IDBR: TYPE A: Automated protein analysis: Gel-to-Mass Spectrometer Coupling Device

卡内基梅隆大学被授予一个奖项，以促进基础生物和医学研究的蛋白质发现领域的发展。细胞含有数千种不同类型的蛋白质。比较蛋白质组学的目标是发现在不同条件、不同遗传背景、不同发育或疾病阶段生长的细胞、组织和生物体之间存在差异的重要蛋白质。这些“差异蛋白”代表了使这些细胞在不同环境下表现不同的原因。因为细胞含有数以千计的蛋白质，单个蛋白质通常通过一种叫做凝胶电泳法的过程在像果冻一样的薄片中彼此分离，这让我们能够发现差异--蛋白质。为了找出哪些基因编码感兴趣的蛋白质，需要将蛋白质从凝胶中移除并注入质谱仪，质谱仪是一台非常准确地确定蛋白质身份的机器。将蛋白质从凝胶转移到质谱仪是一个严重的瓶颈。该项目将建造一种装置，大大提高这一转移过程的速度和效率。这个项目的一个关键要素是向本科生介绍这些复杂的、高度技术性的实验。目标是建立学生作为专业科学家的认同感。这个项目的独特之处在于，学生们作为一个综合团队，在合作项目上工作。在他们的学术生涯中，他们从实习生成长为培训师。他们学到了宝贵的团队管理和沟通技巧。到目前为止，所有从这个小组毕业的学生都进入了STEM职业生涯。这个项目的目标是开发一种设备，其目的是将蛋白质从电泳胶中转移出来，将分离出的蛋白质酶切成多肽，并将得到的多肽转移到质谱仪(MS)中进行蛋白质鉴定。我们将开发一种增强蛋白质识别的自动化设备，使从电泳胶中分离出来的蛋白质能够识别到0.1fmol(10-16摩尔)，这意味着比现有方法提高了100倍。此外，这一自动化系统将把整个过程从每个样品24-48小时加速到大约1小时。从而使研究人员能够以更高的灵敏度和速度进行他们的蛋白质组学实验。这将通过开发一种处理技巧来实现，用于将感兴趣的蛋白质从电泳胶中分离出来。分离出的蛋白质将被消化成末端内的多肽。然后，这些多肽将通过微流控装置引入MS。这一设备将有利于任何生物学家对从电泳胶中分离出的蛋白质进行MS鉴定。

An award is made to Carnegie Mellon University to advance the field of protein discovery for basic biological and medical research. Cells contain thousands of different types of proteins. The goal of comparative proteomics is to discover important proteins that differ between cells, tissues and organisms grown under different conditions, with different genetic backgrounds, or at different stages of development or disease. These "difference-proteins" represent what makes these cells behave differently under different circumstances. Because cells contain thousands of proteins, individual proteins are routinely separated from one another in thin "Jello"-like slabs by a process called gel electrophoresis, which allows us discover the difference-proteins. To figure out what genes encode the proteins-of-interest, the proteins need to be removed from the gel and injected into a mass spectrometer, a machine that very accurately determines the identity of the protein. There is a severe bottleneck in moving proteins from the gel to the mass spectrometer. This project will build a device to greatly increases the speed and efficiency of this transfer process. A key element of this project is to introduce undergraduate students to these complex, highly technical experiments. The goal is to build the students' identity as professional scientists. The unique feature of this program is that the students work as an integrated team to work on collaborative projects. They progress from trainees to trainers as they move through their academic careers. They learn valuable team management and communication skills. All of the students graduating from this group to date have gone onto STEM careers.The goal of this project is to develop a device whose purpose is to couple the transfer of proteins out of electrophoresis gels, to enzymatically digest the isolated proteins into peptides, and to the transfer the resultant peptides into a mass spectrometer (MS) for protein identification. We will develop an automated device for enhanced protein identification that will enable the identification of proteins isolated from electrophoresis gels down to 0.1 fmol (10-16 moles), representing a 100-fold improvement over existing methods. In addition, this automated system will accelerate the entire process from 24-48 hours per sample to about 1 hour. Thus enabling researchers to perform their proteomics experiments with far greater sensitivity and speed. This will be accomplished by developing a disposal tip for electrophoresing proteins-of-interest out the electrophoresis gel. The isolated proteins will be digested into peptides within the tip. The peptides will then be introduced into the MS through a microfluidic device. This device will benefit any biologist doing MS identification of proteins isolated from electrophoresis gels.