Finish up array applications Move on to proteomics Protein microarrays

Applications of DNA microarrays Monitor gene expression –Study regulatory networks –Drug discovery - mechanism of action –Diagnostics - tumor diagnosis –etc. Genomic DNA hybridizations –Explore microbial diversity –Whole genome comparisons - genome evolution –Identify DNA binding sites –Diagnostics - tumor diagnosis ?

Identification of DNA regions bound by a protein. Compare a wild-type strain to a ∆gene (DNA-binding protein). Do not need any prior knowledge of the sequence the protein binds. Iyer et al Nature, 409:

Identifying replication origins in yeast Only 5% of the genome previously screened for replication origins. Used known replication initiation factors to perform ChIP/chip analysis Identified hundreds of additional replication origins in a single experiment.

DNA diagnostics Uses of microarrays is cancer research and diagnosis. –2733 papers published on microarrays and cancer –1038 papers published on microarrays, gene expression, cancer diagnosis –0 since 1997 Gene expression profiling –Identify genes involved in cancer diagnosis. –Identify gene expression patterns that are associated with disease outcome. Gene content analysis –Identify genomic regions that are lost or amplified in tumors.

Gene expression and cancer Hierarchical clustering –Method for analyzing microarray data –Gene level analysis –Experiment level analysis

Vant Veer et al Nature

Why study proteins? They are the machines that make cells function. RNA levels do not always accurately predict protein levels. –Often processes are regulated at the transcriptional level. –Some processes are controlled post- transcriptionally. Most often proteins are the targets of drugs.

Proteomics -large scale analysis of proteins Protein levels - Determining the abundance of proteins in a sample. –2D gel electrophoresis, mass spectrometry, protein microarrays Interacting proteins - determining which proteins come together to form functional complexes. –Yeast 2-hybrid, affinity purification Subcellular localization - site of localization can often provide clues to the function of a protein. –GFP tagging, immunofluorescence microscopy. Protein activity - investigating the biochemical activities of proteins. Structural genomics - high-throughput analysis of the protein structure

From

Proteins Primary structure - sequence –Searching databases –Identifying functional domains Secondary and tertiary structure - 3D folding of proteins. –Proteins have unique 3D structures –Identify functional domains –VAST - online structural tool from NCBI

Western Blot Determine the presence and level of a protein in a cell lysate. ml - review of Northern, Western, and Southern blots. ml

Monitoring protein levels - large scale 2D gel electrophoresis –Old technology - not as useful for lowly expressed proteins. Mass spectrometry –Many new techniques for protein detection and quantitation being developed. Protein microarrays Many developing technologies

Protein microarrays Analysis of thousands of proteins at one time. Many different types –Antibody arrayed - detect many proteins –Proteins arrayed - detect interacting proteins –Proteins arrayed - detect interacting small molecules –Etc.

Templin et al Trend in Biotch. Vol 20

Protein:protein interactions

Protein activity arrays

Small molecule arrays

Why bother with DNA microarrays? Protein microarrays are not as robust –DNA is DNA - all features will behave similarly under single hybridization conditions. –Proteins are unique - will behave differently. Protein microarrays are costly –$ per antibody –$10 per oligo Used for different purposes