BIOLOGY 429 PROTEOMICS LABORATORY Fall 2004

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1 Dr. Karin Sauer Dr. Anna Tan-Wilson BIOLOGY 429 PROTEOMICS LABORATORY Fall 2004 COURSE BULLETIN DESCRIPTION: Proteomics techniques for the analysis of global changes in protein patterns. Methods include sample preparation, 2D/PAGE, image analysis, mass spectrometry, post source decay, and database mining to separate, detect and identify proteins. Techniques will be used in independent research projects that address a wide range of biological questions. Prerequisite: BIOL 301, CHEM 335, and consent of instructor; strongly recommended corequisite: BIOL credits. BACKGROUND INFORMATION: Availability of complete genome sequences has spawned a new approach to biological questions global gene expression analysis. Techniques to determine which genes are expressed at both the mrna and protein levels give a global, integrated picture of what is happening in cells and tissues at different stages of development, differentiation, or environmental conditions. Protein separation and identification by mass spectrometry techniques underlie the differential analysis of protein levels, a very important aspect of the new subdiscipline in biology referred to as PROTEOMICS. This new Proteomics Laboratory course is designed primarily to teach this new approach to biological research in a hands-on research-based class. The focus will be on two-dimensional polyacrylamide gel electrophoresis as the protein separation technique, and on peptide mass fingerprinting analysis as well as sequencing by post-source dissociation on the MALDI-TOF mass spectrometer as the main technique for protein identification. This training will be done in the context of exercises to teach 2D-PAGE and MALDI-TOF MS analysis during the first half of the semester followed by research projects in the second half. FINANCIAL SUPPORT: The State University of New York at Binghamton Dept. of Biological Sciences is able to offer this course through an educational grant from the National Science Foundation. Grant funds allowed the purchase of the Amersham Biosciences Ettan-MALDI- PRO, isoelectric focusing and electrophoresis apparatus. In addition, payment for supplies will be coming from faculty members who sponsor the research projects to be conducted by student pairs during the second half of the semester. PREREQUISITES: Biology 301 (Molecular Genetics) and Chemistry 335 (Organic Chemistry Laboratory, although other advanced level Biology course in the Cell and Molecular Biology area may work). Students commit to a Spring semester doing independent research following up on the research project for the course, so must plan to be enrolled at Binghamton for Spring 2004 as well. To apply, please contact annatan@binghamton.edu COURSE GOALS:?? Gain technical proficiency in the most widely used technique in proteomics, ie. 2D-PAGE, image analysis, peptide mass fingerprinting and protein identification by MALDI-TOF mass spectrometry?? Develop ability to judge the quality of 2D-PAGE data and basis for protein identification critically?? Experience in planning and carrying out a research project to answer a specific research question, the question to be supplied by a faculty research mentor

2 ?? Advance career goals by acquiring technical skill and lab experience that is highly sought in academic and industrial laboratories CLASS MEETINGS: s and s Science III Rm 280 1:10-4:10 P.M. s Science II Rm 134 1:10-2:20 P.M. Also, note that one person in a group will have to come in for a few minutes on specific days, as described in Remarks. SCHEDULE Week by week schedule of lab work Topic Schedule Assignment Remarks Week 1 Project 1 Separation of a mixture of proteins from various sources Week 2 Week 3 (08/30/04) 09/01/04) (09/03/04) (09/06/04) (09/08/04) (09/10/04) (09/13/04) (09/15/04)?? Introduction,?? Preparation of solutions, preparation of protein extracts?? Protein rehydration?? Isoelectric focusing (IEF), casting of 2D-gels?? 2D-image analysis exercise?? Labor Day, no class?? Strip equilibration and 2D/PAGE run?? Coomassie staining of 2Dgels,?? 2D-image analysis exercise,?? Picking of spots from 2Dgels, protein digest?? Rosh Hashanah, no class (15/09 19/09/04) Students have to return 24 hours later to start the IEF (takes 10 min) Stop IEF 21 hours later Stop 2D-gels 6 hours after start (09/17/04)?? Rosh Hashanah, no class (15/09 19/09/04)

3 Week 4 (09/20/04)?? Protein identification by mass spectrometry/peptide mass fingerprinting (09/22/04)?? Protein identification by mass spectrometry/peptide mass fingerprinting,,?? Database analysis Week 5 Project 2 Separation of phosphorylated proteins from Pseudomonas aeruginosa Week 6 Project 2, (09/29/04) (04/27/09) (09/29/04) (10/01/04) (10/04/04)?? Yom Kippur, no class?? Hand in lab report for project 1?? Sample preparation, rehydration?? IEF and casting of 2D-gels?? Peptide mass fingerprinting and database search exercise?? Hand in abstract describing research project?? 2D/PAGE run Students have to return 24 hours later to start the IEF (takes 10 min) Stop IEF 21 hours later Stop 2D-gels 6 hours after start (10/06/04)?? 2D-gel staining with silver nitrate Week 7 Project 2, (10/08/04) (10/11/04)?? 2D-image analysis for project 2 (Melanie software)?? Picking of spots from Melanie-analyzed 2D-gels?? protein digest (10/13/04)?? Mass spectrometry by peptide mass fingerprinting and PSD Week 8 to 14 Independent research projects (04/15/03) 10/18/04-12/03/04?? Protein identification,,?? Evaluation of results?? Hand in lab report for project 2?? Mid-term exam 10/18/04?? Students start working on

4 projects Week 15 12/06/04-12/10/04 independent research projects?? Thanksgiving, no class (24/11-28/11/04)?? Hand in research project report?? Poster presentation RESEARCH PROJECT AND GOAL BY THE END OF THE COURSE Students work in pairs. Placement under the tutelage of a faculty research mentor is done before a student is registered in the class. The faculty research mentor will provide the research question (upon discussion with the instructors to set attainable goals for the duration of the semester). Biological materials will be prepared outside of class time and may be provided by the students registered in the course or by other members of the research laboratory. Students registered in the course must start their work from the point of preparation of protein extracts. By the end of the semester, it is expected that students will have compared two protein samples in duplicate, and identified at least ten proteins. The faculty research mentor will also provide funds to cover the cost of supplies incurred by the student with respect to the research project. COMMITMENT TO INDEPENDENT STUDY IN THE FOLLOWING SEMESTER: By registering in the course, students are expected to continue the research project as independent study projects with their faculty research mentor during the Spring 2005 semester. This additional commitment accomplishes several goals: (1) gives students more opportunity to apply techniques learned in the Proteomics Laboratory course, hopefully to the point of producing publication-quality data; (2) gives faculty research mentors who provide financial support during the research portion of the Proteomics Laboratory course, results that will justify investment of their research grant funds. GRADING: Individual laboratory reports (2) 30% Mid-term test 25% Group research project abstract in consultation with faculty mentor 5% Individual research project report in consultation with faculty mentor 30% Laboratory performance 10% Grading is based on individual performance. ACADEMIC HONESTY: Any breach of academic honesty will be prosecuted fully, according to the guidelines of the Harpur College of Arts and Sciences < In addition, the students concerned will receive a grade of 0 for the assignment or test. INSTRUCTORS CONTACT INFORMATION: Dr. Karin Sauer: ksauer@binghamton.edu Dr. Anna Tan-Wilson annatan@binghamton.edu

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