This work was supported by NSF/DBI-1255991 grant to IML.

1.毛细管微反应器的研制<ol><li>切割100μm的内径（ID）×360微米的外径（OD）的毛细管到7-8厘米的长度，并且在20微米内径x 90微米的OD毛细管3-5厘米的玻璃毛细管劈刀;这两个毛细管末端有一个干净，直切，在显微镜下检查，没有任何突出的毛刺。 </li><li>插入20μm的内径x 90微米的OD毛细管导入100微米内径×360微米外径的毛细管的一端，为〜6mm的长度;在需要的情况下，在显微镜下观察此操作。 </li><li>应用胶水E6000的一小滴周围棉条的最后90微米OD / 100微米的毛细管的交界处，并让胶水固化O / N在室温。 </li><li>切插入20微米ID×90微米OD毛细管的〜10-15毫米的长度;这将是电喷射离子化的发射器。 </li><li> 1/32"OD PEEK，1/16"OD PEEK预减和1/16"OD PTFE浴缸ING在4-5厘米长片;这些将在毛细管工会用于提供无泄漏连接的套管。 </li><li>连接100微米内径×360微米外径的毛细管在PEEK联盟的另一端;使用1/32"PEEK套管用于提供无泄漏的连接。 </li><li>插入/拧紧了一块5厘米长的1/16"PTFE管的〜到工会的另一端。 </li><li>权衡〜4毫克18（5微米）的颗粒在一预清洗/干燥2毫升玻璃小瓶;加入0.5毫升异丙醇，关闭小瓶中并分散在超声发生器浴中浆料。 </li><li>用250微升注射器大约200微升浆撤出，将在PEEK工会的1/16"PTFE管注射器针头，并分配慢慢进浆100微米内径x 360微米OD毛细管;观察显微镜下毛细管颗粒直到2-3毫米填料的长度达到填充;这将是微反应器（ 图1）的20μ。米ID毛细管通过梯形效果保留在微型反应器中的颗粒。15 </li><li>冲洗〜的H 2 O / CH 3 CN 50:50体积/体积50微升溶液中的微反应器，然后用〜的H 2 O / CH 3 CN 98 50微升溶液:2体积/体积。 </li></ol> 2.样品溶液的制备注意:涉及处理的有机溶剂，酸和溶液的制备操作是在通风橱中进行。戴上护目镜，手套和防护服。 <ol><li>用CH 3 OH冲洗和干燥几个玻璃小瓶（4毫升）和聚丙烯管（15毫升）中。 </li><li>制备出10毫升水2 O / CH 3 CN中的溶液（98:2体积/体积）在15毫升聚丙烯管通过混合9.8毫升H 2ö用200μlCH 3 CN;通过超声混合。 </li><li>制备的10ml溶液的 H 2 O / CH 3 CN（50:50体积/体积）在15毫升聚丙烯管通过将5毫升水2 &lt;/分&gt; 0 5毫升CH 3 CN;通过超声混合。 </li><li>通过加入4微升TFA（10％）到4ml 的 H 2 O / CH 3 CN制备4毫升酸化水溶液（TFA 0.01％V / V）的:在一个4毫升的玻璃小瓶（98 2 V / V）;通过超声混合。 </li><li>通过加入4微升TFA（10％）到4ml 的 H 2 O / CH 3 CN（50:50体积/体积）在4毫升的玻璃小瓶中制备4毫升酸化的有机溶液（TFA 0.01％体积/体积）的;通过超声混合。 </li><li>称取16毫克NH 4 HCO 3 与在一个4毫升的玻璃小瓶中的分析微量天平;加入4毫升H型2 O / CH 3 CN（98:2体积/体积）溶液，并通过超声处理分散;这导致在含水缓冲液中的NH 4 HCO 3（pH约7.8）的50mM的溶液。 </li><li>称量5毫克标准蛋白质（ 例如 ，牛血清白蛋白，血红蛋白的α/β，细胞色素C，碳酸酐酶，α-2-HS-糖蛋白等 ），在一个4毫升的玻璃小瓶中的分析微量天平;加4毫升DI水:r和通过超声分散;这通常导致高μM级浓度的溶液。 </li><li>通过用NH 4 HCO 3（50毫摩尔）水性缓冲液的适当体积稀释高μM级浓度溶液制备1 ml蛋白质溶液（1-2微米）;通过超声混合。 </li><li>通过将20微克的测序级胰蛋白酶在170微升的NH 4 HCO 3（50毫摩尔）水性缓冲液制备的胰蛋白酶溶液（5μM）;通过超声分散。 </li></ol> 3.实验装置注:LTQ-MS系统装配有包括它能使接口质谱仪对各种样品输入方法家用内置XYZ级的改性ESI源。 <ol><li>从PEEK工会断开毛细管微反应器并连接到PEEK T恤。 </li><li>插入在将PEEK三通的侧臂1〜2厘米长的Pt丝;使用1/32"PEEK套管以提供无泄漏的连接和绝缘的铂丝。 </li><li>连接为50μm内径x 360微米外径（约0.5米长）样品转移毛细管将PEEK三通的相对端;使用1/32"PEEK套管用于提供无泄漏的连接。 </li><li>固定在XYZ阶段的PEEK T恤和ESI发射〜2毫米位置与质谱仪入口毛细管路程。 </li><li>铂丝连接到质谱仪的ESI电力供给源。 </li><li>连接样品转移毛细管到不锈钢联盟的相对端;使用1/16"PEEK套管用于提供无泄漏的连接。 </li><li>一个4-5厘米长的1/16"PTFE管连接到不锈钢联盟的另一端并插入在PTFE管的250微升注射器针头;启动泵，并设置在所需值的流速（ 例如 ， 2微升/分钟）。 </li><li>输入串联MS数据采集参数到软件包，用于控制MS仪器一ð保存为方法文件，准备安装程序执行的分析;对于LTQ-MS系统，请使用以下数据相关分析参数:180秒，排除质量宽度启用动态排斥±1.5 M / Z;一个MS扫描后跟一个变焦和MS 2次扫描的前5个最强峰，放大扫描宽度±5 M / Z;碰撞诱导解离（CID）设置在隔离宽3 M / Z，标准化碰撞能量的35％，活化Q 0.25，活化时间为30毫秒的参数。 </li></ol> 4.微流控设置<ol><li>准备由玻璃或聚合物基底的微流体装置; 16-18的设备的布局在图2A提供，由一个微反应器（1）（0.13毫米宽×2毫米长×50微米深）连接到入口（2 ），出口（3）和一个侧口（4）;为流体操作互连通道〜110微米，宽50〜深微米;多信道结构（5），由〜1.5-2微米深×10微米宽×100微米长的微通道放置25微米间隔，将作为在微型反应器保持的颗粒的过滤器。 </li><li>通过胶粘专门连接至入口端口，或通过设计容纳件用于流体输送（ 图2B）的聚合物支架使向芯片流体输送连接。16 </li><li>制备如在步骤1.8中所述的C18颗粒（5微米）的浆液;通过使用连接到芯片一侧端口1/16"PTFE管与注射器递送浆料微反应器（4）;密封用环氧树脂胶和固化1小时在90ºC烘箱颗粒负载后口16。 </li><li>在出口插入毛细管（20微米ID×90微米外径×10毫米的长度），与E6000胶水封了，让治愈O / N;这将成为ESI发射器（ <s仲&gt; 6）。 </li><li>连接为50μm内径x 360微米外径（约0.5米长）的样品传输毛细管的芯片吸入口（2）;毛细管的另一端连接到250微升注射器和注射器泵，如在3.6-3.7说明。 </li><li>放置在样品传输线刚好在入口端口一个重量轻的PEEK三通连接器（2），并插入在三通的侧臂上的铂丝，如在3.2描述这将是ESI电极。 </li><li>固定在与位于〜2毫米距离质谱仪入口毛细管与ESI发射的XYZ载物台的微流体装置;如3.8中所述进行分析准备该MS。 </li></ol> 5.上样，蛋白水解消化和洗脱的MS分析注意:所有溶液/样品转移步骤用注射器泵的帮助下进行，并且应当允许一些额外分钟完成，为了补偿死区volum与输送管线和从微反应器相关联的ES;必要的时间将取决于所涉及的流速。 <ol><li>冲洗微反应器和注入的NH 4 HCO 3（50毫摩尔）H中的水溶液2 O / CH 3 CN进行分析准备它（98:2 V / V）在2微升/分钟5分钟;用250微升注射器。 </li><li>通过注入在2微升样品溶液装载于微型反应器的蛋白质样品（1μM）/ min离心5分钟;用250微升注射器。 </li><li>注入的胰蛋白酶溶液（5微米）上吸附的蛋白质在微反应器在2微升/分钟1-3分钟。 </li><li>通过用TFA的酸化水溶液冲洗微反应淬灭水解消化处理（0.01％V / V）的H 2 O / CH 3 CN（98:2 V / V）在2微升/分钟5分钟;用250微升注射器。 </li><li>开始洗脱蛋白质从微反应器由注入的TFA酸化有机溶液消化（0.01％体积/体积）的H 2 O / CH 3 CN（50:50体积/体积），在300升/分钟。 </li><li>打开MS数据采集过程和ESI电压（〜2000Ⅴ）;获得使用步骤3.8中提供的数据采集参数MS数据;观察胰蛋白酶肽的洗脱。 </li></ol> 6.数据处理<ol><li>处理与原始MS数据格式兼容的搜索引擎的串联MS的数据。 <ol><li>过程中使用的生物体特定最小冗余蛋白质数据库中的LTQ-MS RAW文件;上传的原始数据，在搜索引擎，分别设置亲体和碎片离子质量公差为2和1大，并与最多两个遗漏的搜索分裂只使用完全的胰蛋白酶片段;不允许的翻译后修饰;设置的高和中等置信错误发现率（FDR），以1％和3％，分别和不允许的翻译后修饰。 </li></ol></li><li>筛选数据，只选择高可信度肽匹配数据库中的蛋白质类;评价蛋白和肽水平的结果。 </li></ol>

