Microscale analytical methods and sample processing tools for biomolecule characterization

我们的研究小组开发了识别和量化两类生物分子的策略和仪器：蛋白质和糖。我们正在研究通过一种叫做肽图谱的技术来量化生物体液中痕量蛋白质的方法，这种技术可以确定蛋白质是否因疾病而发生了变化。当这项技术与质谱（MS）结合使用时，质谱（MS）测量每个肽的质量，荧光检测（LIF）确定存在多少肽，母体蛋白可以通过其子肽质量来识别和量化。我们使用不溶酶作为一种手段，将大的蛋白质分解成它的组成子肽。这些肽的定位可以通过毛细管电泳和液相色谱等微尺度分离技术来完成，这些技术与质谱和液相色谱相结合。我们的提议描述了我们如何将一种特定的酶（如胰蛋白酶、凝乳胰蛋白酶等）固定到一个毛细管大小的柱（微反应器）中，然后在蛋白质溶液流过固定酶微反应器时，用它将纯化的蛋白质切成肽片段。将使用各种策略来开发和优化：a)从生物样品中分离蛋白质的程序，b)将蛋白质切割成肽片段的微反应器，c)分离，鉴定和定量（即绘图）肽片段，作为确定生物样品中每种蛋白质数量的手段。当来自健康和患病个体的蛋白质不同时，它们可能代表该疾病的重要生物标志物。发现一种生物标志物的意义在于它有开发新疗法的潜力。如果这些蛋白质能在它们的浓度还很低的时候被早期发现，治疗将会更有效。

Our research group develops strategies and instrumentation for identifying and quantifying two classes of biological molecules: proteins and sugars. We are studying ways to quantify trace levels of proteins in biological fluids through a technique called peptide mapping, which can identify whether a protein has been changed as a result of disease. When this technique is used in conjunction with mass spectrometry (MS), which measures the masses of each peptide, and with fluorescence detection (LIF), which determines how much peptide is present, the parent protein can be identified and quantified by its daughter peptide masses. We use enzymes that are insoluble as a means of fragmenting the large protein into its constituent daughter peptides. Mapping of these peptides can be done by microscale separation techniques called capillary electrophoresis and liquid chromatography, which are coupled to MS and to LIF. Our proposal describes how we will immobilize a particular enzyme (e.g., trypsin, chymotrypsin, etc.) into a capillary-sized column (microreactor) and then use it to cut a purified protein into peptide fragments as the protein solution is flowed through the immobilized-enzyme micoreactor. Various strategies will be used to develop and optimize: a) the procedures for isolating proteins from a biological sample, b) the microreactor for cutting the proteins into peptide fragments, c) separating, identifying and quantifying (i.e., mapping) the peptide fragments as a means to determine the amounts of each protein in the biological sample. When proteins from healthy and diseased individuals are different, they may represent important biomarkers for that disease. The significance of discovering a biomarker is its potential for developing new therapies. If such proteins could be detected early, when their concentration is still very low, treatment would be more effective.