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Proteomics Course Syllabus

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					                             Proteomics Course Syllabus

Course Title: Current Proteomics

Credit: 3

Instructors:
      Masaru Miyagi (Course Director), Jinsook Chang, Janna Kiselar, Anne
      Distler, Rob Ewing, Wuxian Shi, Mark Chance, Sayan Gupta

Class time and location: Lecture: Wednesday 1-3 pm
                         Laboratory: Wednesday 1-4 pm
                         Journal presentation: Monday noon-1 pm

Course Description: This course is designed for graduate students across the university
who wish to acquire a better understanding of fundamental concepts of proteomics and
hands-on experience with techniques used in current proteomics. Lectures will cover
protein/peptide separation techniques, protein mass spectrometry, bioinformatics tools, and
biological applications which include quantitative proteomics, protein modification proteomics,
interaction proteomics, structural genomics and structural proteomics. Laboratory portion will
involve practice on the separation of proteins by two-dimensional gel electrophoresis,
molecular weight measurement of proteins by mass spectrometry, peptide structural
characterization by tandem mass spectrometry and protein identification using computational
tools. The instructors’ research topics will also be discussed.

Exam: The exam will be to write a 5-10 pages research proposal applying proteomic
approaches.

Grading Policy:
      - Journal presentation will count for 20% of the final grade
      - Lab reports: 20%
      - Proposal presentation: 20%
      - Final proposal: 40%

Reading Material and Lecture Notes: PowerPoint presentations from each class will be
posted after each lecture. All of the reading material for the course will also be posted on the
course webpage. No textbook is required. We will let everyone know what reading material is
recommended for each upcoming class.

SCHEDULE

See attached.
PHRM 555
Current Proteomics
Spring Semester
Rm. BRB932
Wednesday 1:00 – 4:00pm
Monday or Friday lunch hour

Participating Faculty: Masaru Miyagi (Course Director), Jinsook Chang, Janna Kiselar, Anne
                       Distler, Rob Ewing, Wuxian Shi, Mark Chance, Sayan Gupta

Week 1 (Jan. 16), Masaru Miyagi
Introduction to Proteomics
Objectives: Introduce students to the dynamic nature of proteome and provide overview of the current
               proteomics studies

Lecture outline:
       - what is proteome
       - nature of proteome
       - overview of the tools to study proteome
       - overview of the categories of current proteomic studies

Reading lists:
      1. Is proteomics the new genomics? Cox J, Mann M. Cell. 2007, 130, 395-398
      2. Correlation between protein and mRNA abundance in yeast. Gygi SP, Rochon Y,
               Franza BR, Aebersold R. Mol Cell Biol. 1999 Mar;19(3):1720-30

Week 2 (Jan. 23), Masaru Miyagi and Jinsook Chang
Fundamentals of Protein/Peptide Separation Technique
Objectives: Introduce participants to the techniques used for separating proteins/peptides

Lecture outline:
       - two-dimensional gel electrophoresis (2D-PAGE)
               Property of proteins
               2D electrophoresis
               Protein detection
               2D DIGE
       - high-performance liquid chromatography (HPLC)
               fundamentals of high-performance liquid chromatography
               reverse-phase chromatography
               strong cation exchange chromatography
               multidimensional HPLC

Reading lists:
      1. López JL. Two-dimensional electrophoresis in proteome expression analysis.
               J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Apr 15;849(1-2):190-202.
       2. Barceló-Batllori S, Kalko SG, Esteban Y, Moreno S, Carmona MC, Gomis R.
              Integration of DIGE and bioinformatics analyses reveals a role of the anti-obesity agent
              tungstate in redox and energy homeostasis pathways in brown adipose tissue. Mol Cell
              Proteomics. 2007 Nov 13; [Epub ahead of print]
       3. An automated multidimensional protein identification technology for shotgun proteomics.
              Wolters DA, Washburn MP, Yates JR 3rd. Anal Chem. 2001 Dec 1;73(23):5683-90


Week 3 (Jan. 30), Jinsook Chang
Protein Separation Lab.
Objectives: Provide students with hands-on experience for 2D-PAGE analysis of proteins.

Lab. work outline:
      - Isoelecteric focusing
      - Gel electrophoresis
      - Staining of gel

Week 4 (Feb. 6), Janna Kiselar and Anne Distler
Fundamentals of Mass Spectrometry
Objectives: Introduce students to peptide fragmentation mechanisms and interpretation of mass
              spectra to determine peptide sequences.

