This work has been performed at the Molecular Foundry, Lawrence Berkeley National Laboratory and supported by the Office of Science, Office of Basic Energy Sciences, Scientific User Facilities Division of the US Department of Energy, under Contract No. DE-AC02&#8211;05CH11231. The financial support of F.M. by a ME-Fulbright fellowship and A.S. by Higher Education Commission of Pakistan are gratefully acknowledged.

注:在开始之前，检查所有相关的材料数据表（MSDS）。几个在合成和应用程序使用的化学品是有毒的。请遵守所有适当的安全措施，并使用足够的保护设备（白大褂，全长长裤，闭趾鞋，防护眼镜，手套）。处理液氮氮吸附测量（绝缘手套，面罩）时，请使用所有低温个人防护装备。 1.多孔聚合物的制备巨石散装和毛细管柱格式<ol><li>本体聚合物为整体式表征<ol><li>通过碱性氧化铝柱纯化苯乙烯，二乙烯基苯和甲基丙烯酸，以除去聚合抑制剂。放置10克碱性氧化铝在25毫升一次性塑料注射器玻璃棉纤维包装在注射器尖端的插头。通过该柱渗滤约10毫升的单体。 </li><li>加载单体（50毫克苯乙烯，100毫克二乙烯基苯和50毫克甲基丙烯酸）和孔隙形成剂（300毫克甲苯和300毫克异辛烷）在1毫升的玻璃小瓶中。添加聚合反应引发剂，4毫克的2,2&#39;-偶氮二异丁腈（AIBN，1％，相对于单体）。 </li><li>通过超声均化10分钟。由通过液体鼓泡氮气10分钟除去溶解的氧。密封瓶盖用石蜡膜，并将其放置在水浴中，在60℃下进行6小时聚合该混合物。 </li><li>冷却至室温，并小心地打破小瓶。转移聚合物整料成纤维素提取套管。将抽提套管成索氏提取腔室，并将其组装到一个圆底烧瓶中，包含甲醇的量，这是提取室的至少三倍的体积。装配一个冷凝器到提取室的上部。通过煮沸甲醇进行索氏提取16小时，以确保完全除去未反应的单体和孔隙形成剂。 </li><li>干燥过夜，在真空烘箱中在60℃。确认羧基官能团的存在附加的PCP通过傅里叶变换红外光谱（FT-IR）。测量表面积通过氮气吸附孔率。 </li></ol></li><li>功能化石英毛细管的整体柱的制备<ol><li>切割2米的聚酰亚胺涂层的100微米ID熔融石英毛细管。它连接到一个0.25-0.50毫升玻璃注射器和用丙酮洗涤毛细管。通过用水冲洗毛细管除去丙酮。 </li><li>为了激活毛细管的内部二氧化硅涂层，使用注射泵流到一个的0.2M NaOH水溶液在0.25微升/分钟进行30分钟。用清水冲洗干净，直到出水呈中性。 </li><li>用pH试纸条，检查出水pH。为了质子毛细管的硅烷醇基团，泵送的0.2M aqueo通过毛细管我们HCl溶液以0.25微升/分钟进行30分钟。用清水冲洗干净，直到出水呈中性。冲洗用乙醇。 </li><li>在0.25微升泵20％（W / W）的3-（三甲氧基甲硅烷基）丙基甲基丙烯酸酯（pH值为5用乙酸调节）乙醇溶液/分钟1小时。在此步骤中，二氧化硅毛细管官能乙烯基为了将聚合物整料附着到毛细管内表面上。 </li><li>冲洗，用丙酮，干燥在氮气流中，在室温下过夜，使用前离开。切断毛细管成更短的片长20公分。 </li></ol></li><li>整体毛细管色谱柱的研制<ol><li>制备相同的聚合混合物为在用橡胶隔膜1毫升的玻璃小瓶本体聚合物整料（第1.1节）。加入引发剂1％AIBN相对于单体。通过超声均化10分钟。 </li><li>通过偶联一个非官能化的二氧化硅毛细管用氮气净化的聚合混合物以氮气流。 <ol><li>通过小瓶的橡胶隔片插入氮气流毛细管并沉浸入聚合混合物中，使氮气气泡通过液体。离开瓶盖略显宽松，以避免过压。吹扫10分钟。 </li><li>解除从聚合混合物中的氮气流毛细管到小瓶的顶部空间，并关闭紧瓶盖。通过隔膜加入聚合混合物中插入一个官能毛细管。产生成通过注入顶部空间的氮的毛细压力过量泵通过官能毛细管聚合混合物。 </li><li>从毛细管的流出物收集几滴聚合混合物，以确保其完全填充并用橡胶隔片关闭它。取毛细管出来的小瓶的非常仔细，并关闭毛细管的入口用橡胶隔膜。 </li></ol></li><li>聚合混合在60℃下包含在水浴毛细管6小时TURE。在室温下冷却并切断毛细管的两端的几毫米。通过使用HPLC泵在3微升/分30分钟，冲洗柱，用乙腈除去未反应单体和成孔剂。检查毛细管柱的反压。 </li></ol></li></ol> 2.生长铁benzenetrycarboxylate的（FeBTC）PCP <ol><li>在FeBTC卫生部对本体聚合物为整体式增长的表征<ol><li>磨用研钵和研杵将预先干燥整料。 </li><li>浸入100毫克整料粉末在5毫升2毫的FeCl 3·6H 2 O的乙醇15分钟。用尼龙过滤器（0.22微米），并用乙醇洗涤粉末真空过滤。浸入整料粉末在5ml的乙醇2mM的1,3,5-苯三酸（BTC）15分钟。用尼龙过滤器（0.22微米），并用乙醇洗涤粉末真空过滤。</li><li>根据需要重复步骤2号。最终的金属 - 有机涂层的增长将受到施加的周期数来定义。通常情况下，在10和30个循环被执行。确认的新孔由氮吸附孔率的存在。测量的由热重分析（TGA）的附加金属位点的量。 </li></ol></li><li>在FeBTC卫生部对毛细管整体柱增长磷酸的浓缩<ol><li>使用注射泵。冲洗毛细管整料有2mM的FeCl 3·6H 2 O的乙醇15分钟，2微升/分钟。用乙醇进行15分钟，在2微升/分钟洗净。冲洗毛细管整料具有2mM的BTC在乙醇中15分钟，在2微升/分钟。用乙醇进行15分钟，在2微升/分钟洗净。 </li><li>根据需要重复步骤1。最终的金属 - 有机涂层的增长将通过执行循环的数目来限定。 </li></ol></li></ol> 3.蛋白质的消化和E磷酸肽nrichment <ol><li>蛋白质消化<ol><li>溶解0.5非脂乳毫升在1毫升水中，并将其分成200微升馏分。 </li><li>对于蛋白质的消化添加160微升的1M碳酸氢铵和50微升45毫二硫苏糖醇对每个级分，以裂解二硫键。在恒温15分钟孵育在50℃下。 </li><li>逐渐加入50微升的碘乙酰胺的100mM的水溶液，同时将溶液冷却至室温。碘乙酰胺将阻止新的二硫键的形成。 </li><li>孵育在黑暗中进行15分钟，在室温下进行。加入1毫升去离子水。加入2微克胰蛋白酶消化的蛋白质在恒温在37℃下14小时。 </li><li>通过酸化终止消化，用10微升的1％三氟乙酸，并且将其放置在恒温5分钟，在室温下进行。存储消化的蛋白质在-20 C。 </li></ol></li><li>富集使用毛细管柱卫生部的磷酸化。 <ol><li>冲洗用100μl的4列:含有0.1％三氟乙酸进行10分钟的乙腈的1:1混合物以1微升/分钟的流速。泵通过柱子蛋白质的消化在2微升/分钟进行30分钟。 </li><li>含有0.1％三氟乙酸，10分钟，以1微升/分钟的流速乙腈的1:1混合物:用4再次洗出非磷酸化的肽。与水10分钟，以1微升/分钟的流速洗涤。 </li><li>使用泵送以1微升/分15分钟，250毫pH7的磷酸盐缓冲溶液洗脱磷酸肽。收集在小瓶中的洗脱剂和脱盐使用标准协议19的溶液。制备2毫克/毫升2,5-二羟基苯甲酸使用它作为基质为飞行时间的基质辅助激光解吸/电离质谱（MALDI-TOF-MS）。绘制2微升2,5-二羟基苯甲酸的进该尖端部以洗脱phosphopeptides并直接发现他们到MALDI板上。 </li><li>分析斑点通过MALDI-TOF-MS和通过用水和甲醇冲洗彻底再生柱子。 </li></ol></li></ol>

