我们感谢金融支持从加拿大基因和基因组不列颠哥伦比亚省 (204PRO) 的操作和技术发展 (214PRO) 通过基因组创新网络 (GIN)。我们感谢研究所的麦吉尔大学健康中心拍摄 MALDI TOF 仪用于药物发现平台。H.L.是感谢支持从国家科学和工程研究理事会的加拿大 (NSERC) 博士后。C.H.B 非常感谢支持从前缘养老基金 （叶）。C.H.B.是感谢支持从在麦吉尔大学 （加拿大，魁北克，蒙特利尔） 分子肿瘤学的西格尔麦吉尔椅子。M.X.C.和 C.H.B.都很感激支持沃伦 Y.索珀慈善信托基金和阿尔文 · 西格尔家族基金会往犹太医院 （蒙特利尔，魁北克，加拿大）。

试剂如下所述的数额基于 20 病人血浆样品的测量。议定书 》 提出了以下如下准则的维多利亚大学 &#39; s 人类研究伦理委员会。
 1.昂代我人血浆中 <ol><li>解冻血浆样品 (&#8805; 200 微升) 在一个房间里温度水浴 5 分钟，然后把样品直到完全融化的冰上。</li>
	<li>每个样品的浓度手动来分离 1.1 毫升深孔板 （样板） 井和离心 10 分钟在 2 ° C 和 1278 x g.离心机板转移 200 微升</li>
	<li>使用自动化的液体处理系统连续稀释 500 fmol/微升 Ang I NAT 标准溶液准备与鸡卵清白蛋白的六个校准器解决方案 (0.1、 0.2、 0.6、 1.9、 5.7，17.2 fmol/微升)。</li>
	<li>的每个校验仪对这一代人很好，和 125 µ L 移液管 200 微升缓冲区来样来板。 
	注意: 在磷酸盐缓冲的液 (PBS) 缓冲区需要新鲜准备实验日 CEWA.</li>
	<li>使用自动化的液体处理系统，在一个新的盘拌 125 µ L 的等离子体上清液或 125 µ L 的 PBS CEWA 25 微升的昂我代缓冲区，包含 1 M 三、 0.2 毫米乙二胺四乙酸 (EDTA) 和 1 毫米苯甲基磺酰氟 (PMSF)。 
	注: 准备 Ang 我代缓冲区由搅拌 1 M 三/0.2 毫米 EDTA 缓冲溶液 (适应 pH 5.5 使用醋酸) 和 PMSF 解决方案 （在甲醇中的 100 毫米）。两种解决方案都可以被存储到 1 个月在 4 ° C.混合两个解决方案日实验。</li>
	<li>自动转移到 96 孔板，3 复制每个解决方案，每井 34 微升的解决方案。孵化在 37 ° C 为 3 h.板</li>
</ol> 2。免疫富集的昂我使用抗体包裹小珠 <ol><li>上磁珠 G 蛋白抗体 注: 抗体与珠的共轭手动术 3 h 昂我代期一天的实验。其他分析物，共轭的珠子可能能够存储在 PBS 缓冲含有 0.015%章 (PBSC) 在 4 ° C 为三个月或更长时间，取决于性能和稳定性的抗体。
	<ol><li>珠浆 （足够测量 20 样品和制作 6 点标准曲线） 的转让 110 微升到 1.5 毫升管。洗珠七次 1 毫升的 25%乙腈/外周血干细胞，与三次 1 毫升的外周血干细胞。使用磁性表座颗粒之间每清洗一步珠。在去年华盛顿后移除洗涤缓冲液 
		注: 洗 7 倍 25%乙腈/外周血干细胞是删除不兼容的 MS 添加剂中珠浆，如吐温-20 的关键。如果所选的珠子有没有这种添加剂，可能不需要此广泛洗涤步骤。</li>
		<li>重珠悬在 110 µ L 的外周血干细胞，并添加的抗 Ang 110 µ L 我抗体 (最后抗体浓度: 100 微克/毫升)。吹打，混合的珠子和解决方案，然后孵化它们在室温下 1 h，旋转在 8 rpm。</li>
		<li>洗珠 3 次 1 毫升的外周血干细胞，并在中央政治局常委会的 1100年微升。 
		注: 如果任何珠解决方案孵化期间流入的管帽，降速使用台式离心机在 2680 x g.解决方案</li>
		<li>手动将珠解决方案转移到 96 孔板 （珠标准板）。使用自动化的液体处理系统对分装至同一 96 孔板，每井 120 微升珠</li></ol></li> <li>免疫富集在珠子上 <ol><li>3 h 昂我代期间，放在孵化盘后10 分钟终止代昂 I.冰</li>
		<li>自动稀释 100 倍 SIS 肽原液 (10 皮摩尔/μ L) 用 PBS 缓冲，并进一步自动分装到 96 孔板 PCR 板稳定 isoptope 标准肽稀释。将 1.5 微升的 （含 100 fmol） 的稳定同位素标准 (SIS) 肽溶液转移到孵化板每个井和混合与血浆样品或 PBS 缓冲区中的 CEWA.</li>
		<li>自动将孵化盘的内容转移到珠解决方案，每井 10 毫升，混合。</li>
		<li>以 8 rpm 旋转，同时孵化在 4 ° C 1 h 板。</li>
		<li>珠子三次自动用 5 毫米碳酸氢铵 (AmBic) 溶液冲洗，每次洗涤井每 100 μ L。在最后一次冲洗后, 重 7 微升 5 毫米 AmBic 解决方案在每口井的悬珠。使用一块磁铁把珠拉到底部后最后华盛顿州</li>
	</ol></li></ol> 3。转移到 MALDI 靶板和添加矩阵解法珠 <ol><li>转让 7 微升的珠浆自动到 MALDI 靶板上配有现货 2 600 微米。让珠子干涸。 
	注意: 一个小的 USB 供电风扇可以用来加速干燥过程珠。</li>
	<li>自动添加 2 µ L 的 α-氰基-4-羟基肉桂酸 （本病）-从好到现场对靶板每个样本矩阵的矩阵解法 （包含 3 毫克/毫升本病、 1.8 毫克/毫升柠檬酸铵、 70%乙腈和 0.1%三氟乙酸）.</li>
</ol> 4。信号采集的 MALDI TOF MS 和数据分析 <ol><li>分析样品点与 MALDI TOF 仪器使用积极的反射模式。执行内部校准、 数据平滑和特定供应商的软件自动基线减法。 
	注意: 选择的模式 （正/负线性/反射） MALDI TOF 测量取决于目标多肽或蛋白质。</li>
	<li>计算相对响应比 （Nat/SIS 强度比），并进行比较的标准曲线确定的李安我每个样品中的浓度。计算方程 (1)，在那里的 PRA δ t Ang 我代 表示的时间用于生成 Ang I. 
	PRA = [昂我] / δ t Ang 我代 （1)</li></ol>

