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Chapter 8 Proteomics Using high-throughput methods to identify proteins and to understand their function © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Contents Definition of proteomics Proteomics technologies 2-D gel electrophoresis Mass spectrometry Protein chips Yeast two-hybrid method Biochemical genomics Using proteomics to uncover transcriptional networks © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 What is proteomics? An organism’s proteome A catalog of all proteins Expressed throughout life Expressed under all conditions The goals of proteomics To catalog all proteins To understand their functions To understand how they interact with each other © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 The challenges of proteomics Splice variants create an enormous diversity of proteins ~25,000 genes in humans give rise to 200,000 to 2,000,000 different proteins Splice variants may have very diverse functions Proteins expressed in an organism will vary according to age, health, tissue, and environmental stimuli 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 Example: the calcitonin gene Gene variant #1 Protein: calcitonin Function: increases calcium uptake in bones Gene variant #2 Protein: calcitonin gene-related polypeptide Function: causes blood vessels to dilate © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Posttranslational modifications Posttranslational modifications are defined as any changes to the covalent bonds of a protein after it has been fully translated. Proteolytic cleavage Fragmenting protein Addition of chemical groups to one or more amino acids on the protein © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Chemical modifications Phosphorylation: activation and inactivation of enzymes Acetylation: protein stability, used in histones Methylation: regulation of gene expression Acylation: membrane tethering, targeting Glycosylation: cell–cell recognition, signaling GPI anchor: membrane tethering Hydroxyproline: protein stability, ligand interactions Sulfation: protein–protein and ligand interactions Disulfide-bond formation: protein stability Deamidation: protein–protein and ligand interactions Pyroglutamic acid: protein stability Ubiquitination: destruction signal Nitration of tyrosine: inflammation © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Practical applications Comparison of protein expression in diseased and normal tissues Likely to reveal new drug targets Today ~500 drug targets Estimates of possible drug targets: 10,000– 20,000 Protein expression signatures associated with drug toxicity To make clinical trials more efficient To make drug treatments more effective © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Technologies for proteomics 2-D gel electrophoresis (2-dimensional) Separates proteins in a mixture on the basis of their molecular weight and charge Mass spectrometry Reveals identity of proteins based on computer software that can uniquely identify individual proteins Protein chips A wide variety of identification methods structure, biochemical activity, and interactions with other proteins Yeast two-hybrid method Determines how proteins interact with each other Biochemical genomics (Enzymatic) Screens gene products for biochemical activity © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 2-D gel electrophoresis pH gradient along first axis neutralizes charged proteins at different places pH constant on a second axis where proteins are separated by weight Basic Low MW x–y position of proteins on stained gel uniquely identifies the proteins Acidic High MW Polyacrylamide gel Voltage across both axes © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Differential in gel electrophoresis Label protein samples from control and experimental tissues Fluorescent dye #1 for control Fluorescent dye #2 for experimental sample Mix protein samples together 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 Poor performance of 2-D gels for the following: Very large proteins Very small proteins Less abundant proteins Membrane-bound proteins Presumably, the most promising drug targets © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Mass spectrometry Measures mass-tocharge ratio Components of mass spectrometer Ion source Mass analyzer Ion detector 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) With capillary electrophoresis and liquid chromatography Matrix-assisted laser desorption/ionization (MALDI) Extracts ions from sample surface ESI MALDI © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Mass analyzers used for proteomics Ion trap Ion Trap Captures ions on the basis of mass-to-charge ratio Often used with ESI Time of flight (TOF) Time for accelerated ion to reach detector indicates mass-to-charge ratio Frequently used with MALDI Time of Flight Detector Also other possibilities © 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 Preparation of protein sample Extraction from a 2-D gel Digestion by proteases — e.g., trypsin Mass spectrometer measures mass-charge ratio of peptide fragments Identified peptides are compared with database Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in database 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 Stable isotopes used to label proteins under different conditions Variety of labeling methods Enzymatic Metabolic Via chemical reaction 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 Data from a MALDI experiment Distributions of individual proteins in a slice of rat brain Tissue is coated with UV-absorbing matrix MALDI ion source with laser sampling tissue every 180 mm Mass-spectrum peaks reveal individual proteins Image processing for false-color images Sections of rat brain imaged by mass spectrometry © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Limitations of mass spectrometry Not very good at identifying minute quantities of protein Trouble dealing with phosphorylated proteins Doesn’t provide concentrations of proteins Improved software eliminating human mediated analysis is necessary for highthroughput projects Are only able to identify hundreds of proteins in a single day © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Protein chips Thousands of proteins analyzed simultaneously Wide variety of assays Antibody–antigen Enzyme–substrate Protein–small molecule Protein–nucleic acid Protein–protein Protein–lipid Yeast proteins detected using antibodies © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Fabricating protein chips: physical array that can hold proteins, isolate them from each other, and prevent them from becoming denatured Protein substrates: minipads Polyacrylamide Polydimethylsiloxane or agarose gels Glass Nanowells Proteins deposited on chip surface by robots PDMS UV Quartz mask Glass slide © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Protein attachment strategies Diffusion (pads) Protein suspended in random orientation, but presumably active Adsorption/Absorption Diffusion Adsorption/ Absorption Some proteins inactive Covalent attachment Covalent Some proteins inactive Affinity Orientation of protein precisely