9/14/20151 EECS 730 Introduction to Bioinformatics Introduction to Proteomics Luke Huan Electrical Engineering and Computer Science

9/14/20152 Proteome: Protein complement of a genome Time- and cell- specific protein complement of the genome. Encompasses all proteins expressed in a cell at one time, including isoforms and post-translational modifications.

9/14/20153 Proteome Contrast to genome The genome is constant for one cell and identical for all cells of an organism, and does not change very much within a species The proteome is very dynamic with time and in response to external factors, and differs substantially between cell types. Variable In different cell and tissue types in same organism In different growth and developmental stages of organism Dynamic Depends on response of genome to environmental factors Disease state Drug challenge Growth conditions Stress

9/14/20154 Introduction to proteomics Proteomics is the study of total protein complements, proteomes, e.g. from a given tissue or cell type. Don’t forget that the proteome is dynamic, changing to reflect the environment that the cell is in Definitions Classical - restricted to large scale analysis of gene products involving only proteins Inclusive - combination of protein studies with analyses that have genetic components such as mRNA, genomics, and yeast two- hybrid Examples of important proteomic questions: 1) What proteins are present? 2) What other proteins does a particular protein interact with (networks)? 3) What does a particular protein look like (structure)?

9/14/20155 Genomics vs. proteomics Genomics has provided spectacular amounts of data, but most of it remains uninterpretable at our current level of understanding. In some ways, genomics raises more questions than it answers. The emerging field of proteomics promises to answer some of those questions by systematically studying all of the proteins encoded by the genome.

9/14/ gene is no longer equal to one protein In fact, the definition of a gene is debatable. (ORF, promoter, pseudogene, gene product, etc) 1 gene = how many proteins? There are only 30,000 genes in the human genome, yet there are more than 100,000 proteins in the human proteome. Actually, cataloguing the human proteome requires much more than just 100K proteins. 30,000 genes x myriad of modifications >> 100K protein forms! Modifications include: alternate RNA splicing, chemical modifications, cleavage Chemical modifications include: phosphorylation, acetylation, glycosylation, and many more. 1 gene = 1 protein?

9/14/20157 Why proteomics? Annotation of genomes, i.e. functional annotation Genome + proteome = annotation Protein Function Protein Post-Translational Modification Protein Localization and Compartmentalization Protein-Protein Interactions Protein Expression Studies Differential gene expression is not the answer

9/14/20158 Microarray data doesn ’ t correlate perfectly with protein expression levels Analysis of mRNA transcripts with microarray has provided dynamic information regarding which genes are expressed in cells under a given set of experimental conditions, yielding clues as to which proteins are involved in certain pathways and disease states. However, differences in the half-lives of RNA and proteins, as well as post-translational modifications important to protein function prevent mRNA profiles from being perfectly correlated to the cells’ actual protein profiles.

9/14/20159 Introduction to proteomics Composition of the proteome depends on cell type, developmental phase and conditions Proteome analyses are still struggling to solve the ”basic proteome” of different cells and tissues or limited changes under changing conditions or during processes Current methods can only ”see” the most abundant proteins

9/14/ Types of proteomics Protein Expression Quantitative study of protein expression between samples that differ by some variable Structural Proteomics Goal is to map out the 3-D structure of proteins and protein complexes Functional Proteomics To study protein-protein interaction, 3-D structures, cellular localization and PTMS in order to understand the physiological function of the whole set of proteome.

9/14/ Large-scale protein analysis 2D protein gels Yeast two-hybrid Rosetta Stone approach Pathways

9/14/ D protein electrophoresis and mass spectrometry

9/14/ Two-dimensional protein gels First dimension: isoelectric focusing Electrophorese ampholytes to establish a pH gradient Can use a pre-made strip Proteins migrate to their isoelectric point (pI) then stop (net charge is zero) Range of pI typically 4-9 (5-8 most common)

9/14/ Two-dimensional protein gels Second dimension: SDS-PAGE Electrophorese proteins through an acrylamide matrix Proteins are charged and migrate through an electric field v = Eq / d6  r  Conditions are denaturing Can resolve hundreds to thousands of proteins

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9/14/ Proteins identified on 2D gels (IEF/SDS-PAGE) Protein mass analysis by MALDI-TOF -- done at core facilities -- often detect posttranslational modifications -- matrix assisted laser desorption/ionization time-of-flight spectroscopy

9/14/ Evaluation of 2D gels (IEF/SDS-PAGE) Advantages: Visualize hundreds to thousands of proteins Improved identification of protein spots Disadvantages: Limited number of samples can be processed Mostly abundant proteins visualized Technically difficult Labor-intensive, not really ”high-throughput” methods

9/14/ Yeast-Two-hybrid (Y2H) Aim: Identify pairs of physical interactions among proteins. Solution: Use the transcription mechanism of the cell

9/14/ Yeast-two-hybrid: Principles Recap of biology: Protein vs. domain A protein is composed of modules or domains Domains are individually folded units within the same protein chain. The presence of multiple domains in a protein allow the protein to perform different functions. The central dogma of biology d1d1 d2d2 d3d3 p1p1 d4d4 d5d5 p2p2 TRANSCRIPTION DNA RNA TRANSLATION PROTEIN

9/14/ Yeast-two-hybrid: Principles Normal transcription requires both the DNA-binding domain (BD) and the activation domain (AD) of a transcriptional activator (TA). Transcriptional activator (TA) Protein that is required to activate transcription A DNA-binding domain (BD): binding to DNA, An activation domain (AD): activating transcription of the DNA

