Name: automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays
Uploaded: 2016-11-10T12:30:00.000+00:00
Duration: 8 min 6 s
Description: Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

1.肽序列稀释<ol><li>制备测定缓冲液:汉克斯缓冲补充有25mM的HEPES，0.1％BSA和0.5mM 3-异丁基-1-甲基黄嘌呤（IBMX），pH 7.4的盐溶液（HBSS）。 </li><li>使用批量试剂分配器5微升测定缓冲液系统添加到每个孔5 384孔低体积试验平板。 <ol><li>使用内部软件以5微升体积除一个384孔平板的每一个以及每个制造商的说明建立的分配程序。 </li><li>沉浸在分析缓冲液和总理点胶盒管。 </li><li>放置384孔板载体上的低体积试验板。 </li><li>按开始。 </li></ol></li><li>冲淡一切肽样品，无论存储车辆，在测定缓冲液产生100倍的股票肽。 注意:需要筛选肽可在磷酸盐缓冲盐水（PBS）或二甲基亚砜（DMSO），为被认为适当的（如，DMSO瓦特提供病有与二级修饰如聚乙二醇化）的肽具有有害影响。 </li><li>免除25微升100X肽股成列的声学合格384孔微孔板聚丙烯1-5。该板指定"源板"。确保源板，而不是目的板，是平底，并符合特定声公差由电声乐器的制造商定义。 </li><li>免除25微升100X参考控制入井A23和源板A的A24 </li><li>将10微升检测缓冲液到列11-15和30微升检测缓冲液到源盘A的21-22列</li><li>将10微升分析缓冲液为列6-10和16-20列第二声学合格的384孔微孔板聚丙烯的。该板指定"源板B&#39;。 </li><li>离心机源板A和B在300 XG 1分钟。包括适当的平衡板。 </li><li>使用声fluið分配器集成到自动化机器人系统，制备三个顺序1:从第1列中的源A的100倍的肽股100中间稀释液（在测定缓冲液） 注:编程需要重新格式化板和（由声学流体分配器的制造商提供）剂量反应软件允许源A和源B（参见图1板布局）之间的三声传输。步骤1.9特性的细节运用在受自动机械手控制这个实验室使用的液体分配器（见材料表 ）: <ol><li>载源板和检测板成机器人的板酒店部分。 </li><li>开放式自动化机器人的软件和负载板格式化和剂量反应程序扩展协议。点击"运行"。 注:自动化指示声流体分配器从源头板列1-5转移250 NL到源盘B 6-10列和第二ACOUSTIç流体分配器能够处理更大的液体体积与15微升分析缓冲液混合回填。然后自动化源盘B传送到集成离心机在300 XG 1分钟离心机（一种适当的平衡板是包括所有的离心步骤）。源板B被返回到声学流体分配器250 NL的转移从源盘B的6-10列到源板A的11-15列和用15微升测定缓冲回填混合。自动化传输源板，以集成离心机在300 XG 1分钟离心机。然后源板A被返回到声学流体分配器250 NL的转移从源盘A的11-15列到源盘B的16-20列和用15微升测定缓冲回填混合。自动化然后在300 XG 1分钟源盘B传送到集成离心机和离心机。 注:最后剂量响应协议指示声学分配传送从各4连续稀释源板孔中所需的体积（在源板A和源盘B两者）来构造在测定板一式两份的完整的11点曲线（预填充有5微升测定缓冲液在上面的步骤1.2 ）。自动化转移测定板，以集成离心机和离心机在300×g离心1分钟。 </li></ol></li></ol> 2.细胞的制备<ol><li>在20毫升测定缓冲液37ºC水浴悬浮迅速表达靶小鼠GLP-1受体解冻冷冻保存的中国仓鼠卵巢（CHO）细胞。 </li><li>离心细胞悬浮液，在室温下（RT）200 xg离心5分钟。包括适当的平衡。 </li><li>弃上清，重悬在10毫升分析缓冲液细胞沉淀。 </li><li>稀释细胞库存1:1台盼蓝和使用自动细胞计数器测定活细胞密度。悬浮细胞以每毫升1.6×10 6个细胞测定缓冲液（相当于每逢8000细胞测定板的1）。 </li><li>使用批量试剂分配器5微升细胞悬液加入到测定板（含于5μl测定缓冲液连续稀释的肽）的每个孔中，并孵育在室温30分钟。 </li></ol> 3. HTRF cAMP的检测分析<ol><li>带来HTRF cAMP测定试剂盒RT下在使用前30分钟。 </li><li>准备每个HTRF试剂（穴和D2）分别在裂解液1:20稀释（专利配方，通过cAMP的检测化验的制造商提供）。 注意:注意:裂解液中含有氟化钾（KF），这是有毒的致畸胎13。有关处理，含有KF检测板应密封并焚烧，任何液体废物处置，必须顺着水槽之前，稀释&lt;1 mmol / L的水。 </li><li>使用批量试剂分配器5微升穴试剂加入到测定板的所有孔中。 </li><li>使用批量试剂分配器5微升D2试剂加入到柱纳秒测定板1-22。 </li><li>立即手动移液5微升裂解缓冲液进入测定板的孔A23-D24，得到非特异性结合（NSB）对照孔，和5μld2的试剂的成孔E23-P24。 </li><li>离心机检测板1分钟，在室温200 XG混合井。 </li><li>覆盖试验板，以减少蒸发和光致漂白，并在室温孵育1小时。 </li><li>用在320nm处激发和发射在620nm，并使用板读数器665纳米测量荧光共振能量转移（FRET）信号。 </li></ol> 4.数据分析<ol><li>使用平均NSB从所有孔减去背景。 </li><li>从665纳米/ 620纳米比率计算Δ％˚F如下: 比率=（A 665nm / A 620nm处 ）×10 4 三角洲F =（（采样率-比NSB）/比例NSB））×100 其中，比例NSB =没有D2试剂孔。 </li><li>计算％ACTIVAT未刺激的细胞与细胞之间的离子与最大GLP-1的配体刺激的如下: ％活化=（Δ％FSAMPLE - ％DeltaFunstimulated细胞）/（％DeltaFGLP-1刺激的细胞 - ％DeltaF未刺激细胞）×100 </li><li>通过参考控制1所限定分析通过4参数后勤分析;图作为％活化浓度-响应曲线。 </li></ol>

