我们要感谢来自维多利亚州政府的运营基础设施的支持计划，澳大利亚研究理事会联动项目计划（通过Agilent Technologies），澳大利亚国家卫生和医学研究理事会，维多利亚脑库，合作研究中心心理健康与Neuroproteomics支持设施。

1.缓冲器和样品的制备<ol><li>制成500毫升之缓冲液中，200毫摩尔硝酸铵的pH 7.6 - 7.8（pH值用氢氧化铵（NH 4 OH）调整）与铯（Cs）和锑（Sb）的加入到10ppb的终浓度。使用铯和矛盾的内部标准，以监测在ICP-MS的响应的任何漂移。通过0.22微米的过滤器使用过滤器缓冲区前。 </li><li>液体，组织或细胞培养：取决于样品的种类准备样品。 <ol><li>对于需要均匀化（ 例如 ，组织或细胞）的样品中，确保它们不包含洗涤剂。使用Tris缓冲液的样品制备。另外，使用磷酸缓冲液，但它们可以在metalloproteome随时间或具有冻融循环12产生负面影响。 </li><li>均质化通过手动DOUNCE或超声处理的组织或细胞培养物的样品用于使用Tris缓冲盐水（50毫摩尔Tris pH值8.0，150mM氯化钠5分钟（氯化钠）+乙二胺四乙酸（EDTA）无蛋白酶抑制剂）。 </li><li> 16000 XG 5分钟澄清离心匀浆。收集得到的上清液。保持样品在4℃。 注意：所有样品必须装载到高效液相色谱（HPLC）的小瓶中，以防止颗粒堵塞的尺寸排阻色谱（SEC）柱之前被离心。 </li></ol></li><li>正常化蛋白质装载在整个样品的柱的总金额。确定用微型量紫外分光光度计13在280 nm处的蛋白浓度。 20和150微克蛋白质之间的负载。使用典型注射体积从2 - 80微升取决于样品浓度。 <ol><li>清洁微卷紫外分光光度计用蒸馏水上样前的样品臂。样品臂是其中蛋白质样品加载。 </li><li>对空2微升日的仪器Ë样品缓冲液。然后，将各样品到所述臂的负载2微升并测量在280nm处的吸光度。 </li><li>确定的浓度。粗蛋白的样品，用1 ABS = 1毫克/毫升的消光系数。为了确定蛋白质的样品中的浓度，使用比尔 - 朗伯定律A =εxlxc，其中A是吸光度，ε是消光系数，l是在厘米路径长度和c是浓度。 </li></ol></li></ol>质谱 - 使用电感耦合等离子体金属蛋白标准的2散装分析<ol><li>预热和调使用制造商的协议的仪器。 </li><li>一旦仪器预热和调整，执行批量ICP-MS。 注意：纯化的金属蛋白的储备溶液通常需要测量之前进行稀释。金属stochiometries和估计的蛋白质concentra：标准的金属浓度可以从公知的蛋白质来估计化。这个信息可以被用来确定稀释，这将是适当的下降所产生的标准曲线的范围内。 <ol><li>稀金属蛋白标准，甲状腺球蛋白，铁蛋白，血浆铜蓝蛋白，铜/锌超氧化物歧化酶和维生素B 12，以确保它们落入0的范围内- 500微克/升，用1％的硝酸。到该范围内实现的标准，不使用稀释超过1 20.在水中进行连续稀释液，如果需要的股票稀释此之前。 </li><li>设置注射的顺序。首先，分析的七个校正水平，浓度范围从0 - 500微克/升，接着金属蛋白标准。七个校准级别是0，1，5，10，50，100和500微克/升。 </li><li>选择感兴趣的内容。这里，可以使用铁，铜，锌，钴和我，因为它们是该蛋白质的标准被绑定到的元素。通过打开元素选择器选项卡，并添加eleme选择收购法的要素<html>是NTS和它们各自的同位素质量进行分析。 </li><li>用仪器进行的样品分析和仪器软件自动创建被分析为每个元素的校正曲线。校准曲线由软件通过绘制针对该标准的目的是包含在微克/升的金属量检测到的金属的计数/秒创建。使用校准曲线来确定未知样品中的金属的浓度。 注意：使用校正曲线，该软件决定了在每个金属蛋白标准金属的浓度。 </li><li>从散装分析结果，产生金属蛋白标准，其中包括在哺乳动物组织使用Cu的混合物的三个最丰富的微量元素，Zn超氧化物歧化酶（SOD）在最终稀释至200微克/升铜和锌，铁蛋白（FTN）的2,000微克/升的铁浓度。 </li></ol></li></ol>jove_title“&gt; 3。高效液相色谱系统设置和尺寸排阻色谱柱平衡注意：这部分应在平行于该前一执行，因为它们都需要约1 - 1.5小时内完成。 <ol><li>吹扫的HPLC泵与在步骤1.1制成缓冲液和以5ml /分钟的流速吹扫5分钟的泵。 </li><li>一旦系统吹扫，以0.1毫升/分钟设定的流速和连接大小排阻柱（4.6×300毫米，3微米，150埃）。 