作者感谢教授托马斯·莱纳，雅各布博士霍顿，和Serge Lyaschenko博士有益的对话。

所有上述的体内动物实验是根据经批准的协议，并在纪念Sloan Kettering癌症中心的机构动物护理和使用委员会（IACUC）的道德准则进行。 1.共轭DFO-NCS来J591 <ol><li>在1.7 ml离心管，准备一个2-5毫克/毫升的溶液J591在1ml任1×磷酸盐缓冲盐水（pH 7.4）中或0.5M的HEPES缓冲液（pH 7.4）中的。 </li><li>溶解DFO-NCS在无水DMSO 5-10毫米（3.8-7.6毫克/毫升）间的浓度。声处理或涡流，以促进完全溶解的溶液彻底。 </li><li>通过加入小等分试样的0.1M的Na 2（&lt;10微升）CO 3调整J591溶液的pH至8.8-9.0。 </li><li>一旦抗体溶液是在正确的pH值，添加了对应于一个3-4倍摩尔过量的二官能螯合剂的DFO-NCS溶液的体积。 <ol><li>对于考试PLE，加4-5微升的10mM（7.6毫克/毫升）的DFO-NCS溶液（40.4纳摩尔DFO-NCS），以1ml的2mg / ml的J591抗体溶液（13.3纳摩尔J591）。 DMSO在最终含水反应混合物中的量应不超过2％的体积/体积。 </li></ol></li><li>温育30分钟，在37℃的搅拌加热块为350rpm对反应。 </li><li> 1小时，在37℃后，使用预填充的一次性尺寸排阻脱盐柱使用0.5M的HEPES缓冲液（pH为7.4）作为洗脱剂50,000分子量截止纯化所得免疫。这一步骤将产生完成J591-DFO构建体的溶液2ml。 </li><li>测量J591-DFO的浓度构造上的紫外可见分光光度计。 </li><li>如果构建体的浓度较高是理想的，集中使用离心过滤器单元具有50000分子量截止的J591-DFO溶液。 </li><li>存储完成的J591-DFO免疫偶联物在-20在黑暗中的溶液℃。 </ol> 2.放射性标记J591-DFO 89锆注意：该协议的这一步涉及到处理和操作放射性。在执行这些步骤，或进行任何其他工作与放射性研究人员应该与他们的家机构的辐射安全部咨询。应采取一切可能的措施，以尽量减少暴露于电离辐射。 注意：在适当的放射化学音符维持兴趣，放射​​性的样品中的量应使用的剂量校准器测量和记录前和步骤2.2-2.13在下面的协议之后。这将有助于准确测定放射化学产量和具体活动。 <ol><li>制备0.5-2.0毫克J591-DFO的溶液中加入200μl的0.5M HEPES缓冲液，pH值7.5。 </li><li>吸取的体积&lt;对应于1.0-6.0毫居里（37-222活度）至2ml塑料螺旋盖微量离心管SUP&gt; 89的Zr 4+原液（通常在1.0M的草酸提供）。调节该溶液的体积，以使用1.0M的草酸共300微升。 </li><li>调节89的Zr 4+溶液用的1.0M的Na 2 CO 3的pH值至6.8-7.5。通过加入250微升的1.0M的Na 2 CO 3至89的Zr 4+溶液开始，并随后添加小（&lt;10微升）基的等分试样以达到所需的pH值。 </li><li>加的pH调节的89的Zr 4+溶液所需量的在步骤2.1中制备的J591-DFO溶液。 </li><li>检查放射性标记反应混合物的pH，以确保它落入6.8-7.5的期望的范围内。 </li><li>孵育在室温60分钟的搅拌加热块为350rpm的放射性标记反应。 </li><li>孵育60分钟后，测量RADiolabeling使用无线电型TLC，反应的产率。 <ol><li>为此目的，在硅胶浸渍型TLC条斑的放射性标记反应混合物的1微居里。允许等分试样进行干燥，运行TLC使用50mM的DTPA（pH5.5）中的洗脱液并使用无线电型TLC扫描器分析薄层色谱条89的Zr 4+结合到J591-DFO构建体将出现在原点（R ˚F&lt;0.1），而自由89的Zr 4+阳离子将由DTPA被螯合并会与溶剂前沿（ 的 R F洗脱&gt; 0.9）。 </li><li>通过整合radiochromatogram，曲线下将该区域从R中的曲线在F下0.0-0.1通过的总面积，再乘以100计算出反 ​​应的放射性标记收率。 </li></ol></li><li>如果放射性标记收率是足够（通常&gt; 2毫居里/毫克的理论比活性），淬火用5微升50毫DTPA，pH 5.5的反应。 </li><li>净化产生的免疫USI纳克具有50000分子量截止使用任0.9％的无菌盐水与5毫克/毫升龙胆酸5mg / ml的龙胆酸或0.25M的乙酸钠（pH 5.5）洗脱剂预填充的一次性大小排阻柱脱盐。这一步骤将产生完成89锆DFO-J591放射免疫偶联的溶液2ml。 </li><li>净化后，确认89锆DFO-J591采用无线电TLC的放射化学纯度在2.7步描述。 </li><li>除以活性最初添加到抗体溶液通过放射性的分离与纯化的89的Zr-DFO-J591放射免疫的量的量计算出反 ​​应的总放射性标记收率。 </li><li>除以活性的DFO-J591中的放射性标记反应的初始质量分离与纯化的89的Zr-DFO-J591放射免疫偶联量计算最终比活性。 </li><li>如果浓度较高是理想的，集中第Ë89使用离心过滤器单元具有50000分子量截止锆DFO-J591溶液。 注：在最终纯化步骤中使用的龙胆酸是一个无线电防护剂用于抗体的降解最小化由于辐46虽然89锆DFO-J591放射免疫偶联长达48小时的贮存在4℃。是可能的，这是不推荐的。如果放射免疫偶联是要被存储，则使用0.25M的乙酸钠（pH 5.5）以5毫克/毫升龙胆酸如，以减少次氯酸盐介导的放射分解的风险的存储缓冲器。47 </li></ol> 3. 体内 PET显像与89锆DFO-J591 注意：由于协议第2条，该协议的这一步涉及到处理和操作放射性。在执行这些步骤，研究人员应该与他们的家机构的辐射安全部咨询。所有possib应采取勒步骤，以尽量减少暴露于电离辐射。 <ol><li>在雄性无胸腺裸鼠皮下植入5×10 6的LNCaP前列腺癌细胞，并允许这些（在接种后3-4周），以生长到100-150毫米3异种移植物。44 </li><li>稀释89锆DFO-J591放射免疫，以1.0毫居里/ ml的0.9％无菌生理盐水的浓度。 </li><li>注入200微升89锆DFO-J591溶液（200微居里; 7.4活度）的。到尾静脉移植荷瘤小鼠的48 </li><li>在所需的成像时间点（ 例如，12，24，48，72，96，或120小时后喷射），麻醉的小鼠用2％异氟烷：氧混合气。 </li><li>放置在小动物PET扫描仪的床的鼠标，并用1％异氟烷在扫描期间保持麻醉：氧混合气。在此之前将动物放在扫描仪床上，用脚趾捏法和APPL验证麻醉Ÿ眼药膏鼠标，以防止在麻醉过程中干燥的眼睛。49 </li><li>通过用最少的使用350-700千电子伏的能量窗和6纳秒重合定时窗口40000000同步事件的静态扫描获得的小鼠的PET的数据。50 </li><li>完成收购的图像后，不要离开鼠标无人看管，不把它关在笼子里与其他老鼠，直到它恢复意识。 </li></ol>

