1. Clinical Proteomics MMBS 494.602 Course Title: Clinical Proteomics Coursemaster:Eugene C. Yi, Professor, euyi@snu.ac.kr, 02-740-8926euyi@snu.ac.kr Semester:Spring 2010 Office Hours: by arrangement Class Schedule: Every Thursday 6:00 – 9:00 Place: WCU MMBS classroom, SNU College of Medicine

2. Course Description This course is designed for students who wish to acquire a better understanding of fundamental concepts of proteo mics and its application to real-life biomedical research. Lectures cover protein/peptide separation techniques, pro tein mass spectrometry, bioinformatics tools, biostatistics and biological applications which include quantitative prot eomics, protein modification proteomics, interaction prote omics and protein biomarker development. By the end of this course students should appreciate the s cientific problems involved in the post-genome era biology and understand how the proteomic approaches are being utilized in the context of protein biomarker and therapeutic target discovery.

3. Text and Course Materials We do not have a textbook. Instead, since omics area evolves quite rapidly, a collection of articles from the p ublished literature will be used as a course material. H owever, I do recommend the Liebler text for Introductio n to Proteomics: Tools for the New Biology, Humana P ress Inc., 2002, if you feel you absolutely need a refer ence. Additionally, several review articles below are e xcellent proteomics review resources. PowerPoint presentations from each class will be post ed after each lecture. All of the reading material for the course will also be posted on the course webpage. I wi ll let everyone know what reading material is recomme nded for each upcoming class. References: Please see the hand-out.

4. Grading Policy Grade will be based on attendance (10%), in-class midterm exam (40%), and final paper discussion a nd presentation (50%). Midterm exam will consist primarily of short assay- type questions covered in the lecture. Final exam will be the class presentation format. The purpose of the final exam is to introduce relev ant material not covered in the textbook or regular course lectures, allow students to investigate topic s related to their individual interests, and give stud ents experience in preparing for and leading a disc ussion of current research.

5. Class Schedule, Lectures and Important Dates Please see the hand-out

6. Course introduction & Overview and Current Trend in Proteomics What to expect from this course The birth of proteomics and functional prote omics in post-genomic era How proteomics is applied in real-life scienti fic research Overall proteomics experimental workflows

7. The birth of proteomics

8. A time when genomes are being sequence d and released – Post-genomic era Genome provides a basis for evaluating wh at is possible – blueprint info, static info-but does not provide the critical information abo ut what “is” in a given organism, organ, tiss ue or cell. Genome sequences do not always provide a direct link to biological activity

9. Why proteomics, when we have the genome? The study of the proteome, the complete set of proteins produced by a species, using the t echnologies of large-scale protein separation and identification.proteomeproteins The term proteomics was coined in 1994 by Marc Wilkins who defined it as "the study of proteins, how they're modified, when and wh ere they're expressed, how they're involved i n metabolic pathways and how they interact with one another.“

10. Transcriptome sequencingarrays Genomic DNAmRNA Proteome Quantitative & Qualitative profiling Functionome Catalytic activity Subcelluar locatalization Protein modifications Dynamic protein linkage maps Descriptive protein linkage maps Protein phosphorylation System Interaction between components Measure and define properties Identify systems components Provide functional annotations for the entire proteome Link the linear information into biological system

11. Genomics vs Proteomics Genomics : the systematic study of the entire genome of an organism o Physically map the genome arrangement(assign exact positions in t he genome to the various genes/non-coding regions) o Human Genome Project  Began in 1990, completed in 2003  Total human genome size is in the region of 3.2 gigabases (Gb)  Less than 1/3 of genome is transcribed into RNA and only 5% o f that RNA is believed to encode polypeptides Proteomics : direct examination of the expressed proteins in the cell Systematic and comprehensive analysis of the proteins(proteom ) expressed in the cell and their functions Direct comparison of protein expression Changes in cellular protein profiles with cellular conditions

12. Why bother … Example GABA - the major inhibitory NT in the brain GABA related to Anxiety, mo od disorder, epilepsy, Parkins on, Sleep… Controlling the level of GABA response How? P2P A patch of receptors P2P A patch of receptors Localization A pool of receptors Localization A pool of receptors Signaling Phospho. Endocytosis Signaling Phospho. Endocytosis PTM Ubiquitination of receptors PTM Ubiquitination of receptors Life time Proteosome action Life time Proteosome action No signal at the genome/ transcriptome levels

13. Traditional Biochemical Approach Reductionism Study of biochemical processes is carried out through sequential fractionation/assay cycles of the specific activities that constitute the process This approach requires a lot of effort and it is painfully slow

14. For example, cholesterol biosynthesis Squalene synthase Whole cell lysate assay Microsomal fraction assay The method critically depends on the ability of an assay to follow the purification – purified proteins, timely process (months) – only proteins with measurable activity can be analyzed

15. Current Biochemical Approach Instead of analyzing individu al proteins one at a time, it is possible to study whole syste ms of proteins in cellular eve nts. Obviously, advances in mas s spectrometry techniques an d global network-based data analysis make this possible Integratism

17. Combined traditional & Omic approaches Hypothesis-driven Data-driven coherent New knowledge & refine the model

18. In vitro/ In vivo cell-based assays Bioinformatics Proteomics Transcriptomics CGTCCAAC TGACGTCTA CAAGTTCCT AAGCT Genomics Integrated view of the complex biological systems Integration of Omics validation

