这项工作是由德国研究基金会STE 1765 / 3-1（AS）和德国联邦教育与研究03IPT614A（CG）的支持。我们感谢莱因哈德隆梅尔和佩特拉布斯的出色的技术支持，以及卡琳·约翰逊和克里斯蒂娜Hentzschel与组织和人体测量的执行帮助

（/十分之一百十四协议号EA1）的方案经当地伦理委员会对人类的研究。 1.血液处理<ol><li>根据标准程序或快速的方法，从前臂静脉和流程收集07:00至上午08时00分之间的静脉血后，隔夜快。指示受试者抽血前不能行使或烟雾。 </li><li>对于标准处理，收集冷冻含有EDTA的试管血，离心10 min内的3000 XG在4℃下10分钟。收集上清，并保持在-80°C，直到进一步的处理用放射免疫法。 </li><li>为快速处理，立即稀释血液1:10冰冷缓冲液（pH 3.6）含有0.1M乙酸铵，0.5M氯化钠和酶抑制剂（采血后1分钟之内）（diprotin A，E-64-D，抗蛋白酶，亮肽素，抑糜素，1微克/毫升）。然后，在10分钟内离心以3000 xg离心10分钟，4℃，并收集上清我们ING在大鼠前6详见聚丙烯管吸管。 <ol><li>电荷层析柱（360毫克，55-105微米）用100％乙腈（速率为10毫升/分钟），达到平衡用0.1％三氟乙酸（TFA，速率10ml /分钟）和负载与上清液以1ml恒定速率/分钟使用注射泵。 </li><li>此后，洗涤用3ml 0.1％TFA的墨盒（速率1​​0ml /分钟），并用含0.1％TFA（2毫升/分钟）2毫升70％乙腈缓慢洗脱。 </li><li>采用真空离心并储存在-80°C，直到用放射免疫法进一步处理干燥洗脱的样品。 注:操守聚丙烯（快速处理）和硼硅（放射免疫法）表现出显著下表面结合特性，从 ​​而最大限度地减少对大多数26日前研究肽的肽损失管的所有步骤。 </li></ol></li></ol> 2.测量注:本节步骤应在实验室中进行认证的放射性物质的工作。标准预防措施125我应采取的工作。 <ol><li>放射性标记肽的恢复<ol><li>获得125 I放射性标记的人的肽（ 如酰基生长素释放肽，GLP-1，胰高血糖素，胰岛素，亲吻促动素，瘦素，注射nesfatin-1，PYY 3-36和生长抑素-28）。 </li><li>保持粉末形式的肽，直至实验，然后在0.1％的新鲜稀乙酸（〜每毫升100000的cpm）。 </li><li>为标准的血液处理，之后直接采血到含有管，传送1毫升血液成用50μl含有3,000-6,000的cpm放射性标记的管冷冻EDTA（直接计数的实验开始前）。 </li><li>对于快速处理，含血液进入含9毫升快速缓冲管EDTA转移取血1ml（对于组成见1.3），并含有30,000-60,000 CPM 500微升放射性标记。 radiolabe更高由于1:10稀释使用10倍体积l对于快速处理。 </li><li>此后，过程的样品与步骤1.2到1.3.2说明。 </li><li>对于回收实验，通过真空离心不干燥的样品和在-80℃不存储。相反，评估放射性标记的肽直接事后用γ计数器的恢复。 </li><li>衡量标准样品在整个上清液，而在快速样品分析总体积的1/10到获得的放射性标记的使用量的可比性。为了进行测量，转移上清液在管配合到γ计数器和评估的每分钟计数</li><li>作为一个100％的标准，用两个样品用50μl不进行处理125 I放射性标记肽。在测量中2.1.7所述的相同时间与其他样品。 </li><li>执行实验的五至六倍的每个肽。 </li></ol></li><li>放射性标记的生长素释放肽高效液相色谱法<ol><li>抽回血在C含管和1ml转移到含含15,000-20,000 CPM（实验开始前直接计入）200微升放射性酰基生长素管hilled EDTA。 </li><li>对于快速处理，传输取血1ml到含有9毫升快速缓冲管（用于组成见1.3），并含有15,000-20,000 CPM 200微升放射性酰基生长素。 </li><li>此后，过程的样品与步骤1.2到1.3.2说明。 </li><li>通过反相HPLC进一步分析，直接加载样品到在水中的17％乙腈平衡过的稳定的键C18柱（2.1毫米×50毫米，1.8微米）（均补充有0.1％TFA）纯化。 </li><li> 5分钟的平衡后，使用梯度从17-40％乙腈洗脱的样品在40分钟（流速为1毫升/分钟速率）。 </li><li>收集的每分钟1ml馏分，用γ计数器分析放射性。 </li><li>在一个单独的实验中，负载200微升放射性酰基生长素释放肽含有15,000-20,000CPM直接上柱和步骤2.2.4 2.2.6描述执行HPLC。 </li></ol></li><li>放射免疫分析<ol><li>对于放射免疫分析法，解冻上清（标准处理），并在室温下真空冻干粉（快速法）。 </li><li>紧接放射免疫分析法，双蒸馏水2 O根据血浆（500微升）的原体积重悬浮干燥快速的样品。 </li><li>作为根据制造商的协议使用商业放射免疫测定12,27之前描述评估亲吻促动素和总（包括desacyl和酰基生长素释放肽），以及酰基的生长素释放肽。使用硼硅管，使颗粒稳定形成。 <ol><li>在第一天，孵育在由生产的一段24小时所提供的稀释测定缓冲液和一抗（ 例如抗生长素释放肽）的样品。 </li><li>在第二天，添加125 I示踪剂（ 如 125 I-生长素），VORTEX和孵化为期24小时。 </li><li>第三天，添加沉淀剂，涡旋并孵育如制造商推荐的。然后，离心管在3000×g离心在4℃下20分钟。除去上清液并用γ计数器计数在颗粒放射性</li></ol></li><li>计算desacyl生长素释放肽作为总减去酰基生长素释放肽的差异。通过desacyl生长素释放肽为每个单独的样本除以酰基评估酰基/ desacyl生长素释放比率。 </li><li>处理所有样品 - 如果可能的话 - 在一个批次，避免批间差异。在本实验中的批内变异是亲吻促动素&lt;8％，&lt;总量的7％和酰基生长素释放肽&lt;9％。 </li></ol></li></ol> 3.统计分析<ol><li>确定使用Kolmogorov-Smirnov检验数据的分布。快速数据平均值（SEM）平均值±标准差。 </li><li>评估两者之间的差异经t检验组。评估所有成对多重比较程序（图基事后检验）或双向ANOVA随后的Holm-Sidak方法的多个组之间的差异。 </li><li>考虑P &lt;0.05显著并执行使用统计程序分析。 </li></ol>