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The authors declare no competing financial interests.

在这项工作中所描述的微反应器提供了一个易于实现用于进行蛋白质的酶消化，以使在不到30分钟MS分析和鉴定的实验装置。该系统中，在比常规方法的不同的优点，包括简化，高速，低试剂消耗和低的成本。特别是，没有必要使用昂贵的固定化的胰蛋白酶珠和墨盒。毛细管微反应器的制备是简单的（ 图1A），并且可以从标准用品，在一个色谱实验室通常发现在几分钟内完成。而反相C...

纯化的蛋白质的鉴定和表征经常被使用MS技术实现的。将蛋白质消化酶和它的肽通过MS进一步分析通过使用简单的输注实验装置。蛋白水解消化是必要，用于产生落在在大多数MS分析仪的有用质量范围小的肽片段，并可以通过低能量碰撞诱导解离很容易地分片，以产生氨基酸序列的信息。对分离的蛋白质或简单的蛋白质的混合​​物，对于之前MS检测的肽的色谱分离不再需要。 25-50肽的混合物可以通过与直接注射泵在MS离子源注入样品容易地进行分析。 质谱仪可以执行分析和短的时间框架内确认的蛋白质的序列。随着现代数据采集方法，该方法可以完成瓦特ithin几分钟甚至几秒钟。在完成上一个短的时间尺度了整个过程的限制因素是水解消化步骤。通常，这是执行在几个小时（或O / N），在溶液中，在37ºC，使用基板:的（50-100）酶:1的比率。为了减少酶消化时间为几分钟或几秒钟，固定化酶微反应器，微流控反应器或市售墨盒，已经描述的形式。1-6典型地，所述酶通过共价，非共价/物理吸附，复杂的固定化形成或包封，3,6-酶促过程的增强效率被启用大的表面-体积和酶与底物的比率。固定化反应器的其它优点包括从MS分析酶减少自溶和干扰，提高了酶的稳定性和可重用性。各种方法，使用玻璃或聚合物微制造装置进行了说明，利用由抗体-抗原相互作用固定于磁珠酶，7,8-截留在金纳米颗粒的网络，9包封在二氧化钛-氧化铝溶胶-凝胶10和纳米沸石，11或通过的Ni-NTA或His-标签形成复合物捕获。6可替换地与固定化酶开管毛细管已经开发，以及12另外，增强的蛋白水解切割已经通过使用受控微波照射13或压力辅助或压力循环技术（PCT）的用于降低反应时间以30-120证实分14 尽管固定化酶的反应器的多个优势，商业墨盒成本高，常规使用的微流体设备的可用性是有限的，并且在需要使用微波或PCT技术结果另外的仪器。这项工作的目的是开发circumve的方法NTS这些缺点，并能在每一个实验室很容易地实现与用于制备在几分钟内MS分析进行蛋白的酶裂解的简单而有效的方法来赋予的研究人员。该方法依赖于使用的疏水性，C18-颗粒是在毛细管或微流体装置预加载，并且对这些颗粒随后通过酶消化感兴趣的蛋白质（多个）的酶在输液过程中吸附填充床和捕获蛋白（S）。在这种方法中，基板通过非共价相互作用固定，并且所述酶输注的固定化蛋白质。在蛋白水解消化效率提高了，揭露用于酶处理，减压距离和扩散时间和从粒子表面的蛋白质的大颗粒的表面积，提高质量传递，没有共价连接，可能会影响酶的活性，能快速evaluat不同的酶，可处理性，以及复用电子组合，如果过程中的微流体格式被执行。这种方法表现出与使用标准蛋白和胰蛋白酶的事先到ESI-MS检测水解消化最常用的酶的混合物。在这项研究中用于检测的质谱仪是一个线性阱四极（LTQ）仪器。

