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>&amp;nbsp;Ammonium hydroxide trace metal basis&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>338818</td><td></td></tr><tr><td>&amp;nbsp;Ammonium nitrate&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>256064</td><td>Make fresh 200mM solution on day of experiment</td></tr><tr><td>&amp;nbsp;Antinomy&amp;nbsp;</td><td>Choice analytical&amp;nbsp;</td><td>10002-3&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;Ceruloplasmin&amp;nbsp;</td><td>Sigma</td><td></td><td></td></tr><tr><td>&amp;nbsp;Cesium&amp;nbsp;</td><td>Choice analytical&amp;nbsp;</td><td>100011-1&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;Complete, EDTA free protease inhibitors&amp;nbsp;&amp;nbsp;</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>&amp;nbsp;Conalbumin&amp;nbsp;</td><td>Sigma</td><td>C7786</td><td></td></tr><tr><td>&amp;nbsp;Concanavalin A from Canavalia ensiformis (Jack bean)&amp;nbsp;</td><td>Sigma</td><td>L7647</td><td></td></tr><tr><td>&amp;nbsp;Cu, Zn Superoxide dismutase&amp;nbsp;</td><td>Sigma</td><td>S9697</td><td></td></tr><tr><td>&amp;nbsp;Ferritin&amp;nbsp;</td><td>Sigma</td><td>F4503</td><td></td></tr><tr><td>&amp;nbsp;ICP-MS multielemental calibration standards&amp;nbsp;</td><td>AccuStandard</td><td></td><td>Made up to required concentrations in 1% nitric acid</td></tr><tr><td>&amp;nbsp;Microvolume UV spectrophotometer&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>&amp;nbsp;65% Nitric acid&amp;nbsp;</td><td>Millipore</td><td>100441</td><td>Diluted to 1% for use</td></tr><tr><td>&amp;nbsp;Peek tubing&amp;nbsp;&amp;nbsp;</td><td>Agilent</td><td>5042-6461</td><td></td></tr><tr><td>&amp;nbsp;Size exclusion column BioSEC-3 PLC. column, 4.6 x 300 mm, 3 &amp;mu;m, 150 &amp;Aring;&amp;nbsp;</td><td>Agilent</td><td>5190-2508</td><td></td></tr><tr><td>&amp;nbsp;Sodium Chloride&amp;nbsp;</td><td>Chem Supply</td><td>SA046</td><td></td></tr><tr><td>&amp;nbsp;Tris Hydrochloride&amp;nbsp;</td><td>ICN Biomedicals inc.&amp;nbsp;</td><td>103130</td><td></td></tr><tr><td>&amp;nbsp;Thyroglobulin&amp;nbsp;</td><td>Sigma</td><td>T9145</td><td></td></tr><tr><td>&amp;nbsp;Vitamin B12&amp;nbsp;</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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15.1K 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 ...

科研

化学

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

生物工程

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

生物化学

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

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Quantification of Metal Leaching in Immobilized Metal Affinity Chromatography

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as many of the common di-and trivalent cations used in IMAC resins have an inhibitory effect on enzymes. However, the extent of metal leaching and the impact of various eluting and reducing reagents are poorly understood in large part due to the absence of simple and practical transition metal quantification protocols that use equipment typically available in biochemistry labs. To address this problem, we have developed a protocol to quickly quantify the amount of metal contamination in samples prepared using IMAC as a purification step. The method uses hydroxynaphthol blue (HNB) as a colorimetric indicator for metal cation content in a sample solution and UV-Vis spectroscopy as a means to quantify the amount of metal present, into the nanomolar range, based on the change in the HNB spectrum at 647 nm. While metal content in a solution has historically been determined using atomic absorption spectroscopy or inductively coupled plasma techniques, these methods require specialized equipment and training outside the scope of a typical biochemistry laboratory. The method proposed here provides a simple and fast way for biochemists to determine the metal content of samples using existing equipment and knowledge without sacrificing accuracy.

We present an assay for easy quantification of metals introduced to samples prepared using immobilized metal affinity chromatography. The method uses hydroxynaphthol blue as the colorimetric metal indicator and a UV-Vis spectrophotometer as the detector.

金属浸出在固定金属亲和力色谱中的定量

Contamination of enzymes with metals leached from immobilized metal affinity chromatography (IMAC) columns poses a major concern for enzymologists, as ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

纳米材料获得的蛋白质和代谢物科罗纳的毛细电电量谱方法

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

医学

增强 EV 分离以进行精确的生物流体分析

Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

作者聚焦：在疾病生物标志物发现中推进 EVtrap 用于高通量蛋白质组学 

The present protocol provides detailed descriptions for the efficient isolation of urinary extracellular vesicles utilizing functionalized magnetic beads. Moreover, it encompasses subsequent analyses, including western blotting, proteomics, and phosphoproteomics.

基于化学亲和力的生物流体细胞外囊泡分离用于蛋白质组学和磷酸化蛋白质组学分析

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of ...

用尺寸排阻ICP-MS标准定量分析Metalloproteomic

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此视频中的章节

定量蛋白质组学使用还原二甲基化的稳定同位素标记

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析

多肽和蛋白质量化使用自动免疫-MALDI (iMALDI)

离子交换色谱 (IEX) 与多角度光散射 (ALS) 相结合, 用于蛋白质分离和表征

金属浸出在固定金属亲和力色谱中的定量

纳米材料获得的蛋白质和代谢物科罗纳的毛细电电量谱方法

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

基于化学亲和力的生物流体细胞外囊泡分离用于蛋白质组学和磷酸化蛋白质组学分析

基于化学亲和力的生物流体细胞外囊泡分离用于蛋白质组学和磷酸化蛋白质组学分析