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I., Xie, L., Huang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+of+kisspeptin+levels+in+girls+with+idiopathic+central+precocious+puberty+and+its+significance.">Testing of kisspeptin levels in girls with idiopathic central precocious puberty and its significance.</a> Exp Ther Med. 9 (6), 2369-2373 (2015).</li><li>Hosoda, H., Kojima, M., Matsuo, H., Kangawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ghrelin+and+des-acyl+ghrelin:+two+major+forms+of+rat+ghrelin+peptide+in+gastrointestinal+tissue.">Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue.</a> Biochem Biophys Res Commun. 279 (3), 909-913 (2000).</li><li>Raff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+and+active+ghrelin+in+developing+rats+during+hypoxia.">Total and active ghrelin in developing rats during hypoxia.</a> Endocrine. 21 (2), 159-161 (2003).</li><li>Evans, M. J., Livesey, J. H., Ellis, M. J., Yandle, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anticoagulants+and+storage+temperatures+on+stability+of+plasma+and+serum+hormones.">Effect of anticoagulants and storage temperatures on stability of plasma and serum hormones.</a> Clin Biochem. 34 (2), 107-112 (2001).</li><li>Nabuchi, Y., Fujiwara, E., Kuboniwa, H., Asoh, Y., Ushio, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stability+and+degradation+pathway+of+recombinant+human+parathyroid+hormone:+deamidation+of+asparaginyl+residue+and+peptide+bond+cleavage+at+aspartyl+and+asparaginyl+residues.">The stability and degradation pathway of recombinant human parathyroid hormone: deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues.</a> Pharm Res. 14 (12), 1685-1690 (1997).</li><li>White, A., Handler, P., Smith, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Biochemistry. , (1973).</li></ol>

作者什么都没有透露。

我们之前报道，对于血液处理的快速方法，改善了11/12肽复苏相比，大鼠6个标准的血液处理。在本研究中，我们已经表明，这种方法也适合于在人体中使用。以下快速处理，测试了9 9 125 I标记的肽的回收率相比标准血液处理（在冰上EDTA采血管）的改善。所观察到的改善，从19-39％，这可能是相关的，特别是当该肽的产率是低，这可能被掩蔽时只有细微的差别预期的条件下不等。 </...

