We are grateful to the Pacific Northwest National Laboratory (PNNL) Chemical Imaging Initiative-Laboratory Directed Research and Development (CII-LDRD) and Materials Synthesis and Simulation across Scales (MS3) Initiative LDRD fund for support. Instrumental access was provided through a W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL) Science Themed Proposal. EMSL is a national scientific user facility sponsored by the Office of Biological and Environmental Research (BER) at PNNL. The authors thank Mr. Xiao Sui, Mr. Yuanzhao Ding, and Ms. Juan Yao for proof reading the manuscript and providing useful feedback. PNNL is operated by Battelle for the DOE under Contract DE-AC05-76RL01830.

1.清洁和消毒的SALVI微通道<ol><li>微通道中SALVI消毒<ol><li>抽2毫升70％的乙醇水溶液到注射器，与SALVI的入口端连接注射器，并在10分钟内慢慢地注射1毫升液体。在注射结束取下注射器。接下来，将使用聚醚醚酮（PEEK）工会SALVI的入口和出口。或者，使用注射泵以进行同样的程序。例如，以100微升/分钟设定的流速。 </li><li>保持SALVI微通道在室温下填充有70％的乙醇溶液4小时。 </li></ol></li><li>介绍去离子水（DI）为SALVI <ol><li>抽2毫升无菌DI水到注射器，与SALVI的入口连接注射器，并在10分钟内慢慢地注射1毫升液体。取下注射器，并用PEEK工会连接SALVI的入口和出口。或者，使用注射泵在提到1.1.1一步。 </li></ol></li></ol> 2. SALVI蛋白质膜的固定化<ol><li>在固定的SALVI蛋白质电影<ol><li>抽2毫升纤连蛋白（10微克/ ml，在磷酸盐缓冲盐水，PBS）的溶液到注射器，与SALVI的入口连接注射器，在10分钟内慢慢地注射1毫升的液体，取出注射器，以及连接所述入口并利用PEEK工会SALVI出口。 </li><li>孵育在一个干净的培养皿中填充有纤连蛋白溶液的SALVI，在室温下保持盘在引擎盖12小时。 </li><li>抽2毫升无菌DI水到注射器和带SALVI的入口连接注射器。缓缓注入1ml水在10分钟内，取出注射器，并连接SALVI的入口和出口。 注意:如果装备有光学显微镜，检查SALVI在显微镜下，以确保在SiN膜是前TOF-SIMS分析完好无损。现在SALVI设备已准备就绪TOF-SIMS的alysis。 </li></ol></li></ol> 3.安装SALVI到TOF-SIMS负荷闭锁室注:手套应该在任何时候都处理SALVI设备并安装它到TOF-SIMS阶段，以避免表面分析过程中潜在的污染时佩戴。 <ol><li>摩SALVI到舞台<ol><li>躺在平坦，干净的表面的TOF-SIMS阶段。把SALVI设备上的硅片和罪恶膜朝上的阶段。固定使用四个金属夹紧件SALVI并拧金属片由四个螺丝保持该装置的角落入金属板。 </li><li>轻轻卷起连接到微流体通道的PFTE管。用四个金属片和四颗螺丝保持僵硬的一部分了。确保该装置被定位在上SIMS阶段的开放空间，以便它不会与分析过程中一次离子束干涉。