Proteomics
description
Transcript of Proteomics
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Proteomics
Astrid BruckmannIBL, Leiden University
Workshop Transcriptomics and Proteomics in Zebrafish,Leiden University,13-22 March 2006
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Why Proteomics?Genome - Transcriptome - Proteome
• several levels of regulation from gene to function• Proteins are the ultimate operating molecules producing the physiological effect
Proteome: the protein complement of a genome
from: Graves and Haystead, 2002
Proteomics: large-scale characterization and functional analysis of the proteins expressed by a genome
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Types of proteomics and their application to biology
from: Graves and Haystead, 2002
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Proteomics - the challenge• The Proteome is: - dynamic - highly complex - relative protein abundances in a cell can differ from 105 up to about 1010
• Proteomics aims to analyze the levels and structure of all proteins present in a cell or a tissue including their post-translational modifications
(Honoré and Østergaard, 2003)
• Proteomics approaches include: 1) protein identification 2) protein quantitation or differential analysis 3) protein-protein interactions 4) post-translational modifications 5) structural proteomics
Proteomics is complementary to transcriptomics and metabolomics, integration of different -omics data should lead to a more complete understandingof biological systems at a molecular level
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Proteomics - the classical definition
- High resolution 2D-PAGE first developed in 1975 (O’Farrell and Klose)- Combination with biological mass spectrometry (1990s)- Availability of genome sequences in databases
central role in proteomic studies
Two-dimensional gelelectrophoresis (2D-PAGE) of cell lysates
generates global patterns of protein expression annotation large-scale visualization of differential protein expression
Mass spectrometry+
Peptide mass fingerprintingfor protein identification
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First dimension: Isoelectrofocusing (IEF)
strip containing a pH gradient immobilized on a gel matrix (Garfin et al. 2000)
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7.59.5
7.5pI 4.5 7.5
7.5 7.5pI 4.59.5
9.5
9.5 9.5pI 4.5
pI 3.5pI 3.5pI 4.57.57.57.57.57.5
9.59.59.59.59.5
Position of proteins after IEF
Position of proteins before IEF
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Second dimension: SDS-PAGE
MW• Proteins enter SDS-Polyacrylamide gel and are dissolved according to their molecular mass
• Postelectrophoretic staining of the proteins with: Coomassie, Silver, Fluorescent stains (SYPRO Ruby)
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2D-PAGE based expression proteomics
• posttranscriptional control mechanisms can influence protein expression
• posttranslational modifications of a protein such as phosphorylation, glycosylation, processing of signal sequences or degradation can be visualized
pIMW
• Protein expression profiling: ~ 1000 proteins routinely detectable in a 2D-gel global changes in the proteome readily detectable
SYPRO Ruby stained gel
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Protein identification by peptide mass fingerprinting
(A) The unknown protein is excised from a gel and converted to peptides by the action of a specific protease. The mass of the peptides produced is then measured in a mass spectrometer.
(B) The mass spectrum of the unknown protein is searched against theoretical mass spectra produced by computer-generated cleavage of proteins in the database.