controlled Affinity antibodies © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Classes of capture molecules Agent that interacts with molecules applied to the chip to carry out some kind of assay Different capture molecules must be used to study different interactions Examples Antibodies (or antigens) for detection Proteins for protein-protein interaction Enzyme-substrate for biochemical function analytical microarrays and functional protein chips Antigen– antibody Protein– protein Aptamers: short peptides Enzyme– substrate Receptor– ligand © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Reading out results Fluorescence Most common method Fluorescent probe or tag Can be read out using standard nucleic acid microarray technology Surface-enhanced laser desorption/ionization (SELDI) Laser ionizes proteins captured by chip Mass spectrometer analyzes peptide fragments Atomic-force microscopy Detects changes in chip surface due to captured proteins © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Difficulties in designing protein chips Unique process is necessary for constructing each probe element Challenging to produce and purify each protein on chip Proteins can be hydrophobic or hydrophilic Difficult to design a chip that can detect both Protein’s function may be dependent on posttranslational modification or an interaction with another biological molecule Challenging and constantly improving with new technological advancements © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Regulation of transcription UE TATA box © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Yeast two-hybrid method Goal: Determine how proteins interact with each other Method Use yeast transcription factors Gene expression requires the following: A DNA-binding domain An activation domain A basic transcription apparatus Attach protein1 to DNA-binding domain (bait) Attach protein2 to activation domain (prey) Reporter gene expressed only if protein1 and protein2 interact with each other © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 A schematic of the yeast two-hybrid method © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Results from a yeast two-hybrid experiment Goal: To characterize protein–protein interactions among 6,144 yeast ORFs 5,345 were successfully cloned into yeast as both bait and prey Identity of ORFs determined by DNA sequencing in hybrid yeast 692 protein–protein interaction pairs Interactions involved 817 ORFs interactome on a genome-wide scale © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Caveats associated with the yeast two-hybrid method There is evidence that other methods may be more sensitive (protein chip) Some inaccuracy reported when compared against known protein–protein interactions False positives False negatives © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Subcellular localization of the yeast proteome Complete genome sequences allow each ORF to be precisely tagged with a reporter molecule Tagged ORF proteins indicate subcellular localization Useful for the following: Correlating to regulatory modules Verifying data on protein–protein interactions Annotating genome sequence © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Attaching a GFP tag to an ORF Marker gene: HIS free medium GFP PCR product HIS3MX6 Homologous recombination Chromosome Fusion protein ORF1 NH2 protein ORF2 GFP COOH © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Location of proteins revealed 75% of yeast proteome localized > 40% of proteins in cytoplasm 67% of proteins were previously un-localized Localizations correlate with transcriptional modules cytoplasm nucleus A protein localized to the nucleus © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Pregenomics biochemical assays Methods used to find genes responsible for specific biochemical activity before the inception of genomics Laboriously purify responsible protein Often expensive and time consuming Expression cloning Introduce cDNA pools into cells Look for biochemical activity in those cells Caveat: Often difficult to detect biochemical activity in cell’s biochemical “background” © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Biochemical genomics Genome of an organism is already known Approach Construct plasmids for all ORFs Attach ORFs to sequence that will facilitate purification Transform cells Isolate ORF products Test for biochemical activity © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Biochemical genomics in yeast 6,144 ORF yeast strains made ORFs fused to glutathione S-transferase (GST) for purification purposes Biochemical assay revealed three new biochemical reactions associated with yeast ORFs tRNA ligase pre-tRNA Ligated tRNA tRNA halves Abc1 35 64 Used as screening pools © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Microfluidics Proteomics requires greater automation Microfluidics: a “lab on a chip” Microvalves and pumps allow control of nanoliter amounts Can control biochemical reactions A microfluidics chip © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Microfluidics in action loading mixing compartmentalization purging 500 mm © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Finding transcription-factor targets All yeast transcription factors were used to make yeast strains Use chromatin immunoprecipitation to select factors attached to promoter regions on DNA (ChIP) DNA fragments used on microarray to identify transcription-factor targets (ChIP on chip) antibodies Genomic DNA Over 106 TFs of 141 are active © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Network motifs for transcriptional regulation Autoregulation Multicomponent loop Multi-input motif TF Motif Regulator chain © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Studying proteins with posttranslational modifications Example: tyrosine phosphorylation Traditional method Radioactive labeling with 32P followed by gel electrophoresis or chromatography Problems using mass spectrometry are being overcome to allow high-throughput analysis Better purification techniques Mass spectrometers more capable of detecting phosphorylated peptides © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 HUPO Human Proteome Organization (HUPO) was established in 2002 Mission Consolidate proteomics organizations in different countries into single worldwide body Scientific and educational programs to help spread proteomics knowledge and technology Coordination of public proteomics initiatives Examples of current initiatives Human Liver Proteome Project Human Plasma Proteome Project © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Future prospects The next decade may see the complete deciphering of the proteome of yeast More initiatives, like the Human Liver Proteome Project, are underway Better understanding of disease © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 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 © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Summary III Future prospects Better technology for studying posttranslational modifications ~10 years for completion of yeast proteome? © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458