额外的图像和SALVI fabricat的插图离子和安装在以前的出版物提供。8,9,11 </li></ol></li><li>安装法拉第杯的舞台上， <ol><li>通过螺丝固定在舞台上的法拉第杯。 </li></ol></li><li>加载阶段进入TOF-SIMS负荷闭锁室<ol><li>打开TOF-SIMS负荷闭锁，持有和水平搭建了舞台上装载平台，并关闭装载锁定门。 </li></ol></li></ol> 4. TOF-SIMS数据采集<ol><li>开启真空泵，并确保适当的真空（例如，10 -6毫巴或乇的数量级）在装料锁气室达到。 </li><li>移动SALVI到主腔室，一旦真空后约30分钟稳定化。 注:真空应该低于5×10 -6毫巴真空中在数据采集期间主腔。否则，较高的真空是表示在SALVI系统泄漏。 </li><li>调整TOF-SIMS的与初级离子束和分析器根据制造商的建议的约400纳米的空间分辨率。初级束的电流是1200 pA的，而循环时间下DC模式30微秒。 注意:这个过程基本上遵循制造商的建议。 </li><li>发现使用光学显微镜的微流体通道。使用光学图像中心为基准，并对齐以与次级离子图像中心一致。 </li><li>每次测量前，用1千电子伏O 2 +束（〜40 NA）扫描的SiN窗口上用500×500微米2面积约15秒，以除去表面的污染。此外，使用电子流枪中所有的测量，以弥补地面充电。使用流枪的自动功能在此步骤的默认模式。 </li><li>选择数据采集之前积极或消极的模式。使用25千电子伏的Bi 3+光束作为在所有测量的一次离子束。 注意:步骤类似˚F或正面和负面的数据采集。对空间的兴趣，正模式在细节描述。 <ol><li>深度剖析<ol><li>扫描上的圆形区域中的Bi 3+光束直径为〜2微米与32像素乘32像素的分辨率。 </li><li>为了尽量减少对穿通在SiN膜所需要的时间，使用长脉冲宽度（即，180纳秒，束电流〜1.0 pA的以100微秒的周期时间）的Bi 3+光束。当达到从液面上升次级离子代表的强度穿通。的穿通时间变化从300秒至500秒，这取决于批量在SiN膜。 注意:此差异似乎与给批次根据我们的经验获得的SiN膜。但是，穿通时间并没有真正影响到的数据分析。 </li><li>在SiN膜后穿通，保持相同的电流约200秒获得2D图像重建足够的数据。 </li><li>减少脉冲宽度为80毫微秒的数据收集来获取光谱具有更好的质量分辨率。继续本次收购大约另外200秒。在图1所示的光谱数据从该区域获得的。 </li></ol></li><li> 2D成像和质谱<ol><li>测量深度剖析数据时收集在同一时间的二维成像和质谱数据。在TOF-SIMS仪器保存所有在实验过程中每个点的信息。该数据显示在不同的窗口的数字控制软件（FPanel - 单独图像光谱，FPanel - - 概况和FPanel）。明智期间数据采集检查每个面板数据。 </li></ol></li></ol></li></ol> 5. TOF-SIMS数据分析注意:最初使用IonToF TOF-SIMS仪器软件进行数据分析。 <ol><li>打开一个SIMS（测量资源管理器），然后的alysis软件点击"配置文件"，"光谱"和"图像"按钮，分别用于处理深度剖析，M / Z频谱和图像数据。 </li><li>打开具有扩展名".ita"的数据文件。 </li><li>选择感兴趣的峰，请在"离子"框物种的名称，并在图谱中具有不同的颜色标记它们。使用鼠标滚轮沿x轴拖动。使用Ctrl和滚轮调节峰值的高度和宽度。沿y轴的线性和登录模式之间字母"L"的开关。 </li><li>质量校准之前进行数据重建。否则，就很难选择在质量校准步骤的峰之间的间隔。 <ol><li>选择感兴趣的深度分析数据，并根据深度分布时间序列重构的光谱。 </li><li>点击按钮重建。在"开始重建"窗口中，键入int的扫描范围erest。点击"确定"重建深度分布数据。 </li></ol></li><li>执行质量校正。按"F3"，打开"质量校正"窗口。