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C.H.B 拥有专利的 iMALDI 技术。

相比传统的基于 MS 蛋白质定量，iMALDI 使用抗体来丰富分析物和净化他们从复杂的样品，因此使它能够量化的蛋白质或多肽在低浓度时。IMALDI 协议的关键一步是目标肽免疫富集。为此目的，应选择具有高特异性和亲和力抗体。在 SISCAPA，报道在 10-9米或更好的抗体亲和力将需要实现高灵敏度低吴/毫升21。此外，与高的抗体结合能力的珠子是首选最优浓缩。
<p class="jove_co...

质谱技术已经成为不可或缺的工具，在定量蛋白质组学。质谱分析可以确定目标蛋白质或多肽的群众，因此所得的分析物信号可以高度特异性相比于免疫分析。两种电离方法，电喷雾串联质谱和 MALDI，常被用于检测蛋白质和多肽1，，23，4。在基于 MS 蛋白质定量的主要挑战在于复杂样品在吴/毫升或 pg/mL 的浓度在高丰度蛋白低丰度蛋白的检测及血浆中发现很多候选人蛋白质生物标志物在这范围之内5。这个问题很大程度上造成的人类蛋白质组6复杂性与本质上宽动态范围。
为了克服这些检测，免疫色谱-质谱方法已充实目标蛋白质或多肽从样品溶液在固体的表面，紧接着洗脱物和 MS 测量7，8,9,10.通过免疫浓缩物都从复杂样品纯化，因此从其他分子离子抑制影响降至最低。各种固体支架间磁珠是目前使用最广泛那样高的抗体结合能力的优势和易用性的处理。磁珠与不同的 functionalizations 和大小有被开发和商业化免疫沉淀实验。到目前为止，免疫浓缩制成的小珠已经配套与电喷雾电离 (ESI) 和 MALDI 质谱蛋白质和多肽的测量。稳定同位素标准和捕获由抗多肽抗体 (SISCAPA) 技术，在样品中的蛋白质消化，紧接着孵化与抗体包裹小珠免疫富集。在"古典"SISCAPA，捕获的 proteotypic 多肽是从珠、 洗脱，由液相色谱-质谱 (LC-MS)，或直接输注 ESI 多反应监测-质谱 (ESI-MRM-MS)11，12来衡量。免疫富集 MRM 检测灵敏度提高 3-4 个数量级，达到低吴/mL 范围13。
与电喷雾质谱相比，质谱速度更快，并且不涉及清洗和重新平衡的 LC 列所以没有携带量和污染的问题，使它更加适合高通量研究14。免疫 MALDI 技术得到了我们的实验室要结合质谱免疫富集的敏感和具体量化的多肽和蛋白质 （基于 proteotypic 多肽定量）15，16 ，17。免疫富集，珠 MALDI 靶板上沉积、 矩阵解的方法添加到珠子，和干燥后板是准备 TOF 质谱分析。从珠肽洗脱不执行作为一个单独的步骤，但亲和绑定物洗脱 MALDI 基质解决方案时将其添加到珠点，从而简化样品制备和样品损失最小化。IMALDI 技术已被应用于各种应用程序18，19，最近经过自动化和用于测量血管紧张素我 (和我) 测定血浆肾素活性 (PRA)20。本议定书将演示用于执行自动化的 iMALDI 测定的过程。以 PRA 测定为例，间天 CVs 的少于 10%已通过自动化，与每一天20744 样品的检测能力。
IMALDI PRA 测定表明这份手稿在不需要蛋白质的消化，作为靶分子 (Ang 我) 是一种肽 1295.7 大分子量。在哪里是必要的蛋白质的消化和肽用作完整的蛋白质代孕其他试剂，选定的肽对 iMALDI 应该是独特和特定于目标蛋白，与大量超过 800 的大，所以它可以很容易区分 c从 MALDI 基质溶液的化学噪声。抗多肽抗体所需的多肽免疫富集。IMALDI 试验测量 PRA 的协议包括四个步骤: 1) 昂代我在人血浆;2） 免疫富集昂我使用抗体包被珠;3） 珠转交 MALDI 靶板和添加矩阵解法;和 4） 信号采集20名 MALDI TOF MS 和数据分析。

<table width="892">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Healthy control human plasma</td>
			<td class="xl63" style="width:134pt" width="178">Bioreclamation</td>
			<td class="xl68" style="width:89pt" width="119">HMPLEDTA2</td>
			<td class="xl66" style="width:242pt" width="323"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ammonium bicarbonate</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl69">09830</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ammonium citrate dibasic</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">09833</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">CHAPS (&#62;=98%)</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">C9426</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Albumin from chicken egg white (&#62;98%)</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">A5503</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Ethylenediaminetetraacetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">EDS</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Alpha-cyano-4-hydroxycinnamic acid</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">70990</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Phosphate buffered saline</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">P4417</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Phenylmethanesulfonyl fluoride</td>
			<td class="xl63" style="width:134pt" width="178">Sigma Aldrich</td>