Lecture outline:
       - Sample Introduction
       - Mass Spectrometry Ionization Techniques
       - Mass analyzers
       - Peptide fragmentation mechanism
       - Interpretation of Mass Spectra

Reading lists:
      1. Chapter 4 of “Protein Sequencing and Identification using Tandem Mass Spectrometry”, by
               Michael Kinter and Nicholas E. Sherman, Wiley-Interscience Series on Mass
               Spectrometry

Week 5 (Feb. 13), Janna Kiselar and Anne Distler
Mass Spectrometry Lab. 1
Objectives: Provide students with hands-on experience with Quadrupole-ion trap (Q-Star) and ion trap
(LTQ) instrument for instrument calibration, intact protein analysis, and peptide sequence
determination by ESI-MS/MS.

Lab. work outline:
      Q-Star XL section
      - Instrument calibration and MS/MS of peptide (GluFib MS/MS)
      - Molecular weight determination for intact protein
      - Molecular weight determination for intact protein mixture
      - Sequence unknown peptide and determine sequence
       LTQ (Linear Ion Trap) section
       - principals of instrument design
       - Instrument characteristics (sensitivity, scanning cycle and speed)
       - Applications
       - Instrument calibration
       - Tunning
       - MS, MS/MS in Tune+ software
       - Mw measurements of proteins/peptides by infusion

Week 6 (Feb. 20), Rob Ewing
Introduction to Bioinformatics
Objectives: Introduce participants to the principals of bioinformatic methods in mass-spectrometry
               based proteomics

Lecture outline:
       - Mass-spectrometry data: basics, spectra
       - Sequence data: databases, tools and resources
       - Mass-spectrometry search engines: fundamentals, X! Tandem, Mascot

Reading lists:
      1. Large-scale database searching using tandem mass spectra: looking up the answer in the
               back of the book. Sadygov et al. Nat Methods. (2004) (pdf) [bioinformatics]

Week 7 (Feb. 27), Janna Kiselar and Anne Distler
Mass Spectrometry Lab. 2
Objectives: Provide students with hands-on experience with MALDI-TOF and LC-ESI-MS/MS
experiment for protein identification.

Lab. work outline:
      MALDI-TOF section
      - Instrument calibration
      - Analysis of digest of a single protein (two enzymes)
      - Analysis of digest of protein mixture
      - Protein identification of digests by peptide mass fingerprinting with MASCOT

       LC-MS/MS section
       - Principals of instrument design
       - Nano LCMS experimental set up
       - Data dependent acquision experimental set-up
       - SIM, SRM, MS/MS experimental set up

Week 8 (Mar. 5), Masaru Miyagi
Quantitative Proteomics and Protein Modification Proteomics
Objectives: Introduce students to the techniques and applications of quantitative proteomics and
              protein modification proteomics
Lecture outline:
       - 2D-PAGE based method
       - mass spectrometry based method
       - absolute quantification method
       - post-translational modification proteomics

Reading lists:
      1. Proteolytic 18O-labeling strategies for quantitative proteomics. Miyagi M, Rao KC. Mass
               Spectrom Rev. 2007 Jan-Feb;26(1):121-136
      2. Mapping protein post-translational modifications with mass spectrometry.Witze ES, Old
               WM, Resing KA, Ahn NG. Nat Methods. 2007 Oct;4(10):798-806

Week 9 (Mar. 12), Rob Ewing and Jinsook Chang
Bioinformatics Computer Lab.
Objectives: Provide participants with hands-on experience and understanding of MS bioinformatics
               tools for peptide and protein identification and quantitation

Lab. work outline:
      - Familiarisation with use of peptide fingerprint and Tandem MS search engines, (MS-Fit,
              Mascot)
      - Analysis and interpretation of peptide and protein identification results
      - DeCyder and DeCyder EDA


Week 10 (Mar. 19), Rob Ewing
Interaction Proteomics
Objectives: Introduce participants to the technologies and applications of interaction proteomics

Lecture outline:
       - Principal Technology Platforms
               Yeast 2-hybrid
               Co-immunopurification/Mass-spectrometry
       - Data analysis
               Graph-based visualisation
               Identication of protein clusters and modules
               Data analysis challenges: false positives
       - Protein-protein interaction networks
               Topology
               Network motifs