注意：管与塔之间的湿式连接避免了柱的连接期间被截留的任何空气气泡。 </li><li>连接列设置以测量使用PEEK管280的吸光度的UV检测器的相对端。 </li><li>逐渐增加列的在0.05的增量的流量 - 0.1毫升/分钟直到0.4 ml的最终流速/到达分钟。虽然这是发生，检查油管接头为任何泄漏的列。监控压力系统，以确保它不通过观察在软件色谱图中的压力轨迹超过列要求。 </li><li>离开柱处的所需的流速经5平衡 - 10个柱体积。它是平衡后，连接仪器允许进行样品分析。 </li><li>设置HPLC方法最多以0.4毫升/分钟通过柱泵缓冲液A为15分钟。 </li></ol> 4.设置和运行体积排阻 - 电感耦合等离子体 - 质谱法注：操作步骤可能仪器和型号而有所不同。联系仪器技术专家，以了解更多有关如何配置使用的ICP-MS。 <ol><li>放置ICP - MS进入待机模式改变硬件设置之前。 <ol><li>一旦进入待机模式，关闭了自动进样器通信，改变样品引入到“其他”。 注意：根据软件和硬洁具配置选择“HPLC”为试样导入源都可以使用。联系仪器技术专家学习，如果此选项可用。 </li></ol></li><li>使用PEEK管（ID0.13毫米），从UV检测器的出流直接连接到对ICP-MS的喷雾器。 </li><li>电源备份ICP - MS，让仪器预热10 - 20分钟。 </li><li> MS的HPLC自动进样器的背面 - 等离子体点燃之后，从利用ICP连接远程电缆。做到这一点，一旦等离子闹得沸沸扬扬;否则HPLC系统将自动关闭，因为ICP - MS不上。 </li><li>更改ICP-MS方法来收集LC-ICP-MS数据。 <ol><li>改变来自光谱时间分辨采集（TRA）的采集方法。 </li><li>待分析选择的元素，铁，铜，锌，钴，我以及感兴趣的任何其它元件，并设置积分时间（通常介于0.05 - 秒0.3）。感兴趣的元素之后重新选择，调整采集时间以匹配的运行时间的色谱法（ 例如，900秒，用15分钟色谱运行）。 </li><li>手动调整ICP - MS灵敏度和碰撞单元氦（He）的气体流速，使用Cs和锑包含在缓冲器中流动的色谱缓冲液中。 He流率通常比那些用于散装分析更少〜1毫升/分钟。一旦金属离子计数已经稳定，相对标准偏差（RSD）的值是在5％以下，该系统就可以使用。 </li></ol></li><li>使在HPLC和ICP-MS软件程序都运行样本名单。样品运行列表包含在其中各样品是待注射以及通过该数据将被保存的名称的顺序。检查，在两个程序之间的列表匹配的样本总数。 </li><li>开始为HPLC之前ICP-MS的样品批次，如通过HPLC样品的注入将触发ICP - MS开始收集数据。如果这是不正确的顺序进行它会导致丢失的数据。 </li><li>通过注入从200微克/升的SOD和FTN混合标准的变化量到6000微克/升生成的校准曲线的点上柱注入对Cu及Zn和2000微克/升高达60000微克/升的Fe。注射体积范围为1到30微升。 注意：这些浓度范围包括通常在复杂的匀浆观察铁，铜和锌的浓度。用于仅包含纯化的蛋白质样品的曲线的​​最大范围应作相应的调整。作为与蛋白质相关的，可能需要更小或更大的范围，因为有来自其他因素在样品中污染少的金属量。 </li><li>分析每个未知样品的组织，血浆或细胞培养。 </li></ol> 5.数据分析，处理和可视化<ol><li>存储在逗号分隔值（CSV）文件格式的数据，并加载我按要求n要处理的程序。 </li><li>为了控制仪器漂移，除以每秒的计数每秒的计数或铯或Sb的每个元素。 </li><li>产生用于每个元件的校正曲线。 <ol><li>确定对应于它是由在优选的数据分析软件进行峰积分绑定到每个注射的金属蛋白酶的金属峰下的面积。 </li><li>画出针对于每个运行注入柱的金属的总量，200的金属峰下的面积 - 6000微克/升的铜，锌和2000 - 60,000微克/升的Fe。根据软件协议来执行线性回归分析。 </li><li>使用来自线性回归分析的斜率的结果的一个因素，以每秒计数转换为皮克/秒。由线值的梯度将每个整个色谱每秒的数据点计数。 </li></ol></li><li>图表中的数据对色谱时间皮克/秒。确定感兴趣的峰下的面积。峰下的面积表示该蛋白被绑定到，以pg金属的总量。 </li><li>生成基于已知金属蛋白，并在其洗脱时间的分子量校准。使用这个估算复杂样品中的蛋白质峰的大小。 </li></ol>