请根据具体的样品进行校准峰。21在这项工作中，请使用以下的山峰CH 3 +（M / Z 15），H 3 O +（M / Z 19），C 2 H 5 +（M / Z 29 ），H 5 O 2 +（M / Z 37），C 3 H 7 +（M / Z 43），H 7 O 3 +（M / Z 55），H 9 O 4 +（M / Z 71） H 11 O 5 +（M / Z 99）和H 13 O 6 +（M / Z 109）为正模式。使用下列峰CH 2 - （M / Z 13），O - （M / Z 16）中，OH - （M / Z 17），C 2 H - （M / Z 25），C 3 H - （M / Z 37），和C 4 H - （M / Z 49）为负模式。 <ol><li>选择一个高峰，确保向下箭头指着窗口左下角的高峰期。点击峰值，并在"质量校准"窗口中键入高峰名称，单击"添加"，和感兴趣的离子包括在质量校正。 </li><li>最后，点击"重新校准"按钮。检查峰面积是在正确的地方根据自己的质荷比。在"光谱"菜单，选择并点击"应用质量校准"，"所有谱"或"选中的光谱"，这取决于具体的分析正在进行。 </li></ol></li><li>峰值选择是必要的试样分析，根据文献或其它以前的研究结果确定样品的特征峰。比较在M / Z谱感兴趣的峰的强度。如有必要，在峰列表中添加新的高峰或删除无用的峰值。 <ol><li>添加峰。点击一个高峰，发现在左侧窗口底部的红线。霍尔D中的"Ctrl"键，滚动鼠标水平来定义峰宽，它会自动添加一个峰值到峰值列表。添加一个峰的另一种方法是选择在主光谱窗口的峰，然后点击窗口上方的"加峰"按钮。如果有许多山峰添加，在"光谱"菜单，选择"搜索峰值"，检查相关的规范在新打开的窗口中，然后根据需要添加峰。 </li><li>要导出峰值列表，在"峰列表"菜单，选择"保存..."中的"itmil"格式的峰值列表。使用Ctrl和左按钮沿频谱拖动。在光谱窗口中，选择感兴趣的峰和两条虚线拖动到理想的位置。 </li><li>要导入一个峰值列表，在"大众间隔列表"菜单，选择"导入"，找到文件扩展名为".ITPL"现有的峰值列表文件。点击"打开"。 </li><li>要使用"itmil"高峰期名单中，在"大众间隔列表"菜单上，单击"打开..."，找到该文件，然后单击"打开"。 </li></ol></li><li>应用峰值列表来进一步分析数据。在"光谱"窗口的左下方，有一个名为"大众间隔列表"窗口中。导入的峰列表如下所示的。 <ol><li>右击峰列表文件被使用，其应用到"活性谱"或"所有光谱"。 </li></ol></li><li>查看资料文件。在"配置文件"窗口中，用字母"M"，以适合窗口（全系列）和字母"N"以适应y轴。使用Ctrl并滚动滚轮来调整轮廓宽度。或者使用"/"和"*"的键板来改变y轴范围和高度。 </li><li>使用其他图形软件的初步分析后进一步绘制导出数据。 <ol><li>要导出深度剖面，在"配置文件"窗口中，单击"文件"菜单，然后选择"导出＆＃34 ;,然后选择"另存为"一个名为".txt"文件。 </li><li>要导出为m / z频谱文件，在"光谱"窗口中，单击"文件"菜单，然后选择"导出"，然后选择"另存为"一个名为".txt"文件。 </li><li>要导出图像文件，在"图像"窗口，打印屏幕并保存为图像文件。 </li></ol></li></ol> 6. TOF-SIMS数据绘图和演示注意:使用一个图形工具的SIMS数据使用的工具软件处理后绘制数据。 <ol><li>使用图形工具来导入数据。 </li><li>使使用M / Z作为x轴和峰强度作为y轴，以显示所述重构的频谱的曲线图。一个例子在图1中给出。 </li><li>使用溅射时间作为x轴和峰强度作为y轴，以显示所述重构深度分布时间序列的图。一个例子在Figu给出再2。 </li><li>结合不同的M / Z的重构的二维图像，并形成一个矩阵显示离子映射。一个例子在图2中给出。 </li></ol>