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The authors have nothing to disclose.

这个协议描述自动声学分配的成功应用到在3×10 6个要求小于1微升样品的浓度范围连续稀释的肽样品。这种方法的主要优点是通过经由还原样品暴露于所通常所需的试剂转移和混合试验容器和塑料制品（如枪头）最小化的肽吸附到塑料制品，以提高数据质量。虽然声学分配不能完全排除使用塑料制品的，它确实显著酌减。此外，消除基于尖部-移液还消除了样品的交叉污染，和错误不会在?...

针对GLP-1R是2型糖尿病2的治疗已建立的药物靶标。对于该受体的天然肽激动剂，GLP-1，具有2-3分钟3 的体内半衰期。 GLP-1的其G蛋白偶联靶受体的结果在下游生产通过天然G蛋白偶联的第二信使cAMP对腺苷酸环化酶的活化的结合。累积的cAMP测量提供了可靠的测定法来监测受体激活和筛选活性GLP-1类似物与优选的物理化学性质。这样的测定需要测试样品的连续稀释来构建浓度 - 响应曲线，而这移交肽样品时，是特别复杂。从基于尖连续稀释准备的潜在错误先前已经1,4,5描述。肽将吸附到塑料制品，造成不可靠的效力的估计。肽损失，可以通过T为最小化他纳入缓冲器牛血清白蛋白（BSA）和使用硅化塑料制品的，但蛋白质结合的仍无法预测。特别是，在实验容器GLP-1的结合的变体已经描述6。有在实验室塑料制品中使用可从提示和微量滴定板成水测定缓冲液浸出，并与蛋白质功能7,8干扰该稳定剂进一步复杂化。因此，方法，以减少暴露于塑料制品是必要的，以增加测量的准确度。 声学液体分配器集中的高频声信号到流体样品的表面上，从而导致在精确纳升液滴喷射到相邻测定板9。利用声学弹射的是制药行业的准备和大量的合成化合物库的筛选标准，而且技术已经很好的验证了小moleculES 10。据我们所知，我们是来形容声配药重组和合成肽的制备第一组和我们先前已经报道了更高的精度比传统的基于技巧的方法1。 本文介绍了在完全自动化的处理板的机器人系统由非接触式声学传递肽序列和直接稀释配制的整合。许多围绕样本声传输方法先前已被描述11。我们利用两步法来制备中间库存浓度和以串行稀释肽类似物为全剂量反应曲线的产生。制备的肽温育的细胞表达靶小鼠GLP-1R，并且我们使用市售均相时间分辨荧光（HTRF）测定法来测量这些细胞中的cAMP积累作为肽激动剂ACTIV的读出性。该测定是健壮和适合于高通量384孔格式和常规应用到分析开发和药物筛选项目12。

