Incorporating Mass Spectrometry-Based Protein Identification into Biochemistry Laboratory

生物科学(61)这个项目将基于质谱学的蛋白质鉴定引入一个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层析和离子交换层析来分离Bowman-Birk抑制剂，这是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存在。学生小组正在对分离的抑制剂进行凝胶内蛋白分解，通过肽质量指纹图谱和串联质谱仪(MS/MS)确定它们的肽序列，一旦鉴定出肽序列，就访问整个序列，并确定每个保守区域中的敏感肽键，以区分胰蛋白酶、糜蛋白酶和弹力酶的抑制剂。然后，学生们通过测量因预先与抑制剂孵育而失去的蛋白酶活性来测试他们关于基本键的假设。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，并包括制作免费提供的蛋白质识别在线学习材料。该课程的有效性正在通过学生应用新学概念的能力的前后测试进行评估，并与以课堂讨论形式学习相同概念的控制组学生进行比较。这个项目的智力成果来自于它将前沿技术引入到一个本科实验室，旨在鼓励学生理解与蛋白质结构和功能有关的基本概念，并适当使用现代蛋白质组相关技术。这一项目的更广泛影响包括课程和由此产生的传播材料的设计，以便各种机构能够全部或部分采用这些内容。此外，通过参与一个服务于代表不足的少数族裔的方案(国立卫生研究院通向文凭的桥梁方案)，首席调查员积极鼓励妇女和代表不足的少数族裔学生参加该课程。

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.