作者什么都没有透露。

而施工，放射性标记，成像和89锆DFO-标有放射免疫通常是一个相当简单的过程，它保持了几个重要的考虑因素记在该过程的每个步骤是重要的。例如，也许在该过程的缀合步骤关注最可能的原因是该抗体的缀合反应期间的聚合。此问题是最经常在加入DFO-NCS原液后的缀合反应的混合不良的产品。22当这种情况发生时，DFO-NCS的非均匀分布可能导致过高的水平与当地反应的抗体，其可?...

由于其显着的敏感性，亲和力和选择性，抗体长期以来被认为是有前途的载体用于递送放射性同位素至肿瘤细胞。但是，它们在正电子发射断层扫描（PET）成像中的应用受到阻碍，缺乏适当的正电子发射放射性同位素为其标签1-3之一在放射性免疫的设计中最关键的考虑因素是匹配的物理衰变半寿命放射性同位素与抗体的体内药动学。更具体地说，抗体通常具有相对长的，多天的生物半衰期，因此必须用放射性同位素标记具有可比的物理半衰期。用于PET成像的应用中，抗体历来放射性标记的64铜（叔1/2 = 12.7小时），86 Y（吨1/2 = 14.7小时），或124 I（叔1/2 = 4.18 d）所示。4， 5然而，每个这些放射性同位素拥有妨碍其适用于临床影像显著的局限性。而放射免疫标记86 Y和64铜已被证明有希望的临床前调查，既同位素具有物理半衰期太短是有效用于成像在人类。124 I，与此相反，具有接近理想的物理半衰期为成像用的抗体，但它是昂贵的，并且具有导致相对较低的分辨率临床图像欠佳衰减特性。此外，124 I标记的放射性免疫能受到脱卤体内 ，一种能降低肿瘤-背景活动比率的过程。6,7 该驱动器找到一个正电子发射放射性同位素来取代64铜，86 Y，124我放射免疫起到了推波助澜的最近激增的研究89锆标记的抗体。8-12牛逼他原因89锆问世是直截了当：放射性金属具有近乎理想的化学和物理性质用于诊断的PET放射性免疫使用13 89锆经由89 Y（P，N）上使用一个回旋加速器89 Zr的反应而产生。市售和100％自然丰富89ÿ目标。14,15的放射性金属具有23％的正电子产率，衰变78.4小时的半衰期，并且发射正电子与395.5千电子伏（ 图1）的相对低的能量。 13,16,17要注意的是89锆还发射较高能量是很重要的，909千电子伏γ射线具有99％的效率。虽然这种发射不使劲与发射的511keV光子干扰，但是它对于运输，处理，和剂量需要额外考虑。尽管这样的警告，这些衰减特性最终意味着89锆不仅拥有更有利的ħ用于成像ALF-寿命的抗体比86 Y和64铜，但也可以比124 I产生更高分辨率的图像，其发射正电子与687和975 keV的更高的能量，以及一个数的光子能量与内100-150千电子伏的在511千电子伏的正电子创建的光子。13此外，89的Zr也更安全地处理，以产生更便宜，并且residualizes肿瘤更有效地比其对应物的放射性碘。18,19的一个潜在的89 Zr的限制是，它不具有治疗isotopologue， 比如 ，86 Y（PET）与90 Y（治疗）。这就排除了可以用作剂量球探对他们的治疗对口化学性质相同，替代显像剂的建设。尽管如此，调查结果表明，89锆标记的抗体是有潜力的成像代理人90 Y-和177路标记免疫。20,21 从化学的观点来看，作为一种Ⅳ族金属，89 Zr的存在是为4阳离子在水溶液。