<table><tbody><tr><td>Ion trap ESI-MS</td><td>Thermo Electron</td><td>LTQ</td><td>The LTQ mass spectrometer is used for acquiring tandem MS data</td></tr><tr><td>XYZ stage</td><td>Newport</td><td>Multiple parts</td><td>The home-built XYZ stage is used to adapt the commercial LTQ nano-ESI source to receive input from various sample delivery systems</td></tr><tr><td>Stereo microscope</td><td>Edmund optics</td><td>G81-278</td><td>The microscope is used to observe the microreactor packing process</td></tr><tr><td>Analytical balance/Metler</td><td>VWR</td><td>46600-204</td><td>The balance is used to weigh the protein samples</td></tr><tr><td>Ultrasonic bath/Branson</td><td>VWR</td><td>33995-540</td><td>The sonic bath is used for mixing/homogenizing the samples and dispersing the C18 particle slurry</td></tr><tr><td>Syringe pump 22</td><td>Harvard Apparatus</td><td>552222</td><td>The micropump is used for loading, rinsing and eluting the sample and the enzyme on and from the packed capillary microreactor</td></tr><tr><td>Milli-Q ultrapure water system</td><td>EMD Millipore</td><td>ZD5311595&amp;nbsp;</td><td>The MilliQ water system is used to prepare purified DI water</td></tr><tr><td>Pipettor/Eppendorf (1,000&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-410</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (100&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-406</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Pipettor/Eppendorf (10&amp;nbsp;&amp;micro;l)</td><td>VWR</td><td>53513-402</td><td>The pipettor is used to measure small volumes of sample solutions</td></tr><tr><td>Fused silica capillary (100 &amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP100375</td><td>This capillary is used for the fabrication of the microreactor</td></tr><tr><td>Fused silica capillary (20&amp;nbsp;&amp;micro;m ID x 100 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP020090</td><td>This capillary is used for the fabrication of the ESI emitter</td></tr><tr><td>Fused silica capillary (50&amp;nbsp;&amp;micro;m ID x 360 &amp;micro;m OD)</td><td>Polymicro Technologies</td><td>TSP050375</td><td>This capillary is used to transfer the samples and the eluent from the syringe pump to the capillary microreactor</td></tr><tr><td>Glass capillary cleaver</td><td>Supelco</td><td>23740-U</td><td>This is a tool for cutting fused silica capillaries at the desired length</td></tr><tr><td>Glue</td><td>Eclectic Products</td><td>E6000 Craft</td><td>This glue is used for securing the ESI emitter into the capillary microreactor or the microfluidic chip</td></tr><tr><td>Epoxy glue</td><td>Epo-Tek</td><td>353NDT</td><td>This glue is used to seal the microfluidic inlet hole through which the C18 particles are loaded</td></tr><tr><td>Reversed phase C18 particles (5&amp;nbsp;&amp;micro;m)</td><td>Agilent Technologies</td><td>Zorbax 300SB-C18</td><td>These are C18 particles on which the proteins are adsorbed; the particles were extracted from a 4&amp;nbsp;mm x 20 cm C18 LC column from Agilent</td></tr><tr><td>Syringe/glass (250 &amp;micro;l)</td><td>Hamilton</td><td>81130-1725RN</td><td>The glass syringes are used to load the C18 particle slurry in the capillary microreactor and to deliver the sample and eluents to the microreactor</td></tr><tr><td>Internal reducing PEEK Union (1/16&amp;rdquo; to 1/32&amp;rdquo;)</td><td>Valco</td><td>ZRU1.5FPK</td><td>This union is used to connect the 250 &amp;micro;l syringe to the microreactor for loading the 5 &amp;micro;m particle slurry</td></tr><tr><td>Stainless steel union (1/16&amp;rdquo;)</td><td>Valco</td><td>ZU1XC</td><td>The stainless steel union is used to connect the glass syringe needle to the infusion