			<td class="xl68" style="width:89pt" width="119">78830</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Trifluoroacetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">85172</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">acetonitrile</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">34967</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">water</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">39253</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">acetic acid</td>
			<td class="xl63" style="width:134pt" width="178">Fluka</td>
			<td class="xl68" style="width:89pt" width="119">320099</td>
			<td class="xl66">LC-MS grade</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Tris(hydroxymethyl)aminomethane</td>
			<td class="xl63" style="width:134pt" width="178">Roche Diagnostics</td>
			<td class="xl71">3118169001</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Dynabeads Protein G magnetic beads</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">10003D</td>
			<td class="xl66">2.8 &#956;m, 30 mg/mL</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">anti-Ang I goat polyclonal antibody</td>
			<td class="xl63" style="width:134pt" width="178">Santa Cruz Biotechnology</td>
			<td class="xl68" style="width:89pt" width="119">sc-7419</td>
			<td class="xl66"></td>
		</tr>
		<tr height="84" style="height:63.0pt">
			<td class="xl63" height="84" style="width:204pt;height:63.0pt" width="272">Nat and SIS Ang I</td>
			<td class="xl63" style="width:134pt" width="178">synthesized at the University of Victoria-Genome BC Proteomics Centre</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Automated liquid handling system</td>
			<td class="xl63" style="width:134pt" width="178">Agilent</td>
			<td class="xl68" style="width:89pt" width="119">16050-102</td>
			<td class="xl66">Agilent Bravo robotic workstation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">12321D</td>
			<td class="xl66">Invitrogen DynaMag-2 magnet</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Tube rotator</td>
			<td class="xl63" style="width:134pt" width="178">Theromo Scientific</td>
			<td class="xl68" style="width:89pt" width="119">400110Q</td>
			<td class="xl66">Labquake Tube Rotator</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">Thermo Fisher Scientific</td>
			<td class="xl68" style="width:89pt" width="119">12027</td>
			<td class="xl66">DynaMag-96 side skirted magnet</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">Magnet</td>
			<td class="xl63" style="width:134pt" width="178">VP Scientific</td>
			<td class="xl68" style="width:89pt" width="119">771RM-1</td>
			<td class="xl66">used to pull the beads to the bottom of the well</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl63" height="21" style="width:204pt;height:15.75pt" width="272">MALDI-TOF</td>
			<td class="xl63" style="width:134pt" width="178">Bruker</td>
			<td class="xl68" style="width:89pt" width="119"></td>
			<td class="xl66">Bruker Microflex LRF instrument</td>
		</tr>
	</tbody>
</table>

