Name: quantitative proteomics using reductive dimethylation for stable isotope labeling
Uploaded: 2014-07-01T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

注：此方法之前描述12。 1，蛋白质分离制备1毫克细胞蛋白通过裂解细胞，优选通过物理方法如法国压机，珠击，或超声处理。避免溶菌酶介导的细胞溶解，因为这种酶会混淆质谱测量。 2，三氯乙酸沉淀蛋白加入1倍体积的三氯乙酸（TCA），以4倍体积的冰蛋白和冷却10分钟以沉淀蛋白质。 12,000×g离心5分钟?...

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几点让使用还原二甲基化（REDI标签）一个有吸引力的方法，定量蛋白质组学肽的稳定同位素标记：物美价廉标记试剂（试剂成本低于1美元的样品），反应速度快（〜10分钟），无副产物，高再现性（ 图3,4），稳定的反应产物，能够使用任何蛋白酶，和标记的肽的高电离效率。由雷迪化学标记也是有利的相对于代谢标记，因为它不需要株或细胞系具有特定氨基酸营养缺陷型或生长在合...

测量复杂样品间的许多蛋白质​​的浓度差异是在蛋白质组学面临的核心挑战。越来越多地，这是由标记蛋白在每个样品中不同的同位素标记，结合样品，并用质谱定量浓度差异进行。蛋白质和多肽的稳定同位素标记的方法有几种。15 N标记1和2 SILAC 在体内引入同位素标记的代谢，而ICAT 3的iTRAQ 4，和二甲基化减少5蛋白的提取和消化后添加稳定同位素标记。在这些方法中，还原的二甲基化（雷迪标签）正逐渐成为一种廉价的，可再现的方法来量化在几乎任何类型的样品的蛋白质浓度差异。 雷迪标记包括使肽与甲醛以形成席夫碱，然后将其减压通过氰基。这种反应dimethylates上的N-末端和赖氨酸侧链和monomethylates N-末端脯氨酸游离氨基。这里所描述的协议甲基化的肽中的样品1使用的试剂与氢原子在其天然同位素分布和样品2使用氘化的甲醛和氰基硼氢化（ 图1）一个“重”标签的“光”的标签。关于在6.0377沓轻质和重质的形式，它是采用利用质谱仪的两种形式之间进行区分之间的质量差异的肽的结果各二甲基化的氨基。具体而言，相对于肽的丰度定量为MS1的提取离子色谱图区域的光的比率（MS1峰面积之比）和重版本的每个肽的离子对。一个蛋白的相对丰度的计算方法中的所有蛋白肽中的中位数MS1峰面积之比。在这份报告中，我们描述了一个强大的协议行为ING雷迪标记实验通过LC-MS/MS，包括反相肽固相萃取，柱上的Redi标签，肽分馏由碱性pH反相（BPRP）层析，并用StageTips肽混合物的纯化（ 图2） 。我们讨论使用REDI标记定量蛋白质组学的优点和局限性。

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">trichloroacetic acid</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:136px;">T9159</td>
			<td style="width:319px;">protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">650501</td>
			<td>protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71736</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">S8045</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">43816</td>
			<td>denature, reduce, alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">protease Inhibitor Complete Mini Cocktail</td>
			<td>Roche</td>
			<td>4693124001</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td>alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">H7523</td>
			<td>resuspend, extract, label protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">C5670</td>
			<td>resuspend protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">Lysyl Endoprotease</td>
			<td>Wako Chemicals</td>
			<td style="width:136px;">129-02541</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sequencing grade trypsin</td>
			<td>Promega</td>
			<td style="width:136px;">V5111</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">320099</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">tC18 Sep-Pak C18 cartridges</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">14261</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cyanoborohydride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71435</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">deuterated formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium cyanoborodeuteride</td>
			<td>CDN isotopes</td>
			<td style="width:136px;">D-1797</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18-HPLC column (4.6 x 250 mm, 5 &#181;m particle size)</td>
			<td>Agilent</td>
			<td style="width:136px;">770450-902</td>
			<td>basic pH reversed-phase chromatography</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">399388</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18 Empore Disks</td>
			<td>3M</td>
			<td style="width:136px;">14-386-3&#160;</td>
			<td>STAGE tips</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">methanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">494437</td>
			<td>STAGE tips</td>
		</tr>
	</tbody>
</table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

肽通过还原二甲基化（REDI标签）的稳定同位素标记的方法来准确地量化用质谱样品之间蛋白表达的差异。无论是使用常规（光）或氘（重）形式的甲醛和氰基硼氢化钠加两个甲基对每个游离胺进行REDI标签。在这里，我们展示了一个强大的协议REDI标签和复杂的蛋白质混合物的定量比较。蛋白样品用于比较被消化成肽，标记为运载轻或重的甲基标签，混合，并通过LC-MS/MS共同分析。相对的蛋白丰度，通过比较构成的肽从全MS谱中提取的重链和轻标记版本的离子色谱图的峰面积进行定量。此处描述的方法包括用反相固相萃取的样品制备，肽柱上的Redi贴标，通过碱性pH转肽分馏ersed相（BPRP）层析，并StageTip肽纯化。我们讨论REDI标签的优点和局限性相对于其它方法稳定同位素掺入。我们使用REDI标签作为一种快速，廉价，准确的方法，在几乎任何类型的样品的比较蛋白质丰度突出创新应用。

我们使用酿酒酵母评估的准确度，精密度和雷迪标记的再现性和梭菌C.phytofermentans中全细胞裂解液。我们首先量化C的组合的REDI标记效率C.phytofermentans中的蛋白裂解物纤维素（重标记，H）和葡萄糖（光标签，L）的培养。当过滤，以1％的肽假发现率，该样品含有具有98％的Redi标记效率11194独特的肽序列。未分离S。酵母蛋白溶胞产物同样标记用H或L试剂，混合各种比?...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

定量蛋白质组学使用还原二甲基化的稳定同位素标记

肽的还原性二甲基化（REDI标签）稳定同位素标记是一种快速，廉价的战略精确质谱为基础的定量蛋白质组学。这里我们展示了一个可靠的方法用于制备使用可以应用于几乎任何类型的样品中的Redi途径的蛋白质混合物的分析。

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

规模以上非靶向代谢组学剖析血清超高效液相色谱 - 质谱（UPLC-MS）

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

科研

化学

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Improved In-gel Reductive ÃƒÅ½Ã‚Â²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

改善凝胶还原β-消除综合O连接和磺基糖组学质谱

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

根据环境条件的金纳米粒子的稳定低聚簇的大小控制合成的一种简便方法

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

CN-GELFrEE  - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

定量糖组学和蛋白质组学组合式净化策略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

用尺寸排阻ICP-MS标准定量分析Metalloproteomic

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

高稳定性，功能性毛纳米粒子和木纤维生物聚合物：迈向可持续纳米技术

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

原位金纳米粒子合成快速 Nanoprobe 信号增强

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...