Reading lists:
      1. Mass spectrometry-based functional proteomics: from molecular machines to protein
               networks. Koecher et al. Nat Methods (2007). (pdf) [interaction proteomics - review]
       2. Co-immunoprecipitations revisited: an update on experimental concepts and their
              implementation for sensitive interactome investigations of endogenous proteins.
              Markham et al. Anal Bioanal Chem. (2007) (pdf) [interaction proteomics-methodology]
       3. Large-scale mapping of human protein-protein interactions by mass spectrometry. Ewing et
              al. Molecular Systems Biology (2007) (article) [interaction proteomics-application]
              Towards a proteome-scale map of the human protein-protein interaction network. Rual
              et al. Nature (2005) (pdf) [yeast two-hybrid-application]
       4. Comparative assessment of large-scale data sets of protein-protein interactions. Mering et al.
              Nature (2002) (pdf) [interaction proteomics-data analysis]

Week 11 (Mar. 26), Wuxian Shi
Structural Genomics
Objectives: Introduce students with the high-throughput techniques in structure genomics to generate
               3D structures for all proteins.

Lecture outline:
       - Outline for Structure genomics: high-throughput structure determination (Wuxian Shi)
               Overview of Protein Structure Initiative, Phase 2.
               arget selection: Pfam (protein families) 5000 strategy and targetDB (targetdb.pdb.org).
               Protein preparation.
               Structure determination by NMR and X-ray crystallography: crystallization, data
                       collection (using synchrotron radiation source), structure solution.
               Structure dissemination: structure deposition in PDB and publication.
               Outlook: reducing cost and function annotation.

       - Outline of structure to function and modeling
               Structure to function by fold comparison and functional site comparison
               Homology modeling and ab initio modeling

Reading lists:
      1. Schmid MB. Seeing is believing: the impact of structural genomics on antimicrobial drug
               discovery. Nat Rev Microbiol. 2004 Sep;2(9):739-46
      2. Bonanno JBet al., . New York-Structural GenomiX Research Consortium (NYSGXRC): a
               large scale center for the protein structure initiative. J Struct Funct Genomics. 2005;6(2-
               3):225-32
      3. Zhan C, Fedorov EV, Shi W, Ramagopal UA, Thirumuruhan R, Manjasetty BA, Almo SC,
               Fiser A, Chance MR, Fedorov AA. The ybeY protein from Escherichia coli is a
               metalloprotein. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Nov 1;61(Pt
               11):959-63

Week 12 (Apr. 2), Mark Chance and Sayan Gupta
Structural Proteomics
Objective: To give students a background in structural methods of mass spectrometry sufficient to
              comprehend the literature and design specific experiments in examining structure and
              dynamics
Lecture outline:
       - Deuterium exchange MS.
               hydrogen exchange, equilibrium and dynamics, examples from the literature
       - Foot-printing analysis of macromolecules principles of covalent labeling for DNA, RNA, and
               proteins biophysical analysis (kinetics and thermodynamics) by footprinting, structural
               analysis of DNA and proteins by mass spectrometry
       -Structural proteomics of large cellular complexes structural foot-printing of large complexes
               combining multiple datasets and refinement of structure

Reading lists:
      1. Articles from the Book: Mass Spectrometry Analysis for
               Protein-Protein Interactions and Dynamics, M. Chance Ed. (in press) will be distributed
               at the beginning of the semester, including:

              Chapter 2: Hydrogen Exchange Mass Spectrometry: Principles and Capabilities (John
              Engen and Sebastien Brier, Northeastern University). This chapter will review
              principles of deuterium exchange approaches derived from NMR methods and explain
              how they are used in structural mass spectrometry.

              Chapter 5: Deuterium Exchange Approaches for Examining Protein Interactions: Case
              Studies of Complex Formation (Elizabeth Komives, UCSD). This will demonstrate a
              state of the art application of deuterium MS approaches to understanding the biology of
              protein-protein interactions in PKA and NFkappa-B. It will demonstrate how such MS
              data is used in combination with docking, biochemical, and genetic data to provide a
              comprehensive view of protein complex structure.

              Chapter 8: Cross-Linking as a tool to Examine Protein Complexes: Examples of Cross-
              Linking Strategies and Computational Modeling (Christoph Borchers, UNC).
       2. Takamoto, K., Chance, M.R., “Radiolytic protein footprinting with mass spectrometry to
             probe the structure of macromolecular complexes.” Annu. Rev. Biophys Biomol. Struct.,
              35:251-76 (2006)

Week 13 (Apr. 2)
Researcg Topic 1: Daniela Schlatzer

Researcg Topic 2: TBA

Week 14 (April 9)
Proposal Presentation

				
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