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The authors have nothing to disclose

确保该蛋白质的天然状态装置特别注意使用，并且所需要的样品的存储缓冲器。不是所有的色谱技术或样品制备技术可以采用。重要的是，在整个样品制备和色谱所用的缓冲器是缺乏的金属螯合剂和模仿生理pH和盐浓度使用缓冲剂。其他条件，以避免包括取暖样品或添加蛋白变性（ 如尿素）的。关键是要尽量减少冻融循环的数目。所选择的缓冲器的结合二价金属的能力也很重要，并且是的Tris或硝酸铵缓冲器被选择通过基于磷酸盐缓冲剂的一个原因。 大小排阻相对于其它形式的色谱法的色谱低分辨率和峰容量是这种技术的一个主要限制。然而，尺寸排阻chromatogra的性质温和PHY重要的是要维持该蛋白质的天然状态，因而保留了相对较弱的金属 - 蛋白质结合。以保持蛋白质的天然状态的要求，需要特别注意样品包括限制冻融循环的数目的处理，避免了金属螯合剂（ 例如 ，EDTA）或其离液盐和洗涤剂。 这种低的这种技术影响分辨率量化与特定蛋白相关金属的量，如果它是一个复杂的样品所见将包含一个以上的蛋白质的峰的能力。因此，金属的使用峰积分确定的量将是金属的所有的蛋白质洗脱在该时间点，而不是只是一个特定的蛋白质相关联的总数量的指示。为了克服这一限制的目的蛋白质将必须在天然条件下进一步纯化。这将允许的量化与该蛋白质相关的金属具有更高程度certainity的报告。该技术的另一种潜在的局限性是蛋白质的损失，由于非可逆结合到柱上。为了确定这是否发生一个恢复实验由此分析蛋白质脱柱洗脱的量，以确定是否该匹配喷射量应进行。同样可以通过测量洗脱材料中的金属含量并通过本体的ICP-MS起始材料来完成。列恢复可以根据所使用的条件不同，但它已经表明，从一个尺寸排阻柱蛋白质的完全恢复是可能9。因此，检查是否有正在使用的操作条件下的损失是很重要的。 修改的协议可以与所使用以及分析元件的金属蛋白标准。该型金属蛋白标准我们海关将根据自己感兴趣的内容有所不同。对于元素如铜，铁，锌的蛋白质，SOD和铁蛋白的使用。具有已知stoichometries任何其他金属蛋白也可以使用和几个例子已经在这里显示。 可以从使用​​这种技术产生的一个主要并发症是盐晶体中的ICP-MS的火炬积聚。千毫升缓冲液已经通过系统或当它通过肉眼检查确定该火炬应清洗 - 防止盐晶体的积聚，火炬每500后用蒸馏水洗涤。可能出现的另一个问题是在样品和提取锥的洁净度的快速下降。这些都需要定期清洗以下制造商的协议。 初始样品的准备是在协议中最关键的步骤。如果有任何改变蛋白 - 金属复合物的INFormation产生将是无效的。这是该技术的主要限制之一;除了使用低分辨率的，尺寸排阻柱产生在生物学金属蛋白的真正复杂的有限详细视图。 此处所描述的技术允许生物体的metalloproteome的知识的扩展。散装分析只给出变化的一个样品内的粗指示金属的量。除了需要加以考虑的一般因素，这种技术提供了可用于定量与蛋白质以及确定金属蛋白缔合金属的通过比较得到的迹线的不同的量的工具。利用这种技术可用于鉴定疾病状态之间的差异。在确定金属蛋白就可以进行进一步的调查，以帮助确定它们在疾病过程中发挥的作用。联用ICP-MS的应用程序都有一个成长将来，以确定可用于识别该药物结合的蛋白质具有一个杂原子的药物如铂，碘或铜作为ICP-MS中的作用。