在肥胖1,2世界范围内日益流行的光，研究食物的摄入监管领域越来越重要。而迄今为止，只有一种肽，已知是外周产生并作用于中枢的刺激食物摄入，即生长素释放肽3，在过去的几十年中，范围广泛的肽已发现减少食物摄取， 例如 。瘦素，肽YY（PYY）和也胰高血糖素样肽-1（GLP-1）和胰岛素4，因此，在研究调查饥饿和饱满感肽水平的调节机制经常评估，并在同一时间，假设所研究的肽是稳定的，并在过程中等离子体形成高产量恢复。可是，很多时候不是这种情况下，由于内源性迅速击穿如之前如 。生长素释放肽是从酰基降解为desacyl生长素释放肽5。因此，我们最近描述了血液PROCES的快速方法唱采用ř得出的温度，A cidification老鼠，P rotease抑制， 我 sotopic外源控制和D ilution 6。这种方法改善了11 12的肽测试，并允许正确的循环分子形式的测定标准相比，血液处理（EDTA血冰）6复苏。这种方法已在几个随后的研究7-12被用于检测循环的生长素释放肽以及促肾上腺皮质激素释放因子13的。因此，该方法已被证明为在啮齿类动物的肽的研究是有用的。然而，由于啮齿类动物的研究并不总是平移到另一个物种，该方法应被用于在人血液中的用途确定为好。 本研究的目的是测试在人类中用于血液处理的快速方法相比标准血液处理中，在冰上EDTA采血管，这是广泛推荐14和频繁ūsed的临床和研究环境。我们测试的选择125参与食物摄入，包括设立的肽，以及新的候选的规则I标记的肽的回收最近建议发挥摄食调节作用（对食物摄入的影响在表1中示出）以下的处理用这两种方法。还选择激素来代表不同的长度和电荷（ 表2）的肽。此外，生长素我们调查的分子形式（S）按照标准和快速的方法。最后，我们评估内源性生长激素释放肽（酰基和desacyl生长素释放肽），以及亲吻促动素水平，肽也最近建议发挥食物摄入15,16的下面的快速或标准加工的调节作用。此外，我们还调查了受试者群体，这些肽水平具有广泛的身体质量指数（从10.2-67.6公斤/米2）研究possib的涉及到长期改变体重乐的差异。 <img alt="表格1" src="/files/ftp_upload/53959/53959table1.jpg" /><img alt="表2" src="/files/ftp_upload/53959/53959table2.jpg" />诊断，评估和计划: 研究参与者 所有的研究参与者新住院患者（包含了内入院两天）心身医学科在查理特-Universitätsmedizin柏林，并签署知情同意书。为了避免唯一的女患者被纳入性别产生任何影响。总共有42科目参加了这项研究，并随机分为3组:正常体重（BMI 18.5-25公斤/米2，N = 12），神经性厌食症（BMI &lt;17.5公斤/米2，N = 15）和肥胖（BMI"30公斤/米2，N = 15）。厌食症和肥胖患者根据疾病-10的国际分类诊断和住院体重增加（神经性厌食），或体重减少（肥胖），分别。所有正常体重的患者因躯体症状无相关的躯体疾病住院独占。有胃肠道症状，躯体或胃肠道手术史患者被排除在外。排除标准也包含了&lt;18岁，目前在怀孕和未治疗精神性疾病。以增加或减少体重，分别接收饮食治疗前入院后第2天或3进行采血。人体测量参数进行了评估在同一天。

<table><tbody><tr><td>Diprotin A</td><td>Peptides International, Louisville, KY, USA</td><td>IDP-4132</td><td></td></tr><tr><td>E-64-d</td><td>Peptides International, Louisville, KY, USA</td><td>IED-4321-v</td><td></td></tr><tr><td>Antipain</td><td>Peptides International, Louisville, KY, USA</td><td>IAP-4062</td><td></td></tr><tr><td>Leupeptin</td><td>Peptides International, Louisville, KY, USA</td><td>ILP-4041</td><td></td></tr><tr><td>Chymostatin</td><td>Peptides International, Louisville, KY, USA</td><td>ICY-4063</td><td></td></tr><tr><td>Sep-Pak C18 cartridges</td><td>Waters Corporation, Milford, MA, USA</td><td>WAT051910</td><td>360 mg, 55-105 &amp;micro;m</td></tr><tr><td>Acyl-ghrelin</td><td>Millipore, Billerica, MA, USA</td><td>9088-HK</td><td>Radioactive</td></tr><tr><td>GLP-1</td><td>Millipore, Billerica, MA, USA</td><td>9035-HK</td><td>Radioactive</td></tr><tr><td>Glucagon</td><td>Millipore, Billerica, MA, USA</td><td>9030</td><td>Radioactive</td></tr><tr><td>Insulin</td><td>Millipore, Billerica, MA, USA</td><td>9011S</td><td>Radioactive</td></tr><tr><td>Leptin</td><td>Millipore, Billerica, MA, USA</td><td>9081-HK</td><td>Radioactive</td></tr><tr><td>Kisspeptin-10</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-048-56</td><td>Radioactive</td></tr><tr><td>Nesfatin-1</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-003-26</td><td>Radioactive</td></tr><tr><td>PYY&lt;sub&gt;3-36&lt;/sub&gt;</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-059-02&amp;nbsp;</td><td>Radioactive</td></tr><tr><td>Somatostatin-28</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td>T-060-16&amp;nbsp;</td><td>Radioactive</td></tr><tr><td>ZORBAX Rapid Resolution HT SB-C18 column</td><td>Agilent Technologies, Santa Clara, CA, USA</td><td>822700-902</td><td>2.1 mm x 50 mm, 1.8 &amp;micro;m</td></tr><tr><td>Agilent 1200 LC&amp;nbsp;</td><td>Agilent Technologies, Santa Clara, CA, USA</td><td></td><td>HPLC, several components, therefore no single catalog number</td></tr><tr><td>Kisspeptin&amp;nbsp; RIA</td><td>Phoenix Pharmaceuticals, Burlingame, CA, USA</td><td># RK-048-56</td><td>Radioactive</td></tr><tr><td>Total ghrelin RIA</td><td>Millipore, Billerica, MA, USA</td><td># GHRT-89HK&amp;nbsp;</td><td>Radioactive</td></tr><tr><td>Active ghrelin RIA</td><td>Millipore, Billerica, MA, USA</td><td># GHRA-88HK</td><td>Radioactive</td></tr><tr><td>SigmaStat 3.1</td><td>Systat Software, San Jose, CA, USA</td><td>online download</td><td></td></tr></tbody></table>