capillary</td></tr><tr><td>Microvolume PEEK Tee connector (1/32&amp;rdquo;)</td><td>Valco</td><td>MT.5XCPK</td><td>The Peek tee is used to connect the sample transfer capillary to the capillary microreactor; on its side arm, it enables the insertion of the Pt wire</td></tr><tr><td>Tee connector (light weight)</td><td>Valco</td><td>C-NTXFPK</td><td>This Tee connector is used to apply ESI voltage to the microfluidic chip through the sample transfer line</td></tr><tr><td>Pt wire (0.404 mm)</td><td>VWR</td><td>66260-126</td><td>The Pt wire provides electrical connection for ESI generation and is connected to the mass spectrometer ESI power supply</td></tr><tr><td>PTFE tubing (1/16&amp;rdquo; OD)</td><td>Valco</td><td>TTF115-10FT</td><td>The Teflon tubing is used to enable an air-tight connection between the syringe needle and the stainless steel union&amp;nbsp;</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/16&amp;rdquo; OD)</td><td>Upchurch Scientific</td><td>1565</td><td>The Peek tubing is used as a sleeve to enable an air-tight connection between the stainless steel union and the 50&amp;nbsp;&amp;micro;m ID transfer capillary</td></tr><tr><td>PEEK tubing (0.015&amp;rdquo; ID x 1/32&amp;rdquo; OD)</td><td>Valco</td><td>TPK.515-25</td><td>The Peek tubing is used as a sleeve to enable a leak-free connection between the fused silica capillaries and the Peek Tee</td></tr><tr><td>Clean-cut polymer tubing cutter</td><td>Valco</td><td>JR-797</td><td>This cutter is used to pre-cut the 1/16&amp;rdquo; and 1/32&amp;rsquo; Peek polymer tubing that is used as sleeve for leak-free connections in pieces of ~4-5 cm in length</td></tr><tr><td>Amber vial (2 ml)</td><td>Agilent&amp;nbsp;</td><td>HP-5183-2069</td><td>The vials are used to prepare sample solutions and the C18 particle slurry&amp;nbsp;</td></tr><tr><td>Amber vial (4 ml)</td><td>VWR</td><td>66011-948</td><td>The vials are used to prepare sample solutions</td></tr><tr><td>Polypropylene tube (15&amp;nbsp;ml)</td><td>Fisher</td><td>12-565-286D</td><td>The vials are used to prepare buffer solutions</td></tr><tr><td>Cylinder (100 ml)</td><td>VWR</td><td>24710-463</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Cylinder (10 ml)</td><td>VWR</td><td>24710-441</td><td>The cylinder is used to measure volumes of solvent</td></tr><tr><td>Pipette tips (1,000 &amp;micro;l)</td><td>VWR</td><td>83007-386</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (100 &amp;micro;l)</td><td>VWR</td><td>53503-781</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Pipette tips (10 &amp;micro;l)</td><td>VWR</td><td>53511-681</td><td>The pipette tips are used to measure small volumes of sample solutions</td></tr><tr><td>Glass substrates</td><td>Nanofilm</td><td>B270 white crown, 3&amp;rdquo; x 3&amp;rdquo;</td><td>These are glass substrates for microchip fabrication</td></tr><tr><td>Male nut fitting (1/16&amp;rdquo;)</td><td>Upchurch</td><td>P203X</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Nanoport assembly</td><td>Upchurch</td><td>N-122H</td><td>This fitting is used for connecting transfer capillaries to the microfluidic chip</td></tr><tr><td>Protein standards</td><td>Sigma</td><td>Multiple #</td><td></td></tr><tr><td>Acetonitrile, HPLC grade</td><td>Fisher</td><td>A955</td><td></td></tr><tr><td>Methanol, HPLC grade</td><td>Fisher</td><td>A452</td><td></td></tr><tr><td>Isopropanol, HPLC grade</td><td>Sigma</td><td>650447</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma</td><td>302031</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Aldrich</td><td>A6141</td><td></td></tr><tr><td>Trypsin, sequencing grade</td><td>Promega</td><td>V5111</td><td></td></tr></tbody></table>