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The authors have nothing to disclose.

SALVI是微流体接口，允许动态液面与液 - 固界面的分析通过基于真空手段，如TOF-SIMS和扫描电子显微镜（SEM）。由于小孔的使用情况，以直接在真空暴露液体，SALVI是适用于许多精密聚焦光谱和成像技术没有任何修改; 22可移植性和微流体的多功能性使之成为真正的多模成像平台。不同的特性和相对于其他现有技术SALVI的意义归纳在最近几个评价22-25作为SALVI技术的开发者，我们小组已经积极地将其应用到各种涉及动态液体表面和液-固界面的研究。的新的应用程序的一些例子包括哺乳动物细胞膜，细菌生物膜附着，15,16或纳米颗粒形成作为水性表面氧化的结果。在这种PA每次，我们目前的水合蛋白薄膜吸附在SiN表面上的初始液体TOF-SIMS结果。 这是相当关键小心处理在协议中突出显示的步骤。例如，绘制成的液体在步骤1和2中的注射器时，确保不会有注射器内的任何气泡。因为一旦气泡注入SALVI，他们可能会在真空中引起泄漏。9因此，如果观察到任何气泡，为谨慎起见，由直立设置注射器，轻轻推泡出摆脱他们。此外，这是非常重要的，以使在SALVI在SiN窗口平面优化TOF-SIMS分析中，如在步骤3中所描述的检测区域的这水平度根据TOF-SIMS的设计直接影响到数据的品质。26它要确保一切到SIMS阶段之后目视检查设备。如果在SiN窗口不是水平的，调整螺丝，改变高度所述SALVI装置，以确保一切是在尽可能多的同一平面上。这些微妙的点需要经验和细心，它决定了整个实验的成功和可靠性。 如图1b中，与M / Z 19，37，55，73，91和109具有高的计数的峰值。这表明，有水簇的一个好数字（H 3 O +，H 5 O 2 +，H 7 O 3 +，H 9 O 4 + H 11 O 5 +和H 13 O 6 +）在正二次离子。其他水簇（数据未示出）具有较高的m / z值也有比较强的峰与它们相邻的峰进行比较。这些大量水簇的是从在SiN穿通之后的去离子水的表面射出。在正模式水簇的信号没有在以前的瓦特被观测到的ORK;这是由于二次离子和低计数TOF-SIMS在过去条件非优化8随着优化SALVI和TOF-SIMS采集条件的不断努力，如一次离子束（即，铋+ VS毕3 +），脉冲宽度和深度剖析期间当前的条件下，它现在可以观察到高达44水簇在使用本文提出的条件，正模式。这里显示的结果是令人鼓舞的进一步研究水在各种生物分子和生物系统动力学的作用。 这里作为一个例子来说明在研究生物分子动力学SALVI和TOF-SIMS的优点，纤连蛋白的水合提出。的M / Z 30，44，61，74，81，83，和98的峰，根据前述报告，27,28这比DI笏的强得多属于氨基酸片段呃如图 1b所示。在纤连蛋白样品中的氨基酸片段的强度增加这种差异表明，这些二次离子确实从蛋白质本身。此外，水簇的图 1a中的峰值比在图1b中，这意味着在该蛋白质的吸附表面和界面溶液的水分子的性质可以从那些在纯水中非常不同的那些DI水强得多。 图2a代表深度剖析时间序列两个不同样本:纤维连接蛋白和去离子水在微SALVI。作为在SiN窗口正在通过与长脉冲宽度铋3 +一次离子束轰击收集深度剖析的数据。 如图 2a所示，在SiN窗口是通过在约300秒打孔。前SALVI在SiN窗口砸出通过量小时，选择的次级离子的强度是相当低的。一旦在SiN窗口被冲压穿过的二次离子的强度立即增加，因为液体表面开始被由初级离子束轰击。例如，H +的强度和H 3 O +显示一个大的增加，表明所述水表面的观察。-8,9-虽然强度是穿通或界面处，它们可达到相对稳定后不稳定右经过一段时间后的值（例如，60秒为纤连蛋白和220秒为DI水，分别在图2a）。在长脉冲宽度钻孔的差异并没有真正影响重建M / Z谱（例如，图1）当在这里示出的分析结果中，因为有效M / Z光谱在SiN穿通后获得的。然而，研究人员可以选择使用同样的条件对所有样品FO- [R一致性和简化数据分析过程中选择数据段。为了获得更好的质量分辨率，脉冲宽度随后减少，强度分别在深度剖析实验的后期部分相应地减少。深度轮廓的时间序列中连续收集另一200秒为M / Z频谱分析提供足够的数据。时间的M / Z光谱重建的部分突出显示为绿色的阴影区域。 纤连蛋白已被广泛地用于前细胞培养表面吸附;许多研究已经表明纤连蛋白是负责细胞粘附和细胞的生物材料的相互作用。使用椭圆光度法（羟乙基甲基丙烯酸酯）的刷子19,20,29最近的一项研究，其特征纤连蛋白吸附在聚的厚度。纤连蛋白薄膜被确定为3-12纳米厚。30这种厚度是合理用于在水性SOLU的深度剖面分析灰。-8,9-随着SALVI的发展，吸附在固体表面上的水合蛋白质的直接分析现在是可能的。这种方法潜在地可具有学习与动态固体表面蛋白质的相互作用广泛的应用。 图2b分别显示了从纤连蛋白和DI水样不同的次级离子的选择的2D图像。二维图像从图 2a中绿色阴影区域的深度剖面时间数据，对应于液体表面的SiN之后获得穿通如前所述。选择的氨基酸片段二次离子的图像CH 4 N +，C 5 H 7 O +和 C 4 H 4 NO 2 +（即，M / Z 30，83和98），用于将纤连蛋白薄膜均较亮得多为去离子水。这表明氨基酸片段次级离子观察˚FROM中的2微米直径的光圈内的纤维连接蛋白分子。此外，选择的峰的图像 （例如，M / Z 1，19，37，和55）对应于纤连蛋白的几个水簇比去离子水的显着更亮。这个结果表明，纤维粘连蛋白的水合使得周围的生物分子从在DI水中的水分子不同的水分子。 总之，这项工作提供了关于蛋白质水合诱导的水分子属性改变引人注目的证据。这样的结果可以提供在结构上，构象和生物在液体微吸附在固体表面上的蛋白质的活性的改进的基本理解。

