人类和其他哺乳动物免疫细胞激活的各种状态铺平了道路。不要忘记，使用 DTT 可能会很危险，因此您需要在经过调整的发动机罩下工作才能执行此过程。

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

科研

生物化学

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

人视网膜解剖与 RPE-脉络膜的蛋白质组分析

The human retina is composed of the sensory neuroretina and the underlying retinal pigmented epithelium (RPE), which is firmly complexed to the vascular ...

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Split-BioID ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

用于稳定碳氧同位素分析的牙釉质碳酸盐的取样与预处理

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

全飞头段耗氧率的测定与解释

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...