Plant Proteomics IB 474A/CPSC 468A Steve Huber 197 ERML 265-0909 schuber1@illinois.edu Lecture 1 Intro and Overview Definition of proteomics Student Expectations Survey Housekeeping and course objectives Why study proteomics?

WHAT IS PROTEOMICS? The analysis of complete complements of proteins: identification and quantification; modifications; interactions; and activities. FUNCTION. AND HOW DO THESE CHANGE DURING A BIOLOGICAL RESPONSE? Science 291 (2001) 1221. Peck SC (2005) Update on Proteomics in Arabidopsis. Where do we go from here? Plant Physiol 138: 591-599

Major Proteomics Directions PTMs “Proteomics is an increasingly ambiguous term being applied to almost any aspect of protein expression, structure or function.” Adapted from Human Proteome Organization (www.HUPO.org) Required Reading for Lecture 2: Rose et al.(2004) Plant J 39: 715-733.

Tools of Functional Genomics Colebatch et al (2002) Functional Genomics: tools of the trade. New Phytol 153: 27-36.

Solving the Puzzle of Protein Function Proteomics is a multipotent tool central to research efforts in many fields and disciplines. Maximum functional utility will come from joint efforts.

Overview of lectures—Major Topics Lec 1 Intro and Overview Lec 2 2-Dimensional Electrophoresis Lec 3 Quantitative proteomics (Prof. Yau) Lec 4 UIUC Proteomics Facility tour—meet in 307 Noyes Lec 5 Top-Down Mass spec and IGB tour Lec 6 Post-translational modifications (PTMs): Phosphorylation Lec 7 PTMs-continued Lec 8 Protein-Protein Interactions Lec 9 Pro-Pro continued Lec 10 Proteomes of organs and subcell compartments Lec 11 Abiotic stress Lec 12 Biotic stress Lec 13 Hormone signaling Lec 14 Hot topics; miscellaneous; student presentations? Lec 15 In-class FINAL EXAM FOCUS IS ON EXPERIMENTAL AND STRATEGIC CONSIDERATIONS RATHER THAN INSTRUMENTATION

COURSE OBJECTIVES Appreciate fundamentals of proteomic research Understand protein abundance/PTM in relation to development, nutrition, stress, etc. Enhance presentation skills: Critically evaluate (and present) a current proteomics paper.

GRADING 20% 2 homework problem sets 30% Assigned reading (4 in-class quizzes; pick top 3 of 4 scores) 20% Written essay (journal article evaluation); ‘News & Views’ style with section on application to another significant question. 20% Final exam (last class) 10% Classroom participation 100% Class Presentation; 15 min (same article used for written evaluation); submit PowerPoint slides for distribution to the class before the talk. Instructor and students will grade talks. 10% ATTENDANCE IS EXPECTED; CHEATING AND PLAGIARISM WILL NOT BE TOLERATED

Journal Article Evaluation Paper Rubric (5 page maximum; double spaced) I. Present background and goals of study Background clearly presented 10 points Goals elaborated 5 points II. Describe approaches and methodology Clearly explain principles underlying the work 10 points Identify weaknesses and strengths 10 points Describe general applicability of methods 10 points III. Identify major conclusions Conclusions concisely elaborated 10 points Identify any questionable points of interpretation 10 points Identify any unresolved points 10 points IV. Why the proteomics approach? 10 points Explain unique benefit from the proteomics approach How could this work (concept; technique; approach, etc) be applied to answer another significant biological question? (be as specific as possible; ≤ 1 page) 15 points

RESOURCES Optional textbooks: Introduction to Proteomics. Tools for the New Biology. Daniel C. Liebler (2002) Humana Press, ISBN 0-89603-991-9 Proteomics in Practice. A laboratory manual of proteome analysis. R. Westermeier and T. Naven (2002) Wiley-VCH, ISBN 3-527-30300-6 CURRENT LITERATURE AND WEBSITES ‘Protemics Primer’ (www.spectroscopynow.com) Assigned Reading for Lecture 2: Rose et al. (2004) Tackling the plant proteome: practical approaches, hurdles and experimental tools. Plant J. 39: 715-733.

A Single Gene Can Produce Many Proteins Principle: One gene ≠ one transcript ≠ one protein ONE Genome but MANY Proteomes! Targeting sequence Peck (2005) Plant Physiol 138: 591

Correlation between protein and mRNA in yeast Gygi et al. (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19: 1720-1730.

Diurnal changes of transcript and enzyme activity in Arabidopsis We will discuss AGPase (ADPglucose pyrophosphorylase) and NiA (nitrate reductase) later in the course. Gibon et al. (2004) A robot-based platform to measure multiple enzyme activities in Arabidopsis using a set of cycling assays: comparisons of changes of enzyme activities and transcript levels during diurnal cycles and in prolonged darkness. Plant Cell 16: 3304-3325.

Proteomics Leads to New Biology: Identify and quantitate post-translational modifications (e.g.,O-acetylation; O-glycosylation). Determine localization of proteins Identify signal transduction components (e.g., BSK1; PPDK-RP) Understand plant responses (e.g., to stress; genotypic differences; etc) Anything that can’t be predicted from the genome, or when responses are not controlled transcriptionally.