所述的Zr 4+离子被高度带电的，相对大的（有效离子半径= 0.84埃），并且可以被分类为一“硬”阳离子。因此，它表现出偏爱配体承载多达八个硬的，阴离子的氧供体。容易在89锆标记的放射性免疫中使用的最常见的螯合剂是去铁胺（DFO），一铁载体衍生的，无环螯合剂轴承3异羟肟酸基团。配位体稳定地协调的Zr 4+阳离子迅速干净在RT在生物相关的pH水平，并且将所得的Zr-DFO络合物保持稳定的在盐水中，血清多天的过程中，和全血。22计算研究强烈提示该DFO形成六配位配合物的Zr 4+，其中金属中心配位的三NEUT拉尔和配体的3阴离子氧供体，以及两个外源性配体的水 （ 图2）。23,24放射免疫采用89锆DFO结合支架的体内行为已普遍出色。然而，在一些情况下，成像和急性生物分布研究显示升高的活性水平在注射89锆标记的抗体的小鼠的骨，该数据表明，osteophilic 89的Zr 4+阳离子选自螯合剂释放在体内并且随后矿化在骨。25最近，一些调查到的新的89锆4+螯合剂的发展尤其是配体与八氧捐助者已经出现在文献中。24,26,27不过，目前，DFO是最广泛采用的螯合剂在89大幅锆标记的放射免疫。各种不同生物结合策略已被用来DFO附加到抗体，包括生物正交点击化学，硫醇反应性DFO构建体与抗体的半胱氨酸反应，和活化酯承载DFO构建体与抗体赖氨酸反应。4,28- 30轻松最常见的策略，然而，已使用DFO，DFO-NCS的异硫氰酸酯轴承衍生物（ 图2）。22本市售双功能螯合剂鲁棒且可靠地形成稳定的，共价的硫脲用的赖氨酸键抗体（ 图3）。 在过去的几年中，各种各样的89锆DFO标记放射性免疫已在文献中报道。临床前研究已经特别丰富，具有抗体不等的较为知名的西妥昔单抗，贝伐单抗，曲妥珠单抗和更深奥的抗体，如CD105靶向牛逼RC105和游离PSA靶向5A10 30-36最近，少数使用89锆DFO标记抗体早期临床试验已经出现在文献中。具体而言，在荷兰团体已经发表的试验采用89锆DFO-政制及内地事务U36，89锆DFO-替伊莫单抗，和89锆DFO -曲妥单抗。21,32,37此外，一系列的89其他临床试验锆标记的放射免疫目前正在进行，其中包括使用PSMA靶向89锆DFO-J591前列腺癌成像和HER2靶向89锆DFO，曲妥珠单抗对乳腺癌影像学检查在这里纪念Sloan Kettering癌症中心23， 30此外，虽然放射性标记的抗体仍然是最常用的89锆标记的放射性药物，放射性金属，也越来越多地被使用与其他载体，包括肽，蛋白质，和纳米材料。38-43 </sup&gt; 这89锆DFO标签方法的模块化是 ​​一笔巨大的财富。生物标志物靶向抗体的剧目是不断扩展的，并且在使用这些结构进行体内 PET成像的兴趣日益高涨。因此，我们认为，更加规范的做法和协议的制定可能受益的领域。优异的书面实验方案为DFO-NCS的共轭和89 Zr的放射性标记已经公布由Vosjan 等人 22，我们认为，通过这项工作所提供的视觉演示可进一步帮助调查新到这些技术。在协议中，在另一方面，前列腺特异性膜抗原靶向抗体J591将被用作模型系统，以说明（1）的双功能螯合剂的生物缀DFO-异硫氰酸酯的抗体，89（2）的放射合成和纯化ZR-DFO-单克隆抗体放射免疫，及（3） 在体内 PET成像与89锆DFO-单抗放射免疫偶联在癌症的小鼠模型。23,44,45

