Lecture 27 – Proteomics Based on chapter 9 Functional and Comparative Genomics and web materials Copyright © 2010 Pearson Education Inc.

Contents 1.Definition & Goals of proteomics 2.Proteomics technologies a.2-D gel electrophoresis b.Mass spectrometry c.Protein chips d.Yeast two-hybrid method e.Protein localization 3.Using proteomics to uncover transcriptional networks © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

What is proteomics? 1.A catalog of all proteins expressed throughout the life cycle of the organism. 2.A catalog of all proteins expressed under all conditions in an organism. 3.A catalog of all proteins expressed in all tissues of an organism. 4.Both 1 and 2 5.Both 2 and 3 6.Both 1 and 3 7.All of the above

Which of the following is (are) a goal(s) of proteomics? 1.To catalog all genes in an organism 2.To understand the function of all proteins in an organism 3.To understand how proteins of an organims interact with each other. 4.Both 1 and 2 5.Both 1 and 3 6.Both 2 and 3 7.All of the above

The challenges of proteomics 1.Splice variants create an enormous diversity of proteins a.~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins b.Splice variants may have very diverse functions 2.Proteins expressed in an organism will vary according to age, health, tissue, and environmental stimuli 3.Proteomics requires a broader range of technologies than genomics © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Diversity of function in splice variants 1.Example: the calcitonin gene (Review of eukaryotic transcription regulation) a.Gene variant #1 i.Protein: calcitonin ii.Function: increases calcium uptake in bones b.Gene variant #2 i.Protein: calcitonin gene-related polypeptide ii.Function: causes blood vessels to dilate © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Posttranslational modifications 1.Proteolytic cleavage a.Fragmenting protein b.Examples a.Insulin b.Trypsin c.See previous slide 2.Addition of chemical groups © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Chemical modifications 1.Phosphorylation: activation and inactivation of enzymes 2.Acetylation: protein stability, used in histones 3.Methylation: regulation of gene expression 4.Acylation: membrane tethering, targeting 5.Glycosylation: cell–cell recognition, signaling 6. Hydroxyproline: protein stability, ligand interactions 7.Ubiquitination: destruction signal 8.Others i.Sulfation: protein–protein and ligand interactions ii.Disulfide-bond formation: protein stability iii.Deamidation: protein–protein and ligand interactions iv.Pyroglutamic acid: protein stability v.GPI anchor: membrane tethering vi.Nitration of tyrosine: inflammation © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Practical applications of Proteomics 1.Comparison of protein expression in diseased and normal tissues a.Likely to reveal new drug targets i.Today ~500 drug targets ii.Estimates of possible drug targets: 10,000– 20,000 2.Protein expression signatures associated with drug toxicity a.To make clinical trials more efficient b.To make drug treatments more effective © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Technologies for proteomics 1.2-D gel electrophoresis a.Separates proteins in a mixture on the basis of their molecular weight and charge 2.Mass spectrometry a.Reveals identity of proteins 3.Protein chips a.A wide variety of identification methods 4.Yeast two-hybrid method a.Determines how proteins interact with each other 5.Biochemical genomics a.Screens gene products for biochemical activity © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

2-D gel electrophoresis 1.Polyacrylamide gel 2.Voltage across both axes a.pH gradient along first axis neutralizes charged proteins at different places b.pH constant on a second axis where proteins are separated by weight 3.x–y position of proteins on stained gel uniquely identifies the proteins BasicAcidic High MW Low MW © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Differential in gel electrophoresis 1.Label protein samples from control and experimental tissues a.Cy3 for control b.Cy5 for experimental sample 2.Mix protein samples together 3.Identify identical proteins from different samples by dye color with benzoic acid Cy3 without benzoic acid Cy5 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Caveats associated with 2-D gels 1.Poor performance of 2-D gels for the following: a.Very large proteins b.Very small proteins c.Less abundant proteins d.Membrane-bound proteins 2.Presumably, the most promising drug targets © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Mass spectrometry 1.Measures mass-to- charge ratio 2.Components of mass spectrometer 1.Ion source 2.Mass analyzer 3.Ion detector 4.Data acquisition unit A mass spectrometer © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Ion sources used for proteomics Proteomics requires specialized ion sources  Electrospray Ionization (ESI)  Matrix-assisted laser desorption/ionization (MALDI) ESI MALDI © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Mass analyzers used for proteomics Detection methods  Ion trap  Time of flight (TOF) Ion Trap Time of Flight Detector © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

A mass spectrum © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Identifying proteins with mass spectrometry 1.Preparation of protein sample a.Extraction from a gel b.Digestion by proteases — e.g., trypsin 2.Mass spectrometer measures mass-charge ratio of peptide fragments 3.Identified peptides are compared with database a.Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database b.Match of experimental readout to database PMF allows researchers to identify the protein © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Stable-isotope protein labeling 1.Stable isotopes used to label proteins under different conditions 2.Variety of labeling methods a.Enzymatic b.Metabolic c.Via chemical reaction 3.Relative abundance of labeled and nonlabeled proteins measured in mass spectrum © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Limitations of mass spectrometry 1.Not very good at identifying minute quantities of protein 2.Trouble dealing with phosphorylated proteins 3.Doesn’t provide concentrations of proteins 4.Improved software eliminating human analysis is necessary for high-throughput projects © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Protein chips 1.Thousands of proteins analyzed simultaneously 2.Wide variety of assays a.Antibody–antigen b.Enzyme–substrate c.Protein–small molecule d.Protein–nucleic acid e.Protein–protein f.Protein–lipid Yeast proteins detected using antibodies © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Fabricating protein chips 1.Protein substrates a.Polyacrylamide or agarose gels b.Glass c.Nanowells 2.Proteins deposited on chip surface by robots © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Difficulties in designing protein chips 1.Unique process is necessary for constructing each probe element 2.Challenging to produce and purify each protein on chip 3.Proteins can be hydrophobic or hydrophilic a.Difficult to design a chip that can detect both © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Yeast two-hybrid method 1.Goal: Determine how proteins interact with each other 2.Method a.Use yeast transcription factors b.Gene expression requires the following: i.A DNA-binding domain ii.An activation domain iii.A basic transcription apparatus c.Attach protein 1 to DNA-binding domain (bait) d.Attach protein 2 to activation domain (prey) e.Reporter gene expressed only if protein 1 and protein 2 interact with each other © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Yeast two-hybrid method Reporter Gene

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Yeast two-hybrid method

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey Yeast two-hybrid method

Subcellular localization of the yeast proteome 1.Complete genome sequences allow each ORF to be precisely tagged with a reporter molecule 2.Tagged ORF proteins indicate subcellular localization a.Useful for the following: i.Correlating to regulatory modules ii.Verifying data on protein–protein interactions iii.Annotating genome sequence © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Attaching a GFP tag to an ORF Fusion protein Chromosome PCR product COOH NH 2 Homologous recombination GFP HIS3MX6 ORF1 ORF2 protein GFP © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

Location of proteins revealed 1.75% of yeast proteome localized a.> 40% of proteins in cytoplasm 2.67% of proteins were previously unlocalized 3.Localizations correlate with transcriptional modules A protein localized to the nucleus nucleus cytoplasm © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458