a rapid method for blood processing to increase the yield of plasma peptide levels in human blood

研究食物摄入管制领域越来越重要。这通常包括肽调节食物摄入量的测量。为正确的测定的肽的浓度的，它应该是血处理过程中是稳定的。然而，这不是对几种肽它们很快被内源性肽酶降解的情况。最近，我们开发了一种血液处理方法采用ř得出的温度，A cidification，P rotease抑制， 我 sotopic外源控制和D ilution（RAPID）对大鼠使用。这里，我们已经建立了这种技术的使用在人类和调查恢复，分子形式和食物摄取调节激素的循环浓度。的快速方法显著改善了125 I标记的生长抑素-28（+ 39％），高血糖素样肽-1（+ 35％）的回收，酰基生长素释放肽和胰高血糖素（+ 32％），胰岛素和亲吻促动素（+ 29％ ），注射nesfatin-1（+ 28％），瘦素（+ 21％）相比，标准处理（冰血EDTA，P &lt;0.001），肽YY 3-36（+ 19％）。高效液相色谱表明内源性酰基生长素释放肽的洗脱在快速处理后的预期位置，而标准处理后的酰基生长素释放肽的62％被降解导致​​较早峰值可能表示desacyl生长素释放肽。快速加工后在正常体重受试者的血液酰基/ desacyl生长素释放比例为1:3相比1:23以下标准处理（p值= 0.03）。同时内生亲吻促动素水平的标准相比，处理后快速走高（+ 99％，P = 0.02）。快速血液处理方法，可以在人类中使用，产生较高的肽水平并且允许进行正确分子形式的评估。

快速血液处理增加在人体血液中的125 I放射性标记的肽的标准相比，血液处理的产率。 标准血液处理（在冰上的EDTA血）后，放射性标记的肽的回收在9/9肽（ - K范围从48-68％， 图1A）为〜60％。快速处理提高了产率在所有125 I标记的肽，即在促生长素抑制素-28（+ 39％， 图1A），胰高血糖素样肽-1（+ 35％， 图1B?...

Watch this Scientific Journal Video about A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood at JoVE.com

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

对于血液处理快速的方法，以提高血浆肽水平的人血产量

快速血液处理方法，可以在人类中使用，并产生更高的肽水平以及允许对正确分子形式的评估。因此，这种方法将在肽研究的宝贵工具。

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

a-rapid-method-for-blood-processing-to-increase-yield-plasma-peptide

Research

JoVE Journal

Immunology and Infection

8.7K 次观看.  Charité Universitätsmedizin Berlin, Campus Benjamin Franklin. 快速血液处理方法，可以在人类中使用，并产生更高的肽水平以及允许对正确分子形式的评估。因此，这种方法将在肽研究的宝贵工具。 

视频: A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

科研

遗传学

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

多尺度孔梯度，聚电解质和间接监测改性金属植入物<em&gt;在体内</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

生物工程

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 ...

化学

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

生物学

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.

生产自体富血小板血浆以促进体外人成纤维细胞扩增

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

使用工程 HEK 细胞筛选激活 MRGPRX2 的肽

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

免疫与感染

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Enrichment of Astrocyte-Derived Extracellular Vesicles from Human Plasma

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.

<meta charset="utf8"></head><body>从人血浆中富集星形胶质细胞衍生的细胞外囊泡

This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.

从人血浆中富集星形胶质细胞衍生的细胞外囊泡

Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a ...