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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.5K 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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

自动声胶肽受体激动剂的效价测定测定的连续稀释

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

科研

生物化学

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

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

使用自动工作站进行质谱测量的等离子体样品制备

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

生物学

A Spin-Tip Enrichment Strategy for Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological matrices is often necessary due to their low stoichiometry in comparison to non-modified analytes. Most enrichment workflows of PTMs on peptides in bottom-up proteomics workflows, where proteins are enzymatically digested before the resulting peptides are analyzed, only enrich one type of modification. It is the entire complement of PTMs, however, that leads to biological functions, and enrichment of a single type of PTM may miss such crosstalk of PTMs. PTM crosstalk has been observed between protein glycosylation and phosphorylation, the two most common PTMs in human proteins and also the two most studied PTMs using mass spectrometry workflows. Using the simultaneous enrichment strategy described herein, both PTMs are enriched from post-mortem human pancreatic tissue, a complex biological matrix. Dual-functional Ti(IV)-immobilized metal affinity chromatography is used to separate various forms of glycosylation and phosphorylation simultaneously in multiple fractions in a convenient spin tip-based method, allowing downstream analyses of potential PTM crosstalk interactions. This enrichment workflow for glyco- and phosphopeptides can be applied to various sample types to achieve deep profiling of multiple PTMs and identify potential target molecules for future studies.

Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreatic tissues.

一种同时分析人胰腺组织中N-糖肽和磷酸肽的自旋尖端富集策略

Mass spectrometry can provide deep coverage of post-translational modifications (PTMs), although enrichment of these modifications from complex biological ...

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 ...

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 ...

生物工程

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. ...

Preparation of Peripheral Blood Mononuclear Cell Pellets and Plasma from a Single Blood Draw at Clinical Trial Sites for Biomarker Analysis

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of treatment, and help guide dose and schedule setting of therapeutics. From a single blood draw, peripheral blood mononuclear cells can be isolated and processed to analyze and quantify protein markers, and plasma samples can be used for the analysis of circulating tumor DNA, cytokines, and plasma metabolomics. Longitudinal samples from a treatment provide information on the evolution of a given protein marker, the mutational status and immunological landscape of the patient. This can only be achieved if the processing of the peripheral blood is carried out effectively in clinical sites and samples are properly preserved from the bedside to bench. Here, we present an optimized general-purpose protocol that can be implemented at clinical sites for obtaining PBMC pellets and plasma samples in multi-center clinical trials, that will enable clinical professionals in hospital laboratories to successfully provide high quality samples, regardless of their level of technical expertise. Alternative protocol variations are also presented that are optimized for more specific downstream analytical methods. We apply this protocol for studying protein biomarkers against DNA damage response (DDR) on X-ray irradiated blood to demonstrate the suitability of the approach in oncology settings where DDR drugs and/or radiotherapy have been practiced as well as in preclinical stages where mechanistic hypothesis testing is required.

This protocol details clinically implementable preparation of high quality PBMC and plasma biosamples at the clinical trial site that can be used for translational biomarker analysis.

在生物标志物分析临床试验场单次抽血时，准备外周血单核细胞颗粒和血浆

Analysis of biomarkers in peripheral blood is becoming increasingly important in clinical trials to establish proof of mechanism to evaluate effects of ...

医学

Isolation and Cryopreservation of Highly Viable Human Peripheral Blood Mononuclear Cells From Whole Blood: A Guide for Beginners

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This detailed protocol describes the equipment, supplies, and steps for isolating, cryopreserving, and thawing high-quality and highly viable PBMCs from whole blood cells suitable for downstream applications such as flow cytometry and RNA-sequencing. Protocols for processing plasma and buffy coat from the whole blood in parallel and concurrently with PBMCs are also described. This easy-to-follow step-by-step protocol, which utilizes density gradient centrifugation to isolate PBMCs, is accompanied by a checklist of supplies, equipment, and preparation steps. This protocol is suitable for individuals with any prior experience with laboratory techniques and can be implemented in clinical or research laboratories. High-quality cell viability and RNA sequencing resulted from PBMCs collected by operators with no prior laboratory experience using this protocol.

This protocol presents an accessible guide for collecting, storing, and thawing peripheral blood mononuclear cells suitable for downstream analyses and workflows like flow cytometry and RNA sequencing. Plasma and buffy coat collections are also demonstrated.

从全血中分离和冷冻保存高活力的人外周血单核细胞:初学者指南

Peripheral blood mononuclear cells (PBMCs) are commonly used in biomedical research on the immune system and its response to disease and pathogens. This ...