peptide and protein quantification using automated immuno-maldi (imaldi)

质谱 (MS) 是量化复杂样品中的蛋白质与优秀测定特异性由于每个目标分子质量电荷比直接检测的最常用的技术之一。然而，基于 MS 的蛋白质组学，像大多数其他分析技术，有偏向于测量高丰度分析物，所以它难实现检测限制的低吴/毫升或 pg/mL 的复杂样品和这许多的浓度范围是疾病相关蛋白等人血浆的关键问题之一。为协助低丰度分析物的检测，免疫富集，只纳入测定浓缩和纯化前 MS 测量，显著提高了检测灵敏度分析物。在这项工作，免疫-Matrix-Assisted 激光解吸电离 (iMALDI) 技术是提出了一种定量的蛋白质和肽的关键问题之一，基于免疫浓缩制成的小珠，紧接着 MALDI 质谱测量不事先洗脱。抗多肽抗体是对磁珠，官能化和培养与样品。洗完后，珠子直接转嫁 MALDI 靶板和信号测定 (MALDI Tof) 仪器 MALDI 时间后矩阵解的方法已被应用于珠。样品制备程序简化相比其他免疫色谱-质谱测定和 MALDI 测量是快速。整个样品制备自动化与液体处理系统，以提高的测定的重现性和更高的吞吐量。在这篇文章，iMALDI 法用于确定肽血管紧张素我 (Ang 我) 用于临床作为读出的血浆肾素活性的筛选原发性醛固酮增多 (PA) 的血浆中的浓度。

李安的测量自动化的 iMALDI 程序我是图 1中所示。关于抗肽磁珠，丰富了目标肽 （内源性肽或从消化蛋白质肽） 和珠子然后转给 MALDI 测量的靶板。整个过程就简化相比需要更多肽洗脱步骤其他免疫色谱-质谱技术。IMALDI 检测自动化允许高通量分析的大量样品间天变异系数 (CV 10 20以下)。代表性的光谱测定人血浆样品中的我<strong class=...

Watch this Scientific Journal Video about Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI at JoVE.com

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

提出了在使用自动的免疫-MALDI (iMALDI) 技术的复杂生物体液蛋白质定量的协议。

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 ...

peptide-and-protein-quantification-using-automated-immuno-maldi-imaldi

Research

JoVE Journal

Chemistry

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

7.9K 次观看.  University of Victoria-Genome BC Proteomics Centre. 提出了在使用自动的免疫-MALDI (iMALDI) 技术的复杂生物体液蛋白质定量的协议。

视频: Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

科研

化学

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

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

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

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

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

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

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

使用氢交换质谱分析蛋白质动力学

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

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

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

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

分裂和池合成及肽叔酰胺图书馆表征

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

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

一个主干环肽库作为潜在的抗寄生虫治疗微波辐射的发展

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE  - 透明天然凝胶洗脱液体馏分偷天陷阱电泳

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

表征，气相炭用苯聚羧酸的定量和特定化合物同位素分析（BPCA）

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

毛细管电泳监视肽接枝壳聚糖膜实时

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...