水化是该结构，1构象，2和蛋白质的生物活性的3至关重要。他们周围的蛋白质没有水分子不会有可行的生物活性。具体地，水分子与表面和蛋白质的内部结构进行交互，和蛋白质的不同水合状态使这种相互作用是不同的。4蛋白与固体表面的相互作用是在纳米技术，生物材料和组织工程的过程影响的基本现象。研究早就表明，作为蛋白质遇到表面，可能会发生的构象变化。 TOF-SIMS已经设想作为具有研究蛋白质-固体界面的电位的技术。5-7理解在固体表面上，这可能提供了它们的结构的机构的基本理解蛋白的水合是重要的，构象和生物人的活动。 然而，主要的表面分析技术大多是基于真空和挥发性液体直接的研究应用，由于挥发性液体的真空环境下迅速蒸发是困难的。我们开发了一种真空兼容微流体接口，系统分析在液体真空接口（SALVI），以使液体表面，并使用时间飞行二次离子质谱法（TOF-SIMS）液-固相互作用的直接观察。8- 11独特的方面包括以下内容:1）的检测窗的直径为使液体表面的直接成像的2-3微米的孔，2）表面张力被用于保持所述孔中的液体，以及3）SALVI是在多个平台上的分析的便携式11,12 SALVI由氮化硅（SIN）膜作为检测区域，并提出聚二甲基硅氧烷（PDMS）的微通道。这是FABRicated在洁净室，并在制造和关键设计因素已在先前的论文和专利被详细描述。8-12 TOF-SIMS的作为分析工具中的应用进行了使用各种水溶液和复杂液态混合物，其中一些的证明含有纳米颗粒。13-17具体地说，SALVI液体TOF-SIMS允许动态活生物系统 （即生物膜），单细胞，和固体电解质界面的液-固界面的探测，原位冷凝阶段打开新的机会研究包括使用TOF-SIMS液体。然而，目前的设计不允许气液相互作用爱好。这是未来发展的一个方向。 SALVI已被用于研究水合蛋白质膜在这项工作中，第一次。 纤连蛋白是一种常用的蛋白二聚体，由18由一对的二硫键连接的两个几乎相同的单体，的我旗下著名其结合细胞的能力。它19,20被选择作为模型系统来说明水合蛋白薄膜可以使用SALVI液体TOF-SIMS方法来探测动态。该蛋白质溶液引入到微通道。 12小时温育后，形成在SiN膜的背面侧水合蛋白质膜。去离子水是用来冲洗掉蛋白质出台后的通道。信息来自于使用动态TOF-SIMS的SALVI微纤连蛋白水合蛋白质分子收集。 DI水还研究了作为控制与水合纤维连接蛋白薄膜得到的结果进行比较。水合蛋白膜和DI水之间观察明显的差异。这项工作表明，关于在液体环境表面蛋白吸附可以使用该新型SALVI和液体TOF-SIMS方法进行研究。该视频协议的目的是人谁是感兴趣的人士提供技术指导在利用与TOF-SIMS SALVI的各种应用此新的分析工具，并减少在液体处理不必要的错误以及TOF-SIMS数据采集和分析。