<table><tbody><tr><td>Hanks&amp;rsquo; Balanced Salt solution</td><td>Sigma-Aldrich</td><td>H8264</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A9418</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I7018</td><td>Prepared as a 0.5 M stock in DMSO</td></tr><tr><td>GLP-1 (7-36) amide</td><td>Bachem</td><td>H-6795</td><td>Prepared as a 1 mg/ml stock in PBS, referred to as &#039;100x reference control&#039;</td></tr><tr><td>Test peptides</td><td>Produced in-house at MedImmune</td><td></td><td>Supplied at various concentrations in DMSO or PBS as appropriate</td></tr><tr><td>100x peptide stock</td><td>Produced in-house at MedImmune</td><td></td><td>Test peptide diluted into assay buffer to 100x final required concentration</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250-061</td><td></td></tr><tr><td>Cedex XS Cell Analyzer</td><td>Innovatis</td><td></td><td></td></tr><tr><td>Corning 384 well plates, low volume</td><td>Sigma-Aldrich</td><td>4514</td><td></td></tr><tr><td>Echo Qualified 384-Well Polypropylene Microplate</td><td>Labcyte Inc.</td><td>P-05525</td><td></td></tr><tr><td>Echo Qualified Reservoir</td><td>Labcyte Inc.</td><td>ER-0055</td><td></td></tr><tr><td>Echo 550 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 2.5 nl</td></tr><tr><td>Echo 525 Liquid Handler</td><td>Labcyte Inc.</td><td></td><td>Droplet transfer volumes in increments of 25 nl</td></tr><tr><td>ACell Benchtop Automation&amp;nbsp;</td><td>HighRes Biosolutions</td><td>MC522</td><td></td></tr><tr><td>Cellario Lab Automation Scheduling software for Life Science Robotics</td><td>HighRes Biosolutions</td><td></td><td></td></tr><tr><td>MultidropCombi Reagent Dispenser</td><td>ThermFisher Scientific</td><td>5840300</td><td>Referred to as &#039;bulk reagent dispenser&#039;</td></tr><tr><td>HTRF cAMP Dynamic 2 kit</td><td>Cisbio Bioassays</td><td>62AM4PEJ</td><td></td></tr><tr><td>EnVision Multilabel Reader</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

automated acoustic dispensing for the serial dilution of peptide agonists in potency determination assays

与小分子药物的发现，筛选肽激动剂要求的肽，以产生浓度 - 响应曲线的连续稀释。筛选的肽，得到复杂的附加层作为常规的基于针尖 - 样品处理方法暴露肽塑料制品的一个大的表面面积，提供用于通过吸附肽损失增加的机会。防止过度暴露于塑料制品通过加入塑料降低肽损失，因而在效价的预测最小误差，我们先前已经描述了非接触式的声学分配用于肽激动剂1的体外高通量筛选的好处。这里我们讨论了在利用在小鼠胰高血糖素样肽1受体（GLP-1R）筛选肽激动剂的例子中微滴定板的肽系列稀释的非接触声学制备一个完全集成的自动化解决方案。我们的方法允许高-throughput细胞为基础的分析来筛选激动剂，很容易扩展到支持增加的样品通量，或允许检测板拷贝数量增加（ 例如 ，对于更多的靶细胞系面板）。

<html>我们经常使用两步法通过声传递稀释肽。用于第一步骤中，用自动化对准的声学分配装置被用来创建四个库存肽跨两个源板中间稀释液（ 图1a，b） 中 。对于第二个步骤中，我们使用的声分配器从源板A和B，以进一步稀释库存稀释创建为每个测试肽（ 图1c）11点浓度范围。每种肽的浓度范围是在重复烧制，左跨越测定?...

Watch this Scientific Journal Video about Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays at JoVE.com

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

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. ...

automated-acoustic-dispensing-for-serial-dilution-peptide-agonists

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

7.6K 次观看.  MedImmune. 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. 

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

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 ...

科研

化学

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

生物工程

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

基于无标签阻抗的 GPCR 检测中用于提高吞吐量的逐步加量协议

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

生物化学

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

可扩展的高通量选择从噬菌体展示合成抗体库

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

免疫与感染

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

用于重组G蛋白偶联受体高通量筛选的"双加法"钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Here, we describe a proteomics workflow for characterization of the cell surface proteome of various cell types. This workflow includes cell surface protein enrichment, subsequent sample preparation, analysis using an LC-MS/MS platform, and data processing with specialized software.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

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

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 ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

数字核酸定量实验分析的简单堆积读数

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...