基本金属在发挥正常的生物学功能，包括第二信使途径，代谢途径和细胞器的功能至关重要的作用。所有蛋白质的30％被认为是金属蛋白1和所有酶2的50％。金属酶利用这些微量金属元素作为辅助因子催化化学反应，稳定蛋白质结构和监管的角色，如第二信使。一些在问候神经变性研究最多的微量元素是铜，铁和锌3。他们被认为是参与了许多疾病的途径由dyshomeostasis在那里可以有负面影响。例如超氧化物歧化酶（SOD）直接影响寿命和家族型肌萎缩性侧索硬化症（ALS）4的转基因小鼠模型的表型的金属状态。在阿尔茨海默氏病，metalloproteomics技术已被用来发现在转的p中金属状态下降lasma 5。这些研究突出的重要作用金属蛋白可以在疾病玩。 金属蛋白直接从生物组织的研究是一个发展的领域。尽管一些金属酶已被鉴定，大部分仍然未鉴定的或未知的6。一的测量金属蛋白的主要挑战是维持蛋白质7的天然状态的要求。经典自下而上蛋白质组学技术依赖于蛋白质的消化成肽。这个过程扰乱金属及其蛋白质的非共价相互作用。因此，没有关于蛋白质的金属状态信息获得的。 克服这个问题的一种方式是通过使用尺寸排阻色谱，用感应耦合等离子体配对-质谱8,9-（SEC-ICP-MS）。这将生成有关的蛋白质的近似大小，以及信息的任何ASSOC金属iated与它10。此外，大小排阻是一个温和的色谱技术，可以保存的酶或蛋白 - 蛋白复合物的天然状态。使用感应耦合等离子体的一个优点 - 质谱（ICP-MS）是该技术的定量性质。使用一组的金属蛋白标准能够提供金属蛋白的绝对定量从生物样品9,11。这是通过在一系列金属浓度注入已知金属蛋白产生的标准曲线来实现。 这个协议表示如何可以用于各种金属蛋白标准来实现的例子。在本文中，我们的目标是为那些在生物领域主要调查金属，包括铁（Fe），铜（Cu），锌（Zn），碘（I），和钴（Co）创建标准曲线。