fast enzymatic processing of proteins for ms detection with a flow-through microreactor

绝大多数质谱（MS）的基础的蛋白质分析方法涉及一个酶消化步骤之前检测，通常与胰蛋白酶。这一步是必要的小分子量肽的产生，一般用MW &lt;3000-4000道尔顿，落入质谱仪器的有效扫描范围内的。传统的协议包括在37ºCO / N酶消化。最近的进展已经导致的各种策略，典型地涉及使用的微反应器与固定化酶或范围减少所需蛋白水解消化的时间到几分钟互补物理过程（的例如微波或高的发展压力）。在这项工作中，我们描述一个简单和成本效益的方法，可以在任何实验室来实现用于实现蛋白质的快速酶消化。的蛋白（或蛋白混合物）被吸附在C18键合反相PERFormance在毛细管柱预装液相色谱（HPLC）二氧化硅粒子和胰蛋白酶在含水缓冲液被注入过的时间很短的周期的颗粒。以使上线MS检测时，胰蛋白酶肽洗脱与在MS中离子源直接增加有机含量的溶剂体系。这种方法避免了使用高价的固定化酶颗粒，并且并不需要任何辅助完成的过程。蛋白消化完整样品分析可以在不到〜3分钟分别完成和〜30分钟。

蛋白水解消化方法的一个代表性的结果上的蛋白质的混合物同时进行，与上述微反应器（ 图1或2），在表1中提供。表包括标识特定蛋白质的独特肽序列，交叉相关评分（Xcorr）（ 即 ，表征实验到理论匹配相应串联质谱的质量得分），错过分裂的数量，所述肽电荷状态（z）的和质量/电荷（质子化肽的M / Z）。在蛋白质水平，该表提供了蛋白...

Watch this Scientific Journal Video about Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor at JoVE.com

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

蛋白质的MS检测快速酶法处理与流通型微反应器

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

fast-enzymatic-processing-proteins-for-ms-detection-with-flow-through

Research

JoVE Journal

Bioengineering

7.9K 次观看.  Virginia Tech. A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

视频: Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

一种简单、健壮且高通量的单分子流动拉伸试验研究分子沿 DNA 运移的方法

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

研究蛋白质折叠的微流体混合器

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

生物工程

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

用质谱和电镜分析 Biolayer 干涉传感器上的动态蛋白复合物的组装和释放

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

A new static platform is used to characterize protein structure and interaction sites in the native cell environment utilizing a protein footprinting technique called in-cell fast photochemical oxidation of proteins (IC-FPOP).

具有可移动XY阶段的平台孵化器:实现蛋白质细胞内快速光化学氧化的新平台

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

数字微流体进行自动蛋白质组学处理

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

生物学

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

大蛋白复合物结构质谱分析

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

基于电润的数字微流体平台，用于自动酶链接免疫吸附测定

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

毫秒氢/氘交换质谱法用于生理条件下α-突触核蛋白结构动力学的研究

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...