Incorporating Mass Spectrometry-Based Protein Identification into Biochemistry Laboratory

生物科学（61）本项目将基于质谱的蛋白质鉴定引入高级生物化学实验室，这是所有生物化学专业的必修课，也是生物学专业的选修课。 学生们正在使用亲和和离子交换色谱分离鲍曼-伯克抑制剂，一组稳定的丝氨酸蛋白酶抑制剂，在容易获得的种子中大量发现。学生小组正在对分离的抑制剂进行凝胶内蛋白水解消化，通过肽质量指纹图谱和串联质谱法（MS/MS）鉴定其肽序列，一旦鉴定出肽序列，则访问整个序列并确定每个保守结构域中的敏感肽键，以区分胰蛋白酶，胰凝乳蛋白酶和弹性蛋白酶的抑制剂。然后，学生们通过测量与抑制剂预孵育后失去的蛋白酶活性来测试他们关于必需键的假设。该项目正在加强学生对蛋白质结构-功能和进化关系的学习，包括制作免费提供的关于蛋白质鉴定的在线学习材料。通过对学生应用新学到的概念的能力进行的前后测试，与以讲座-讨论形式学习相同概念的对照组学生进行比较，来评估课程的效果。该项目的智力成果源于其将前沿技术引入本科实验室，旨在鼓励学生了解有关蛋白质结构和功能的基本概念以及现代蛋白质组学相关技术的适当使用。这一项目的更广泛影响包括，课程和由此产生的传播材料经过设计，可以全部或部分被各种机构采用。 此外，通过参与一个为代表性不足的少数民族服务的方案（国立卫生研究院通往学士学位方案的桥梁），主要调查员积极鼓励妇女和代表性不足的少数民族学生报名参加该课程。

Biological Sciences (61) This project introduces mass spectrometry-based protein identification into an upper division biochemistry laboratory, a course required of all Biochemistry majors and an elective for Biology majors. Students are using affinity and ion exchange chromatography to isolate Bowman-Birk inhibitors, a set of stable serine protease inhibitors found in abundance in easily available seeds. Student groups are performing in-gel proteolytic digestion on the isolated inhibitors, identifying their peptide sequence by peptide mass fingerprinting and tandem mass spectrometry (MS/MS), and, once the peptide sequences are identified, accessing the entire sequence and determining the susceptible peptide bonds in each conserved domain that distinguish inhibitors of trypsin, chymotrypsin and elastase from one another. Students then are testing their hypotheses concerning essential bonds by measuring protease activity that is lost by preincubation with inhibitor. The project is reinforcing students' learning of protein structure-function and evolutionary relationships and includes production of freely available online learning materials on protein identification. The effectiveness of the course is being assessed through pre- and post- tests of students' ability to apply their newly learned concepts as compared to a control group of students who are learning the same concepts in a lecture-discussion format. The intellectual outcomes of this project stem from its introduction of forefront techniques into an undergraduate laboratory designed to encourage student understanding of basic concepts concerning protein structure and function and of the appropriate use of modern proteomic related techniques. The broader impacts of this project include the fact the course and the disseminated material stemming from it are designed so that they can be adopted in whole or in part by a variety of institutions. In addition, through involvement with a program that serves underrepresented minorities (the NIH Bridges to the Baccalaureate Program) the Principal Investigators are actively encouraging women and underrepresented minority students to enroll in the course.