Genomics II:The Proteome

Using high-throughput methods to identify proteins and to

understand their function

Protein Complementation

• Enzymatic complementation -galactosidase reconstitution

• Fluorescence complementation– GFP or YFP reconstitution– FRET (fluorescence resonance energy

transfer)

Enzymatic Complementation

Blue=DAPI

Red=BGAL

Blue=DAPI

Green=BGAL

Red=Actin

Bimolecular Fluorescence Complementation

FRET (fluorescence resonance energy transfer)

Summary I

• Goals of proteomics – Identify and ascribe function to proteins under all

biologically plausible conditions

• Proteomics methods– 2-D gel electrophoresis for separating proteins on the

basis of charge and molecular weight– Mass spectrometry for identifying proteins by measuring

the mass-to-charge ratio of their ionized peptide fragments– Protein chips to identify proteins, to detect protein–protein

interactions, to perform biochemical assays, and to study drug–target interactions

Summary II

• Proteomics methods (continued)– Yeast two-hybrid method for studying protein–protein

interactions– Biochemical genomics for high-throughput assays

• Some accomplishments of proteomics– Example: yeast

• Yeast two-hybrid method reveals interactome• Transcriptional regulatory networks deduced• Biochemical genomics uncovers new ORF

functions• Subcellular localization of proteins

Linkage and Mapping

Linkage and Mapping

Linked alleles tend to be inherited together

Crossing over produces new allelic combinations