25 and 27 February, 2004

Chapter 6C

Proteomics

Structural and Functional Characterization in the Post-

genomic era.

Overview• Proteomics examines the collection of proteins present in a

given type of cell at a given time.• Proteomics depends on genome sequences, but differs in

the sense that the genome is static but the proteome changes over time.

• Global analysis of proteomes involves measuring a large number of phenotypes or concentrations simultaneously.

• The link between structure and function is tight, but not well understood.

• Interactions between proteins form networks of functionality. Defining these networks is the most challenging goal of proteomics.

Pathway and localization information are available for the mTn mutant sets.

Uptag/Downtag deletion strategy generates unique tags at deletion site. Unique tags are flanked by sites for PCR amplification.

Phenotype assignment using tagged deletion strategy

Make a microarray containing UPTAG / DOWNTAG sequence. Each strain hybridizes to only one place on the microarray.

–Grow mixtures of hundreds of strains in the same flask under different conditions.

•Measure which strains grew by intensity of unique microarray hybridization of the PCR amplification of tags from all the strains present in the flask before and after growth.

–Time zero = red, 6 hours later = green–No difference --> Yellow, Defect = Red

RNAi• Small dsRNA complementary to a gene will make

expression of the protein product of that gene impossible.

• Allows conditional knockouts in adult eukaryotes, especially C. elegans, where RNAi works even if the small dsRNA is eaten.

• RNAi is one of the most significant advances in functional genetics.

• New - first reported in 1998, just beginnning to be applied.

A high-throughput method to determine three dimensional crystal structure and relate it to function is a long-term goal of proteomics: an example from the Archaea

Aquaporin structure and function

Protein interactions• Hard to detect since the rules are totally opaque

– Interaction depends on structure in complex ways.– Empirical evidence is required.

• Coimmunoprecipitation• High-throughput strategies

– Protein microarrays– 2-hybrid interaction trap

See Math Minute 6.2 for a discussion of graph theory.

Which proteins are present? Two-dimensional gel electrophoresis.

Problems:Sample prep.Spot detectionQuantificationIdentification

Assignment: Discovery Questions 1, 2, 5 ,6 ,9-13, 19, 20, 22-27, 35-39