Plant Proteomics IB 474A/CPSC 468A

Steve Huber197 ERML

265-0909

schuber1@illinois.edu

Lecture 1 Intro and Overview• Definition of proteomics

• Student Expectations Survey

• Housekeeping and course objectives

• Why study proteomics?

Science 291 (2001) 1221.

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?

Peck SC (2005) Update on Proteomics in Arabidopsis. Where do we go from here? Plant Physiol 138: 591-599

Major Proteomics Directions

Adapted from Human Proteome Organization (www.HUPO.org)

PTMs

Required Reading for Lecture 2: Rose et al.(2004) Plant J 39: 715-733.

“Proteomics is an increasingly ambiguous term being applied to almost any aspect of protein expression, structure or function.”

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.

Lec 1 Intro and OverviewLec 2 2-Dimensional Electrophoresis Lec 3 Quantitative proteomics (Prof. Yau) Lec 4 UIUC Proteomics Facility tour—meet in 307 NoyesLec 5 Top-Down Mass spec and IGB tourLec 6 Post-translational modifications (PTMs): Phosphorylation Lec 7 PTMs-continuedLec 8 Protein-Protein InteractionsLec 9 Pro-Pro continuedLec 10 Proteomes of organs and subcell compartments

Lec 11 Abiotic stressLec 12 Biotic stressLec 13 Hormone signaling Lec 14 Hot topics; miscellaneous; student presentations?

Lec 15 In-class FINAL EXAM

Overview of lectures—Major Topics

FOCUS IS ON EXPERIMENTAL AND STRATEGIC CONSIDERATIONS RATHER THAN INSTRUMENTATION

COURSE OBJECTIVES

1. Appreciate fundamentals of proteomic research

2. Understand protein abundance/PTM in relation to development, nutrition, stress, etc.

3. Enhance presentation skills: Critically evaluate (and present) a current proteomics paper.

GRADING20% 2 homework problem sets30% 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 participation100%

ATTENDANCE IS EXPECTED; CHEATING AND PLAGIARISM WILL NOT BE TOLERATED

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%

I. Present background and goals of study Background clearly presented 10 pointsGoals elaborated 5 points

II. Describe approaches and methodology Clearly explain principles underlying the work 10 points

Identify weaknesses and strengths 10 pointsDescribe general applicability of methods 10 points

III. Identify major conclusionsConclusions concisely elaborated 10 points Identify any questionable points of interpretation 10 points Identify any unresolved points 10 points

IV. Why the proteomics approach? 10 pointsExplain unique benefit from the proteomics approach

Journal Article Evaluation Paper Rubric

V. 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

(5 page maximum; double spaced)

RESOURCESOptional 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

Peck (2005) Plant Physiol 138: 591

Targeting sequence

Principle:One gene ≠ one transcript ≠ one protein

ONE Genome but MANY Proteomes!

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

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.

We will discuss AGPase (ADPglucose pyrophosphorylase) and NiA (nitrate reductase) later in the course.

Proteomics Leads to New Biology:

1. Identify and quantitate post-translational modifications (e.g.,O-acetylation; O-glycosylation).

2.Determine localization of proteins

3. Identify signal transduction components (e.g., BSK1; PPDK-RP)

4.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.