9/14/ Yeast-two-hybrid: Principles The binding domain and the activation domain do not necessarily have to be on the same protein. In fact, a protein with a DNA binding domain can activate transcription when simply bound to another protein containing an activation domain this principle forms the basis for the yeast two-hybrid technique

9/14/ Major components of a Yeast-two-hybrid experiment: Bait protein – the protein of interest (X): with a DNA binding domain attached to its N-terminus Prey protein – its potential binding partner (Y): fused to an activation domain A reporter gene (R): a gene whose protein product can be easily detected and measured Yeast-two-hybrid: Principles Protein X interacts with protein Y X and Y form a functional transcriptional activator the reporter gene is transcribed Use the reporter produced as a measure of interaction between X and Y

9/14/ Yeast two-hybrid transcription The yeast two-hybrid technique measures protein-protein interactions by measuring transcription of a reporter gene. If protein X and protein Y interact, then their DNA-binding domain and activation domain will combine to form a functional transcriptional activator (TA). The TA will then proceed to transcribe the reporter gene that is paired with its promoter.

9/14/ Yeast two-hybrid screens Screen a library of proteins for potential binding partner Identifying interacting proteins in a pairwise fashion Feasible at a large scale (genome scale) Z A bait prey Reporter Gene Bait Protein Binding Domain Prey Protein Activation Domain Bait-prey model

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9/14/ red = cellular role & subcellular localization of interacting proteins are identical; blue = localiations are identical; green = cellular roles are identical

9/14/ Y2H Identify proteins that are physically associated in vivo. Use yeast S. cerevisiae as a host Disadvantage The fused proteins must be able to fold correctly and exist as a stable protein inside the yeast cells Advantage Yeast is closer to higher eukaryotics than in vitro experiments or those systems based on bacterial hosts Weak and transient interactions Often the most interesting in signaling cascades Are more readily detected in two-hybrid since the reporter gene strategy results in a significant amplification. Always a trade-off between the identification of weak interactions and the number of false positives

9/14/ <4% Low overlap among independent experiments Uetz et al Ito et al proteins <23% Uetz et al Ito et al interactions High false positives and false negatives in yeast-two hybrid data Two sets of independent experiments Ito et al PNAS 1999 Uetz et al Nature 2000

9/14/ False positives Proteins with transcription activation activity (bait works by itself) Proteins that normally never see each other (e.g. due to the time/space constraints) are expressed together and may be sticky Proteins are expressed at high levels and this promotes promiscuous interaction Another protein bridges the two interacting partners

9/14/ False negatives Proteins become toxic upon expression in yeast Proteins are toxic when expressed and targeted into the yeast nucleus. Proteins proteolyse essential yeast proteins or proteins essential for the system like the DNA binding domain or the activation domain. Proteins don’t get into the nucleus (membrane protein esp.) Proteins are not modified correctly in heterologous environment

9/14/ Final Remark on Y2H Although the outcome of a screening often results in many new hypotheses, they still need to be validated by other techniques. There is enough reason to remain sceptic about two- hybrid screenings but the most convincing argument in favor of the two-hybrid is the number and speed Referred to as functional screens Interacting proteins might give a functional hint if at least one of the partners has a known functional commitment in a well understood signaling pathway.

9/14/ Analysis of protein complexes Aim: Identification of complexes and their sub units. Solution: a two step method Isolation of only relevant complexes Identification of complex units.

9/14/ Affinity chromatography/mass spec Major methods High throughput mass spectrometric protein complex identification (HMSPCI) Tandem affinity purification (TAP) Again, bait – prey model Very sensitive method Identify multi-protein complexes Not really possible in yeast two-hybrid

9/14/ Methods 1. Attach tags to bait proteins Introduce DNA encoding these into cells Cells express modified proteins Proteins form complexes with other proteins in vivo Cells have to express modified protein properly Tag can interfere with protein folding and function Overexpressed protein may be toxic to cell Kumar and Snyder, 2002

9/14/ Methods 2. Bait proteins and associated proteins are precipitated on an affinity column Tag sticks to column along with protein complex Elute other proteins Elute tagged protein 3. Resolve proteins on an SDS- PAGE gel Separate by charge & weight 4. Cut out protein bands Proteins of same size will be in same band 5. Digest protein bands with trypsin Results in segments of proteins

9/14/ Methods Mass spectrometry to analyze protein composition: 6. Samples are vaporized and ionized 7. Ions enter mass analyzer and are separated by mass to charge ratio 8. Ions are detected and a signal generated 9. Compare signal to database to identify proteins in complex

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9/14/ Affinity chromatography/mass spec Data on complexes deposited in databases

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9/14/ Affinity chromatography/mass spec False positives: sticky proteins Bait protein GST

9/14/ Affinity chromatography/mass spec False negatives: Bait must be properly localized and in its native condition Affinity tag may interfere with function Transient protein interactions may be missed Highly specific physiological conditions may be required Bias against hydrophobic, and small proteins Bait protein GST

9/14/ The Rosetta Stone approach Marcotte et al. (1999) and other groups hypothesized that some pairs of interacting proteins are encoded by two genes in many genomes, but occasionally they are fused into a single gene. By scanning many genomes for examples of “fused genes,” several thousand protein-protein predictions have been made.

9/14/ Yeast topoisomerase II E. coli gyrase B E. coli gyrase A Fig Page 256 The Rosetta Stone approach

9/14/ Function Prediction from Interaction It is possible to deduct functions of a protein through the functions of its interaction partners. A difficult task: Within-class, cross-class interactions Available methods based on protein interaction Neighboring counting method Methods based on χ 2 -statistics Markov Random Fields Simulated annealing