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The authors have nothing to disclose.

原聚合物整料包含能够结合金属羧酸官能团。协调对原始材料的初始金属位点，我们能够以生长PCP涂层（ 图1A），结合了许多附加的金属位点成形的微孔网络。这使得所提出的卫生部材料萃取或纯化步骤如涉及金属物质，如固定的金属离子亲和层析（IMAC）技术的吸引力。使用对磷酸化肽的富集的毛细管柱的一般方法示于图1B。 大量粉末巨石的准备使?...

多孔配位聚合物（的PCP）是基于由有机配位体与重复在1，2或3个维度，可以是无定形或结晶1-3延伸协调实体联金属中心配位化合物。在最近几年中，此类多孔材料已引起了广泛的关注，因为它们的高孔隙率，宽化学可调性，并且它们的稳定性。主治医师已探索了广泛的应用，包括气体储存，气体分离，和催化3-6，和最近，主治医师的第一分析应用已经描述7。 因为它们的增强的化学功能和高孔隙率的PCP已针对其巨大潜力的纯化过程和色谱分离的提高，以及一些关于本主题的报告已经发表7-13。然而，初级保健医生的表现并不在目前的equivaleNT水平可能存在的色谱材料，由于通过这些固体填充床的大颗粒间的空隙快速扩散，因为它们通常是不规则形状的颗粒或晶体的形态。这个不规则分布填料导致低于预期的性能，以及高柱背压和不希望的峰形形貌14,15。 为了通过颗粒间的空隙，以解决快速扩散的问题，并伴随地提升的PCP的用于分析应用的性能的基础上，一个大孔聚合物整料16的混合材料的发展，它包含大孔的表面上的PCP会是可取的。聚合物整料是自包含的，单件材料，可以通过它们的气孔维持对流，这使得它们的最有效的替代品对珠粒填料，并已成功地由几个C商品化1<html> ompanies 17,18。多孔聚合物整料通常是基于一个单体的聚合和在致孔剂，这是典型的有机溶剂二元混合物的存在下，交联剂上。获得的单片材料具有microglobular结构和高的孔隙率和流动渗透性。 一种简单的方法来统一这些材料以制备含有五氯酚聚合物整料是基于在整体件的聚合混合物中直接加入作为合成的PCP的。这种方法导致的PCP大多埋在聚合物支架，和不活跃的最终材料14,15的进一步应用。不同的合成方法显然需要以，例如，开发的PCP，或晶体金属 - 有机骨架（MOFs）其中大部分包含在晶体内的孔是从聚合物整料的大孔可访问的均匀的膜。 吨"&gt;此处我们报告一个简单协议，用于基于与合适的官能团为的PCP的连接，可以很容易地实现为大孔聚合物载体上的金属 - 有机聚合物混合材料（卫生部）的制备方法的自包含单-piece聚合物整料与最佳的性能流通应用的列格式的聚合物合成过程之后是一个简单的室温溶液系&#160;方法上的整体件19-20的孔的内表面长出的PCP涂层。作为第一个例子，我们描述内的大孔聚（苯乙烯 - 二乙烯基苯 - 甲基丙烯酸）整料的制备的铁（III）benzenetricarboxylate（FeBTC）配位聚合物膜构成。这个方法是有效的用于制备的散装粉末以及毛细管柱和所描述的协议是很容易实现的其他的PCP。作为MOPHs作为功能材料的流动THROU的电位的例子GH应用，我们应用了开发FeBTC卫生部其中包含一个致密的涂层的Fe（III）的中心，以从消化蛋白质混合物利用磷酸和Fe的结合亲和力富集磷酸（三）。该开发协议21包括三个主要部分:大孔有机聚合物整体支持的准备;整料的孔的表面上的PCP涂层的增长;应用磷酸的富集。

<table><tbody><tr><td>Polyimide-coated capillaries</td><td>Polymicro Technologies</td><td>TSP100375</td><td>100 &amp;mu;m i.d.</td></tr><tr><td>3-(trimethoxysilyl)propyl methacrylate, 98%</td><td>Sigma-Aldrich</td><td>440159</td><td></td></tr><tr><td>Styrene, 99%</td><td>Sigma-Aldrich</td><td>W323306</td><td>Technical grade</td></tr><tr><td>Divinylbenzene, 80%</td><td>Sigma-Aldrich</td><td>414565</td><td></td></tr><tr><td>Methacrylic acid, 98%</td><td>Mallinckrodt</td><td>MK150659</td><td></td></tr><tr><td>Toluene, &amp;ge;99.5%</td><td>EMD chemicals</td><td>MTX0735-6</td><td></td></tr><tr><td>Isooctane, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>650439</td><td></td></tr><tr><td>2,2&#039;-azobisisobutyronitrile, 98%</td><td>Sigma-Aldrich</td><td>441090</td><td></td></tr><tr><td>Aluminium oxide (basic alumina)</td><td>Sigma-Aldrich</td><td>199443</td><td></td></tr><tr><td>Iron (III) chloride hexahydrate, 97%</td><td>Sigma-Aldrich</td><td>236489</td><td></td></tr><tr><td>1,3,5-benzenetrycarboxylic acid, 95%</td><td>Sigma-Aldrich</td><td>482749</td><td></td></tr><tr><td>Acetonitrile, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>360457</td><td></td></tr><tr><td>Ammonium bicarbonate, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>9830</td><td></td></tr><tr><td>Trifluoroacetic acid, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>302031</td><td></td></tr><tr><td>Ethanol, &amp;ge;99.8%</td><td>Sigma-Aldrich</td><td>2854</td><td></td></tr><tr><td>Iodoacetamide, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>I1149</td><td></td></tr><tr><td>Dithiothreitol, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>43819</td><td></td></tr><tr><td>Monobasic sodium phosphate dihydrate, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>71505</td><td></td></tr><tr><td>Dibasic sodium phosphate dihydrate, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>Phosphoric acid, &amp;ge;85%</td><td>Sigma-Aldrich</td><td>438081</td><td></td></tr><tr><td>2,5-dihydroxybenzoic acid, &amp;ge;99%</td><td>Sigma-Aldrich</td><td>85707</td><td></td></tr><tr><td>Trypsin</td><td>Sigma-Aldrich</td><td>T8003</td><td>Bovine pancreas</td></tr><tr><td>&amp;beta;-casein</td><td>Sigma-Aldrich</td><td>C6905</td><td>Bovine milk</td></tr><tr><td>ZipTip pipette tips</td><td>Merck Millipore</td><td>ZTC18S096</td><td>C18 resin</td></tr></tbody></table>