<table>
	<tbody>
		<tr>
			<td>Agilent 1290 Infinity Binary Pump</td>
			<td>Agilent</td>
			<td>G4220A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1290 Infinity Autosampler</td>
			<td>Agilent</td>
			<td>G4226A</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1200 Series Autosampler Thermostat</td>
			<td>Agilent</td>
			<td>G1330B</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1290 Infinity Thermostatted Column Compartment</td>
			<td>Agilent</td>
			<td>G1316C</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 1290 Infinity Variable Wavelength Detector</td>
			<td>Agilent</td>
			<td>G1314E</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 7700 ICP-MS</td>
			<td>Agilent</td>
			<td>G3282A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ammonium hydroxide trace metal basis&#160;&#160;&#160;</td>
			<td>Sigma</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ammonium nitrate&#160;</td>
			<td>Sigma&#160;</td>
			<td>256064</td>
			<td>Make fresh 200mM solution on day of experiment</td>
		</tr>
		<tr>
			<td>&#160;Antinomy&#160;</td>
			<td>Choice analytical&#160;</td>
			<td>10002-3&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ceruloplasmin&#160;</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Cesium&#160;</td>
			<td>Choice analytical&#160;</td>
			<td>100011-1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Complete, EDTA free protease inhibitors&#160;&#160;</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Conalbumin&#160;</td>
			<td>Sigma</td>
			<td>C7786</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Concanavalin A from Canavalia ensiformis (Jack bean)&#160;</td>
			<td>Sigma</td>
			<td>L7647</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Cu, Zn Superoxide dismutase&#160;</td>
			<td>Sigma</td>
			<td>S9697</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Ferritin&#160;</td>
			<td>Sigma</td>
			<td>F4503</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;ICP-MS multielemental calibration standards&#160;</td>
			<td>AccuStandard</td>
			<td></td>
			<td>Made up to required concentrations in 1% nitric acid</td>
		</tr>
		<tr>
			<td>&#160;Microvolume UV spectrophotometer&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;65% Nitric acid&#160;</td>
			<td>Millipore</td>
			<td>100441</td>
			<td>Diluted to 1% for use</td>
		</tr>
		<tr>
			<td>&#160;Peek tubing&#160;&#160;</td>
			<td>Agilent</td>
			<td>5042-6461</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Size exclusion column BioSEC-3 PLC. column, 4.6x300mm, 3&#956;m, 150&#197;&#160;</td>
			<td>Agilent</td>
			<td>5190-2508</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Sodium Chloride&#160;</td>
			<td>Chem Supply</td>
			<td>SA046</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Tris Hydrochloride&#160;</td>
			<td>ICN Biomedicals inc.&#160;</td>
			<td>103130</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Thyroglobulin&#160;</td>
			<td>Sigma</td>
			<td>T9145</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Vitamin B12&#160;</td>
			<td>Sigma</td>
			<td>V2876</td>
			<td></td>
		</tr>
	</tbody>
</table>

standards for quantitative metalloproteomic analysis using size exclusion icp-ms

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

使用金属蛋白标准允许尺寸排阻柱进行校准。 图1A显示了洗脱曲线的标准甲状腺球蛋白铁蛋白，血浆铜蓝蛋白，铜/锌超氧化物歧化酶和基于它们结合到金属维生素B 12（铁，钴，铜，锌和I）。 图1B示出了通过的空隙体积除以基于蛋白质标准的分子量和它们的洗脱时间的大小排阻柱校准曲线，在洗脱体积的格式（Ve的提交）列（VO）。用于生成标准曲线的蛋白刀豆，伴白蛋白，血浆铜蓝蛋白，铁蛋白，超氧化物歧化酶和甲状腺球蛋白。 图2A示出铁蛋白的洗脱在一定范围的2,000 -铁60000皮克注射上柱和图2D是使 ​​用对所述回归分析进行EAK区域; 图2B和2C是用于铜/锌超氧化物歧化酶为Cu和Zn和2E和2F的洗脱曲线使用峰面积分析产生的回归。回归分析的结果被用来在计数/秒的原始数据转换为皮克/秒这样的金属与蛋白质相关联的量可以定量。该转换是通过将线性回归（铜例如 ，334.6（计数/秒）×（秒/ PG））的斜率除以计数/秒进行。 如前所述，该技术可用于识别复杂生物样品中的金属蛋白。人脑和血浆已经经受这种技术和图3和4分别显示获得的结果。通过SEC-ICP-MS分离人脑示于图3A-3C，其每一个代表不同的金属<html>息（铜，锌或铁）。 图4A-4C示出得到的迹线时的人血浆进行这种技术。的样品的复杂性和数量将影响那些看出峰的数目。如所预期的等离子体是通过几个金属蛋白，包括铜蓝蛋白和转铁为主。 <img alt="图1" src="/files/ftp_upload/53737/53737fig1.jpg" /> 图1. 大小排阻色谱的校准-电感耦合等离子体-质谱法使用已知金属蛋白 （A）的洗脱曲线用于基于它们各自的金属的金属蛋白标准。 （B）中对蛋白质标准甲状腺球蛋白（i）中，铁蛋白分子量校正曲线（ii）中，铜蓝蛋白（iii）中，伴清蛋白（ⅳ），铜/锌超氧化物歧化酶（v）和伴刀豆球蛋白A（ⅵ）。“目标=”_空白“&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53737/53737fig2.jpg" /> 60000微克/升- 图2.铁和铜/锌超氧化物歧化酶金属蛋白作为标准使用过注射范围为2000 确定在复杂生物样品中的铜，铁或锌伴有金属蛋白量 （A）洗脱天寒铁蛋白。铁。 （B）和对铜/锌超氧化物歧化酶在200喷射范围（℃）洗脱资料- 6000微克/升铜和锌，分别的。 （D），（E）和（F）显示了金属铁，铜和锌的回归分析结果，分别为。 <a href="https://www.jove.com/files/ftp_upload/53737/53737fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.铜，铁，人脑。（A）铜迹（B）铁痕迹锌Metalloproteome（C）锌痕迹。蛋白质标准，为每个金属洗脱被黑迹用图形表示下其分子量图所示。 <a href="https://www.jove.com/files/ftp_upload/53737/53737fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53737/53737fig4.jpg" /> 图4.铜，铁，锌Metalloproteome人血浆。（A）铜迹（B）微量铁（C）锌痕迹。的蛋白质标准，为每个金属洗脱通过在黑色的跟踪其分子量示图形下方示。 <a href="https://www.jove.com/files/ftp_upload/53737/53737fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS at JoVE.com