<table border="0" cellpadding="0" cellspacing="0" width="676">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:217px;">Name of the Material/Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td style="width:179px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-SCN-Bn-DFO</td>
			<td>Macrocyclics</td>
			<td>B-705</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">[89Zr]Zr-oxalate</td>
			<td>Various, including Perkin-Elmer</td>
			<td>-</td>
			<td>Caution: Radioactive material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>17-0851-01&#160;</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td>EMD Millipore</td>
			<td>UFC805024</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica Gel Impregnanted RadioTLC Paper</td>
			<td>Agilent Technologies</td>
			<td>SGI0001</td>
			<td>Cut into strips 0.5 cm wide</td>
		</tr>
	</tbody>
</table>

the bioconjugation and radiosynthesis of 89zr-dfo-labeled antibodies

特殊的亲和力，特异性和选择性的抗体使其成为肿瘤靶向PET放射性药物非常有吸引力的载体。由于其多天的生物半衰期，抗体必须贴有正电子发射放射性核素具有相对长的物理衰变半衰期。传统上，正电子发射同位素124 I（叔1/2 = 4.18 d）中，86 Y（吨1/2 = 14.7小时），和64的Cu（叔1/2 = 12.7小时），已被用于标记抗体PET成像。最近，然而，该领域已经见证了在基于抗体的PET成像剂的使用的正电子发射放射性金属89 Zr的显着增加。89锆是一种近乎理想的放射性同位素用于PET成像的免疫，因为它具有一个物理半-life（吨1/2 = 78.4小时），它与抗体的体内药动学相容并发射相对低的烯产生高分辨率的图像红绿黄正电子。此外，抗体可直接地使用铁载体衍生的螯合剂去铁胺（DFO）标记89锆。在这个协议中，前列腺特异性膜抗原靶向抗体J591将被用作模型系统，以说明（1）的双功能螯合剂的生物缀DFO-异硫氰酸酯的抗体，（2）的放射和89 ZR-纯化DFO-单抗放射免疫偶联，和（3） 在体内 PET成像与89锆DFO-单抗放射免疫偶联中的癌症的小鼠模型。

在这个协议中DFO-NCS的与抗体缀合的第一个步骤通常是相当坚固和可靠的。一般地，纯化的，螯合剂改性的免疫可以在&gt; 90％的产率获得，并且使用DFO-NCS的3摩尔当量在最初的缀合反应将产生一定程度的-的标记的约1.0-1.5螯合剂DFO的/单克隆抗体。 89锆放射性标记和纯化步骤的过程中也同样简单。在上述协议中列出的浓度&gt; 80％的放射性标记的产量和&gt; 2.0毫居里/毫克因此具体活动是在室温...

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The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

该生物耦合和重新布置<sup&gt; 89</sup&gt;锆DFO标记的抗体

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

the-bioconjugation-and-radiosynthesis-of-89zr-dfo-labeled-antibodies

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Unit 1

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Fundamentals of Chemistry

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17.0K 次观看.  Memorial Sloan Kettering Cancer Center. Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described. 

视频: The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

折纸灵感的图案和可重构颗粒自组装

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

科研

化学

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and cleaves intracellular proteins that are essential for neurotransmitter release. BoNT is comprised of a heavy chain (HC), which mediates host cell binding and internalization, and a light chain (LC), which cleaves intracellular host proteins essential for acetylcholine release. While therapies that inhibit toxin binding/internalization have a small time window of administration, compounds that target intracellular LC activity have a much larger time window of administrations, particularly relevant given the extremely long half-life of the toxin. In recent years, small molecules have been heavily analyzed as potential LC inhibitors based on their increased cellular permeability relative to larger therapeutics (peptides, aptamers, etc.). Lead identification often involves high-throughput screening (HTS), where large libraries of small molecules are screened based on their ability to modulate therapeutic target function. Here we describe a FRET-based assay with a commercial BoNT/A LC substrate and recombinant LC that can be automated for HTS of potential BoNT inhibitors. Moreover, we describe a manual technique that can be used for follow-up secondary screening, or for comparing the potency of several candidate compounds.

The botulinum neurotoxin type A light chain (BoNT/A LC) is a metalloprotease that enters motor neurons, cleaves its substrate SNAP-25, and disrupts neurotransmission, thereby resulting in flaccid paralysis. Utilizing a high-throughput-compatible FRET-based assay, large libraries of small molecules can be screened for their impact on BoNT/A LC enzymatic activity.

Botulinum neurotoxin (BoNT) is a potent and potentially lethal bacterial toxin that binds to host motor neurons, is internalized into the cell, and ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

分裂和池合成及肽叔酰胺图书馆表征

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

核磁共振作为鱼油补充剂的血脂的快速评价一个强大的工具

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

寡 peptoids 的合成及质谱分析

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...