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，在4℃下，去除上清。在4℃下重悬沉淀中加入1ml冰冷的丙酮和12,000×g离心5分钟去除上清，颠倒离心管在板凳上，以干燥沉淀15分钟。店铺蛋白小球于-80℃。 3，使蛋白质变性和还原二硫键回复暂停蛋白质〜2 mg / ml的500微升变性和还原缓冲液（或者4M尿素或3％SDS的50mM HEPES pH值为8.5，5mM DTT的）。任选地，包括在缓冲液中的蛋白酶抑制剂。孵育蛋白30分钟，在56℃，然后在室温下10分钟。 4。Aklylate游离巯基，以不可逆扰乱二硫键形成水准备新鲜0.3M的碘乙酰胺。注意！碘乙酰胺是剧毒。加入25微升0.3M的碘乙酰胺（15mM的终浓度），以500μl的蛋白孵育20分钟在黑暗中在室温下进行。通过加入10μl的300mM的DTT（5mM的终浓度DTT）淬灭碘乙酰胺。存储烷基化的蛋白质于-80℃。 5，蛋白质的消化 TCA沉淀的蛋白质（如步骤2中所述），并重新悬浮于1ml的50mM HEPES（pH值8.2），1M尿素。准备赖氨酰endoprotein的原液酶（赖氨酸-C）在水中的2微克/微升的浓度，并添加5微升的蛋白质溶液。为16小时，在室温下孵育混合物。确保最终的赖氨酸-C浓度为10纳克/微升和蛋白质对lysC的比率（W / W）为1/50至1/200。重悬20微克测序级胰蛋白酶在40微升50mM的乙酸中，添加5微升（10微克胰蛋白酶）对赖氨酸-C消化物，并孵育6小时，在37℃下使用相同浓度的蛋白酶和蛋白酶与蛋白质的比例为赖氨酸-C作为用于胰蛋白酶。 6，反相肽提取<ol><li>通过加入三氟乙酸（TFA）至0.5％的最终浓度（pH≈2）酸化肽。附加C18柱，以提取歧管。除了使用肽的装载和洗脱尽可能高的流速所有步骤。 </li><li>湿塔用6ml乙腈（ACN）。用6ml 80％ACN，0.1％TFA洗柱，然后平衡用6毫升的0.1％TFA。不要让列步骤之间的干运行。 </li><li>停止真空压力和负载500的肽的微克到柱上以大约1毫升/分钟的流速。一旦肽已结合在柱上，重新启动真空和洗涤用6ml 0.1％TFA，然后用3毫升的柠檬酸缓冲液（0.09M的柠檬酸，0.23，1M的Na 2 HPO 4，pH 5.5）中。 注意:较高的肽量可以被标记，但C18柱结合能力至少应为2倍左右的肽量更高，以避免样品损失。 </li></ol> 7，柱头肽标记通过还原二甲基化（REDI标签） 如氰化氢在贴标过程中释放在低浓度下，化学罩执行此步骤。 <ol><li>准备将12ml"轻"和"重"REDI缓冲区，以甲基化肽的游离胺。光REDI缓冲由0.8％甲醛和0.12 M氰基硼氢化钠携带氢在日EIR天然同位素分布在柠檬酸缓冲液。重REDI缓冲由0.8％氘代甲醛和0.12 M在柠檬酸缓冲液氘代氰基硼氢化钠。 </li><li>孵育含有肽柱中加入10ml或者轻或重的Redi缓冲区以1毫升/分钟流速的含肽柱，并重复以确保完整的标签。洗柱用6毫升含0.1％TFA，然后用1毫升0.5％的乙酸。 </li><li>停止真空并用1毫升40％ACN，0.5％乙酸，然后用1毫升80％ACN，0.5％乙酸使用约0.5ml /分钟的流速洗脱第一个标记的肽。如果需要，测量单个样品的标记效率通过质谱混合重型和轻型样品前（参见"代表业绩」）。拌1点01重链和轻-标记的肽样品，通过质谱法进行定量。 </li></ol> 8，独立的肽混合物用pH值基本反相（BPRP）色谱&lt;/ P&gt; 碱性pH值反相（BPRP）层析，肽混合物分离成多个级分，这是通过LC-MS/MS独立地分析，以增加蛋白质的覆盖范围。 <ol><li>通过施加增加乙腈浓度的梯度在10mM碳酸氢铵（pH8）中的C18-HPLC柱分级分离肽混合物。开始用5％（V / V）乙腈5分钟，加至35％的ACN 60分钟，然后以90％的ACN 1分钟。降低ACN 5％至重新平衡为9分钟的列之前保留在90％ACN 4分钟。收集馏分96等体积在96 - 孔板（A1至H12）。使用UV检测器在220nm处，而肽的洗脱从柱（10-70分钟对这里所描述的条件下）监测分馏。 </li><li>从孔A1，C1，E1和G1（级分A1）结合级分，从井B1，D1，F1和H1（馏分B1）中，从井A2，C2，E2和G2（级分A2），并相应的剩余部分。取出瑟尔t使用真空离心机。从馏分重悬肽A1，B2，A3，B4，A5，B6，A7，B8，A9，B10，A11，B12和在130微升的1M urea/0.5％的TFA和纯化，如步骤9中所述使用StageTips。店级分B1，A2，B3，A4，B5，A6，B7，A8，B9，A10，B11，A12和在-20℃。 </li></ol> 9。净化肽由走走停停萃取（StageTips） 由包装200微升移液器吸头具有两个磁盘C18为1.07毫米的内径（ID）的制备C18-StageTip 7微柱。把舞台提示到Eppendorf管。使用微量离心与130微升甲醇，然后130 UL 80％ACN，0.5％醋酸冲洗技巧。平衡StageTips用130微升0.1％TFA。转肽混合物以StageTips和洗涤用130微升0.1％TFA，然后加入40μl的0.1％TFA，然后加入40μl乙酸0.5％。第一洗脱的肽用20微升40％乙腈，0.5％乙酸，然后用20微升80％乙腈，0.5％乙酸。合并洗脱液一第二干燥，真空过滤。 10。微细的LC-MS/MS <ol><li>溶解肽在1-5微升5％的甲酸，5％乙腈至约1微克/微升的浓度。解决〜1微克的肽上的100微米×20厘米C18-反相HPLC柱，用6-22％乙腈的0.125％甲酸梯度施加在75或100分钟的〜300升/分钟的流速。 </li><li>通过使用LTQ的Orbitrap Velos的12或类似的液相色谱-质谱联用平台与质谱仪提供高分辨率和高质量精度鉴定的肽。操作质谱仪的数据依赖模式，在轨道阱分析仪获得了完整的MS扫描（分辨率为60,000）。产生线性离子阱MS / MS谱图在全质谱检测到的20种最丰富的离子。对于MS / MS设置自动增益控制（AGC）的目标，以1×10 6的完整的MS和2000。设置最大的离子积累倍至1,000毫秒对于MS和150毫秒的MS / MS分析。从进一步选择从MS / MS进行20-60秒不包括零碎肽前体离子。 </li></ol> 11，MS / MS数据采集通过使用这些参数（ 表1）比较，MS / MS谱图RAW文件到一个理论数据库用算法如SEQUEST 8识别的肽。 表1。肽数据库搜索参数。 <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> 常规参数 </td><td>全胰蛋白酶消化高达2错过分裂</td></tr><tr height="19"><td> 25 ppm的母离子耐受性</td></tr><tr height="19"><td> 1.0大碎片离子耐受性</td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> 静态修改 </sTRONG&gt; </td><td> 57.02146 Da于半胱氨酸，carboxyamidomethylation </td></tr><tr height="19"><td> 28.03130达赖氨酸和肽的N-末端，光二甲基化标签</td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> 动态修改 </td><td> 15.99491达蛋氨酸，氧化</td></tr><tr height="19"><td> 6.03766沓上赖氨酸和肽的N-末端，重二甲基化标签</td></tr></tbody></table><ol><li>滤波器的肽使用的开放阅读框架的一个数据库中的实际和扭转方向1％的错误发现率的方法，如目标诱饵9策略。 </li></ol> 12。肽定量计算重型和轻型双MS1的领域提取离子色谱（MS1峰面积）和肽信噪（S / N）比10。包括肽对只有当他们的平均信号 - 河内SE比上述五。量化的肽的相对丰度的两个样本为相同的肽（MS1峰面积比）的重链和轻版本MS1峰面积的比值在。计算的相对丰度蛋白的中位数MS1峰面积比为在该蛋白质的所有肽。

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作者宣称没有竞争的经济利益或其他的利益冲突。

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

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。

定量蛋白质组学使用还原二甲基化的稳定同位素标记

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

quantitative-proteomics-using-reductive-dimethylation-for-stable

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

16.1K 次观看.  Genoscope. 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。 

视频: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

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

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

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

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

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

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis 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

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

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 ...

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

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

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

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags 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

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

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

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

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)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

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

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

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 ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

稳定笼 Aziridinium 离子的制备及其开环 Azaheterocycles 的合成

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...