<table><tbody><tr><td>ToF-SIMS</td><td>IONTOF</td><td>TOF.SIMS 5</td><td>Resolution: &gt;10,000 m/&amp;Delta;m for mass resolution; &gt;4,000 m/&amp;Delta;m for high spatial resolution&amp;nbsp;</td></tr><tr><td>System for Analysis at the Liquid Vacuum Interface (SALVI)</td><td>Pacific Northwest National Laboratory</td><td>N/A</td><td>SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum-based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level.</td></tr><tr><td>PEEK Union</td><td>Valco</td><td>ZU1TPK</td><td>for connecting the inlet and outlet of SALVI</td></tr><tr><td>5 Axes Sample Stage</td><td>IONTOF</td><td>N/A</td><td>Stage is self-made for mounting SALVI in ToF-SIMS</td></tr><tr><td>Barnstead Nanopure Water Purification System</td><td>Thermo Fisher Scientific</td><td>D11921</td><td>ROpure LP Reverse Osmosis filtration module (D2716)</td></tr><tr><td>Syringe</td><td>BD</td><td>309659</td><td>1 ml</td></tr><tr><td>Pipette</td><td>Thermo Fisher Scientific</td><td>21-377-821</td><td>Range: 100 to 1,000 ml</td></tr><tr><td>Pipette Tip</td><td>Neptune</td><td>2112.96.BS</td><td>1,000 &amp;micro;l</td></tr><tr><td>Centrifuge Tube</td><td>Corning</td><td>430791</td><td>15 ml</td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td>1 mg/ml</td></tr><tr><td>Ethanol</td><td>Thermo Fisher Scientific</td><td>S25310A</td><td>95% Denatured</td></tr><tr><td>Gibco PBS</td><td>Thermo Fisher Scientific</td><td>10010-023</td><td>pH 7.4</td></tr></tbody></table>

in situ characterization of hydrated proteins in water by salvi and tof-sims

使用分析系统在液体真空接口（SALVI）和时间飞行二次离子质谱法（TOF-SIMS）的水溶液中的蛋白质的生物分子的原位表征这项工作表明。纤连蛋白的蛋白质膜被固定在形成SALVI检测区域的氮化硅（SIN）膜。在TOF-SIMS分析中，进行了分析的三种模式包括高空间分辨率质谱，二维（2D）成像和深度剖析。质谱在积极和消极模式获得的。还分析去离子水作为参比样品。我们的结果表明，在水中的纤连蛋白膜具有单独与水相比更明显的和更强的水簇的峰。的氨基酸的片段的特征峰，可以在水合蛋白质TOF-SIMS光谱观察到。这些结果表明，在表面上的蛋白分子的吸附，可以研究动感中LLY使用SALVI和TOF-SIMS在首次的液体环境。