preparation of highly porous coordination polymer coatings on macroporous polymer monoliths for enhanced enrichment of phosphopeptides

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process.
The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

在有机聚合物整料的细孔表面的PCP生长的示意图示于图1。在该图中，我们示出了初始的Fe（III）原 ​​子保留在原聚合物整料的细孔表面配位的羧酸官能团。使用协议本文所述附加的有机配位体和Fe（III）离子被添加到表面，塑造聚合物整料内的多孔协调网络。 图1还示意性地示出了使用所制备的毛细管卫生部列作为用于流通支持富集磷酸肽。表面积和孔分布的测量，用?...

Watch this Scientific Journal Video about Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides at JoVE.com

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

对磷酸增强富集制备高度多孔配位聚合物涂层的孔聚合物石柱的

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous ...

preparation-highly-porous-coordination-polymer-coatings-on

Research

JoVE Journal

Chemistry

10.0K 次观看.  E. O. Lawrence Berkeley National Laboratory. A procedure for the preparation of porous hybrid separation media composed of a macroporous polymer monolith internally coated by a high surface area microporous coordination polymer is presented.

视频: Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides

Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.

We have used plasma enhanced chemical vapor deposition to deposit thin films ranging from a few nm to several 100 nm on nano-sized particles of various materials. We subsequently etch the core material to produce hollow nanoshells whose permeability is controlled by the thickness of the shell. We characterize the permeability of these coatings to small solutes and demonstrate that these barriers can provide sustained release of the core material over several days.

等离子体聚合空心颗粒的封装和渗透特征

In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma ...

科研

工程学

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

化学修饰肽核酸对单链 rna 双链 rna 的序列特异和选择性识别

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

遗传学

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

使用聚苯乙烯<em&gt;块</em&gt; - 聚（丙烯酸）涂层的金属纳米颗粒作为单体他们均聚物和共聚物聚合

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

化学

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

磷酸浓缩结合无标签定量质谱法研究前列腺癌的 Phosphoproteome

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

癌症研究

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

自组装多胺基多肽 Amphiphiles (PPAs) 及相关生物材料的简易合成协议

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled protein polymerization process is always a challenge. Here, we have developed an enzymatic methodology for constructing polymerized protein step by step in a rationally-controlled sequence. In this method, the C-terminus of a protein monomer is NGL for protein conjugation using OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases) 1) while the N-terminus was a cleavable TEV (tobacco etch virus) cleavage site plus an L (ENLYFQ/GL) for temporary N-terminal protecting. Consequently, OaAEP1 was able to add only one protein monomer at a time, and then the TEV protease cleaved the N-terminus between Q and G to expose the NH2-Gly-Leu. Then the unit is ready for next OaAEP1 ligation. The engineered polyprotein is examined by unfolding individual protein domain using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Therefore, this study provides a useful strategy for polyprotein engineering and immobilization.

Here, we present a protocol to conjugate protein monomer by enzymes forming protein polymer with a controlled sequence and immobilize it on the surface for single-molecule force spectroscopy studies.

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

生物化学

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

利用毛细管区电泳串联质谱技术进行大尺度自上而下蛋白质组学

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

生物学

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltrimethoxysilane (APTMS) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTMS. The deposition of APTMS and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Preparation of Polypentafluorophenyl acrylate Functionalized SiO2 Beads for Protein Purification

A protocol for the preparation of poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads is presented. The poly(PFPA) functionalized surface is then immobilized with antibodies and used successfully for the protein separation through immunoprecipitation.

聚五氟丙烯酸酯 (五氟丙烯酸酯) 功能化 sio2 珠的蛋白质纯化

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

信息承载Peptoids及其序列定向动态共价自组装的合成

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...