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&#160;</td>
			<td>EMD Millipore</td>
			<td>ZD5311595&#160;</td>
			<td>The MilliQ water system is used to prepare purified DI water</td>
		</tr>
		<tr>
			<td>Pipettor/Eppendorf (1000 &#181;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 &#181;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 &#181;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 &#181;m ID x 360 &#181;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 &#181;m ID x 100 &#181;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 &#181;m ID x 360 &#181;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 &#181;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 mm x 20 cm C18 LC column from Agilent</td>
		</tr>
		<tr>
			<td>Syringe/glass (250 &#181;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&#8221; to 1/32&#8221;)</td>
			<td>Valco</td>
			<td>ZRU1.5FPK</td>
			<td>This union is used to connect the 250 &#181;L syringe to the microreactor for loading the 5 &#181;m particle slurry</td>
		</tr>
		<tr>
			<td>Stainless steel union (1/16&#8221;)</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&#8221;)</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&#8221; 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&#160;</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8220; ID x 1/16&#8221; 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 &#181;m ID transfer capillary</td>
		</tr>
		<tr>
			<td>PEEK tubing (0.015&#8221; ID x 1/32&#8221; 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&#8221; and 1/32&#8217; 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&#160;</td>
			<td>HP-5183-2069</td>
			<td>The vials are used to prepare sample solutions and the C18 particle slurry&#160;</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 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 (1000 &#181;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 &#181;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 &#181;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&#8221; x 3&#8221;</td>
			<td>These are glass substrates for microchip fabrication</td>
		</tr>
		<tr>
			<td>Male nut fitting (1/16&#8221;)</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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></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

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

微流体平台精密小批量来样加工及它的使用尺寸分离生物颗粒的声微型

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

与FRET流式细胞仪灵敏地检测Proteopathic播种活动

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

动脉粥样硬化的快速检测的氧化铁纳米颗粒微波驱动综合

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.