一对夫妇的代表性结果提交给验证了该协议的优点。通过使用SALVI微流体接口，初级离子束（铋3 +）可以直接轰击在DI水中水合纤连蛋白膜。因此，液体表面的分子化学映射可以成功地获取。 图1a 和 1b分别示出了水合纤连蛋白膜和DI水的正TOF-SIMS质谱，。感兴趣的峰值包括水簇（ 即 M / Z 19，37，55，73，91和109）和氨基酸片段（ 即 M / Z 30，44，61，74，81，83和98）由红色箭头表示。除了氨基酸片段的特征峰，其中已使用干蛋白质样品被广泛报道，水簇的峰中观察到的在去离子水中水合纤连蛋白薄膜的质谱。而且，这些水簇的峰的强度是从那些在DI水中的质谱大不相同。这个结果提供了周围的水分子的性质的，并且在表面上在它们的吸附发挥作用水合蛋白质分子内的直接证据。 图2a示出的深度轮廓的时间序列水合纤连蛋白膜和DI水。作为样品是在SiN膜下，在SiN膜通过冲压之前选择的第二离子的强度是可以忽略的（即 ，280秒，纤连蛋白和340秒为DI水）。一旦在SiN膜被冲压通过，这些二次离子的强度显著增加。当使用长脉冲宽度，质量分辨率相对较低。因此，短脉冲宽度的过程使用具有更好的质量分辨率，以获得数据图像和光谱重建所强调如图 2a。经过一段时间后（即 ，60秒为纤连蛋白和220秒为DI水），减小的脉冲宽度是用来获得具有更好的质量分辨率M / Z光谱。最后M / Z谱从200秒测量重构突出绿色阴影区域中的深度分布时间序列。 图2b示出了从水合纤连蛋白膜和DI水，其对应于在图2a中的绿色的阴影区域的二维图像。这些2D图像代表选择的次级离子的计数从样品:图像越亮，二次离子计数越高。这种直观的图像给出不同的样品之间选择的次级离子的清晰的比较，这是在数据分析有帮助的。根据我们的经验，在在SiN穿通时间的不同而变化批次在SiN膜。然而，它不影响重动态TOF-SIMS分析sults。 <img alt="图1" src="/files/ftp_upload/53708/53708fig1.jpg" /> 图1:在SALVI微通道水合纤连蛋白膜和DI水的TOF-SIMS阳性光谱的比较 （ 一 ）纤连蛋白的重构M / Z频谱。 （二）DI水光谱。 <img alt="图2" src="/files/ftp_upload/53708/53708fig2.jpg" /> 图 2: 深度剖面和水合纤连蛋白和去离子水的2D图像 （a）在该正模式的深度剖析时间序列纤连蛋白和DI水。 （二）的2D图像重构对应于绿色阴影200秒的时间段沿深度分布的时间序列（a）中。几个特征质量，以相关的比例收取，以水分子团簇（例如，M / Z 1，19，37，55）显示，除了已知的氨基酸片段（ 例如 ，M / Z 30，83，98）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53708/53708fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS at JoVE.com

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

这项工作提出了液体处理和样品引入用于原位时间飞行二次离子质谱法的蛋白质的生物分子的水溶液中分析一个协议到一个微通道。

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

in-situ-characterization-hydrated-proteins-water-salvi-tof

Nanyang Technological University

Research

JoVE Journal

Chemistry

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

8.2K 次观看.  Pacific Northwest National Laboratory. 这项工作提出了液体处理和样品引入用于原位时间飞行二次离子质谱法的蛋白质的生物分子的水溶液中分析一个协议到一个微通道。 

视频: In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

无细胞DNA完整性分析尿样中

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

科研

癌症研究

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+ binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (&#964; &#60; 20 &#956;sec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Here we report an application of the photothermal beam deflection technique in combination with a caged calcium compound, DM-nitrophen, to monitor microsecond and millisecond dynamics and energetics of structural changes associated with the calcium association to a neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator.

亚毫秒构象变化蛋白的光热光束偏转已解决

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

化学

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

在质谱选择离子软着陆准备好定义的表面原位二次离子质谱和红外光谱

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

应用实时 PCR 技术检测血清和血浆拷贝数

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

水在油乳剂:与疏水性颜料装配水溶性叶绿素结合蛋白的新系统

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

掺硼金刚石电极质量和应用，以评估<em&gt;原位</em&gt;本地pH值通过水电改造

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as well as synthesis of essential metabolites. These organelles consist of the following three key sub-compartments. The envelope, characterized by two membranes, surrounds the organelle and controls the communication of the plastid with other cell compartments. The stroma is the soluble phase of the chloroplast and the main site where carbon dioxide is converted into carbohydrates. The thylakoid membrane is the internal membrane network consisting of grana (flat compressed sacs) and lamellae (less dense structures), where oxygenic photosynthesis takes place. The present protocol describes step by step procedures required for the purification, using differential centrifugations and Percoll gradients, of intact chloroplasts from Arabidopsis, and their fractionation, using sucrose gradients, in three sub-compartments (i.e., envelope, stroma, and thylakoids). This protocol also provides instructions on how to assess the purity of these fractions using markers associated to the various chloroplast sub-compartments. The method described here is valuable for subplastidial localization of proteins using immunoblotting, but also for subcellular and subplastidial proteomics and other studies.

Here, we describe a method to purify intact chloroplasts from Arabidopsis leaves and their three main sub-compartments (envelope, stroma, and thylakoids), using a combination of differential centrifugations, continuous Percoll gradients, and discontinuous sucrose gradients. The method is valuable for subplastidial and subcellular localization of proteins by immunoblotting and proteomics.

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

生物化学

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.

在水中水合蛋白原位表征SALVI和TOF-SIMS

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