19. Proteomics - Type of Studies Which Proteins, when, where, with whom? Protein Mining – Catalog proteins, genome annotation and gene function pre diction in a tissue or cell etc. Differential Protein Expression Profiling – Identification of proteins in a s ample as a function of a particular state: differentiation, stage of development, disease state, response to drug or stimulus Subcellular location / Secretion Mapping protein modifications – Characterization of PTM (phosphorylation, glycosylation, ubiquination. oxidation etc) Protein Interactions – Ligand-Receptor & Oligomerization and protein-DNA Network-based analysis – Identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multiprotein complexes

20. Protein Mining – Catalog proteins, genome annotation and gene function pre diction in a tissue or cell etc. Cell surface proteins Cytosolic proteins Nucleus proteins Organelle (mitochondria, peroxisome) proteins

21. Differential Protein Expression Profiling – Identification of proteins in a sample as a function of a p articular state: differentiation, stage of development, disease state, response to drug or stimulus NormalDiseased Which proteins are up or down regulated ? Biomarkers or drug targets

22. Subcellular location / Secretion Cell surface proteins (membrane and extracellular) Cytosolic proteins Nucleus proteins Organelle (mitochondria, peroxisome) proteins Secreated proteins Most eukaryotic proteins are encoded in the nuclear genome and synthesized in th e cytosol. Proteins must be localized at their appropriate subcellular compartment to perform their desired function.

23. Mapping protein modifications – Characterization of PTM (phosphorylation, glycos ylation, ubiquination, oxidation, sumolylation etc) Post-translational modifications are translational independent processes that are known to play a fundamental role in regulating the activity, location and function of a wide range of proteins.

24. Protein Interactions – Ligand-Receptor & Oligomerization and protein-DNA The interactions between proteins are important for numerous biological functions Signal transduction (exterior -> interior) Cell-Cell contact Plays a fundamental role in many biological pr ocesses and in many diseases (e.g. cancers) Carrying another protein (cytoplasm -> nucleus or vice versa) Protein modifications (kinase) Transcription regulations

25. Network-base Analysis – Identification of proteins in functional networks: biosynthetic pathways, signal transduction pathways, multiprotein complexes Network Analysis DNA damage module Inflammation module … “Identification of key disease-associated modules” Marker set = … [ ] extraction

26. System 1System 2 Integrated Proteomics Workflow Database Search # Rank/Sp (M+H)+ deltCn C*10^4 Ions Reference Peptide # Rank/Sp (M+H)+ deltCn C*10^4 Ions Reference Peptide --- ------- ------ ------ ------ ---- ---------- ------- --- ------- ------ ------ ------ ---- ---------- ------- 1. 1 / 1 1994.3 0.0000 4.4675 17/26 G3P_RABIT (R)VPTPNVSVVDLTC*R 1. 1 / 1 1994.3 0.0000 4.4675 17/26 G3P_RABIT (R)VPTPNVSVVDLTC*R 2. 2 /403 1995.1 0.6126 2.7366 13/34 SLTRNGL (E)LGKPVLTANQVTIWEGLR 2. 2 /403 1995.1 0.6126 2.7366 13/34 SLTRNGL (E)LGKPVLTANQVTIWEGLR 3. 3 / 3 1995.0 0.5952 2.6591 16/36 FLP_LACCA (N)IANPNVYTETLTAATVCTI 3. 3 / 3 1995.0 0.5952 2.6591 16/36 FLP_LACCA (N)IANPNVYTETLTAATVCTI 4. 4 /209 1995.0 0.5895 2.6335 14/36 A42912 (Y)LALLPSDAEGPHGQFVTDK 4. 4 /209 1995.0 0.5895 2.6335 14/36 A42912 (Y)LALLPSDAEGPHGQFVTDK 5. 5 /381 1995.1 0.5514 2.4634 13/38 H69373 (L)ALLVLVAPAMAAGNGEDLRN 5. 5 /381 1995.1 0.5514 2.4634 13/38 H69373 (L)ALLVLVAPAMAAGNGEDLRN Differentially expressed proteins vs Cell Surface proteins Secreted proteins Phospho-proteins 188 62 49 38 28 18 14 7 4 Integrated into a specific protein network or pathway Post-database search bioinformatics data validation # Rank/Sp (M+H)+ deltCn C*10^4 Ions Reference Peptide # Rank/Sp (M+H)+ deltCn C*10^4 Ions Reference Peptide --- ------- ------ ------ ------ ---- ---------- ------- --- ------- ------ ------ ------ ---- ---------- ------- 1. 1 / 1 1994.3 0.0000 4.4675 17/26 G3P_RABIT (R)VPTPNVSVVDLTC*R 1. 1 / 1 1994.3 0.0000 4.4675 17/26 G3P_RABIT (R)VPTPNVSVVDLTC*R 2. 2 /403 1995.1 0.6126 2.7366 13/34 SLTRNGL (E)LGKPVLTANQVTIWEGLR 2. 2 /403 1995.1 0.6126 2.7366 13/34 SLTRNGL (E)LGKPVLTANQVTIWEGLR 3. 3 / 3 1995.0 0.5952 2.6591 16/36 FLP_LACCA (N)IANPNVYTETLTAATVCTI 3. 3 / 3 1995.0 0.5952 2.6591 16/36 FLP_LACCA (N)IANPNVYTETLTAATVCTI 4. 4 /209 1995.0 0.5895 2.6335 14/36 A42912 (Y)LALLPSDAEGPHGQFVTDK 4. 4 /209 1995.0 0.5895 2.6335 14/36 A42912 (Y)LALLPSDAEGPHGQFVTDK 5. 5 /381 1995.1 0.5514 2.4634 13/38 H69373 (L)ALLVLVAPAMAAGNGEDLRN 5. 5 /381 1995.1 0.5514 2.4634 13/38 H69373 (L)ALLVLVAPAMAAGNGEDLRN Curated dataset System-specific network Systematic identification of functional proteins Mass spectrometry