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

用尺寸排阻ICP-MS标准定量分析Metalloproteomic

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

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15.0K 次观看.  The Florey Institute of Neuroscience and Mental Health. Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins. 

视频: Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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化学

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

SLP1聚合物和单层从金交互<em&gt; Lysinibacillus球形</em&gt; JG-B53 - QCM-D，ICP-MS和原子力显微镜的工具生物分子金属研究

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

利用毛细管电泳从植物组织量化有机酸：一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

通过动态光散射显微镜混浊解决方案粒度分布的测定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

光子温度计的制作与测试

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Nanosponge Tunability in Size and Crosslinking Density

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

Nanosponge 可调大小和交联密度

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

电压在动态光散射粒子尺寸分析中的应用

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry (CE-ICP-MS) for Quantification of Iron Redox Species (Fe(II), Fe(III))

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative conditions. Excessive iron results in free redox-active Fe(II) and can cause devastating effects within the cell like oxidative stress (OS) and death by lipid peroxidation known as ferroptosis (FPT). Therefore, quantitative measurements of ferrous (Fe(II)) and ferric (Fe(III)) iron rather than total Fe-determination is the key for closer insight into these detrimental processes. Since Fe(II)/(III) determinations can be hampered by fast redox-state shifts and low concentrations in relevant samples, like cerebrospinal fluid (CSF), methods should be available that analyze quickly and provide low limits of quantification (LOQ). Capillary electrophoresis (CE) offers the advantage of fast Fe(II)/Fe(III) separation and works without a stationary phase, which could interfere with the redox balance or cause analyte sticking. CE combined with inductively coupled plasma mass spectrometry (ICP-MS) as a detector offers further improvement of detection sensitivity and selectivity. The presented method uses 20 mM HCl as a background electrolyte and a voltage of +25 kV. Peak shapes and concentration detection limits are improved by conductivity-pH-stacking. For reduction of 56[ArO]+, ICP-MS was operated in the dynamic reaction cell (DRC) mode with NH3 as a reaction gas. The method achieves a limit of detection (LOD) of 3 &#181;g/L. Due to stacking, higher injection volumes were possible without hampering separation but improving LOD. Calibrations related to peak area were linear up to 150 &#181;g/L. Measurement precision was 2.2% (Fe(III)) to 3.5% (Fe(II)). Migration time precision was &#60;3% for both species, determined in 1:2 diluted lysates of human neuroblastoma (SH-SY5Y) cells. Recovery experiments with standard addition revealed accuracy of 97% Fe(III) and 105 % Fe(II). In real-life bio-samples like CSF, migration time can vary according to varying conductivity (i.e., salinity). Thus, peak identification is confirmed by standard addition.

Setup of Capillary Electrophoresis-Inductively Coupled Plasma Mass Spectrometry CE-ICP-MS for Quantification of Iron Redox Species FeII, FeIII

This iron redox speciation method is based on capillary electrophoresis-inductively coupled plasma mass spectrometry with sample stacking combined with short analysis in one run. The method quickly analyzes and provides low limits of quantification for iron redox species across a diverse range of tissues and biofluid samples.

为铁氧化还原物种（Fe（II）、Fe（III）定量设置毛细管电泳-电感耦合等离子体质谱仪（CE-ICP-MS）

Dyshomeostasis of iron metabolism is accounted in the pathophysiological framework of numerous diseases, including cancer and several neurodegenerative ...