from Graves and Haystead, 2002
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Mass spectroscopy for protein identification
MALDI-TOFMatrix assisted laser desorption/ionisation time-of-flight
MALDI-TOF spectrum
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Generation of protein expression reference maps
• Link protein information with DNA sequence information from the genome projects, comprehensive 2D-gel databases constructed for different cell types Listed at: WORLD-2DPAGE: http//www.expasy.org/ch2d/2d-index.html
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• 2D-gel electrophoresis combined with mass spectrometry to get qualitative and quantitative protein behavioural data• Most frequently used method in proteome analysis
from: Pandey and Mann, 2000
2D-PAGE based differential expression proteomics
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Workflow of differential expression proteomics• Sample preparation• Isoelectrofocusing (1.dimension)• Equilibration incl. reduction, alkylation• SDS-PAGE (2. dimension)• Staining• Imaging• Spot detection and matching• Normalization and quantification• Analysis • Cutting of selected spots• Trypsin digestion in-gel• Identification with mass spectroscopy• Database comparison
Steps to be practised during the workshop
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2D-PAGE: critical points
• Samples must be run at least in triplicate to rule out effects from gel-to-gel variation → statistics• Standardized procedures needed to obtain a high reproducibility of 2D-gels
Currently possible to run 12 gels in parallel
• Sample preparation as simple as possible
• Isoelectrofocusing conditions (patience)
• Staining: fluorescent stains for high sensitivity and high linear range of detection
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from Kolkman et al. 2005
• Proteins are labeled prior to running the first dimension with up to three different fluorescent cyanide dyes (Unlu et al.1997)
• Allows use of an internal standard in each gel which reduces gel-to-gel variation,
reduces the number of gels to be run
• Adds 500 Da to the protein labelled
• Additional postelectrophoretic staining needed
Difference in-gel 2D-PAGE system (DIGE)
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Limitations and challenges of gel-based approaches
• Dynamic range detectable on 2D-gels: 104, protein expression levels of a cell can vary between 105 (yeast) and even 1010(humans)
enrichment or prefractionation strategies needed to reach less abundant proteins
• Resolution of 2D-gels has its limits use narrow pH range gels and combine
• Protein extraction and solubility during IEF can be a problem for poorly water-soluble proteins e.g. membrane proteins or nuclear proteins
• Challenges for further development in gel-based proteomics: improve sample preparation to be able to analyze extreme proteins
(extremely basic or acidic, extremely small or big, extremely hydrophobic),
sensitivity, dynamic range, automation
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- 100%
- 80%
- 60%
- 40%
- 20%
Proteome coverage
Copies/cell
- 106
- 105
- 104
- 103
- 102
- 101
2D-gel based proteomics: the state-of-the-art versus the challenge
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Other proteomic approaches
• Liquid chromatography coupled to mass spectrometry - Shotgun multidimensional protein identification technology MudPIT (Link et al. 1999) - ICAT: isotope coded affinity tags (Gygi et al. 1999), cysteine biased - iTRAQ (Ross et al. 2004): amine specific labelling of peptides, quantification possible with tandem mass spectroscopy
• Peptide and protein arrays (Lueking et al. 1999)
• Yeast two-hybrid system (Fields and Song, 1989)
• Phage display (Zozulya et al. 1999)
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ICAT for measuring differential protein expression
ICAT consists of a biotin affinity group, a linker region that can incorporate heavy (deuterium) or light (hydrogen)atoms, and a thiol-reactive end group for linkage tocysteines.
Proteins are labeled on cysteine residues with either thelight or heavy form of the ICAT reagent. Protein samplesare mixed and digested with a protease. Peptides labeledwith the ICAT reagent can be purified using avidinchromatography. ICAT-labeled peptides can be analyzed by MS toquantitate the peak ratios and proteins can be identifiedby sequencing the peptides with MS/MS.
from: Graves and Haystead, 2002
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2D-PAGE in functional proteomicsTypical question:
• Identify specific proteins in a cell that undergo changes in abundance, localization, or modification in response to a specific biological condition
• Often combined with complementary techniques (protein biochemistry, molecular biology and cell physiology)
If: - Monitoring quantitative changes in the biological process of interest - Quantitatively looking at protein modifications Then:
2D-gel based proteomics is the method of choice
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Zebrafish samples used for 2D-GE Experiment
• Treatment startet at high oblong stage of development• Samples taken from 70-90% epiboly stage
Phenotypic differencesbetween untreated and treated zebrafish embryos
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Workflow of Differential Expression Proteomics• Sample preparation• Isoelectrofocusing (1.dimension)• Equilibration incl. reduction, alkylation• SDS-PAGE (2. dimension)• Staining• Imaging• Spot detection and matching• Normalization and quantification• Analysis • Cutting of selected spots• Trypsin digestion• Identification with mass spectroscopy• Database comparison
Steps to be practised during the workshop
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2D-Gelelectrophoresis Practical3 different protein samples from: a) untreated embryos b) ethanol treated embryos c) selenium treated embryosExperiment 1:7cm IPG strips 3-9 NL Passive rehydration/loadingper sample 4 replicates, 12 strips run at the same time, has already been done Start with equilibration and proceed to second dimension
Experiment 2:7cm IPG strips 3-9 NL Passive rehydration/loadingper sample 2 replicates, 6 strips to be run7cm IPG strips 7-10 Anodic cup loading to improve resolution of basic proteinsper sample 2 replicates, 6 strips to be runStart with performing first dimension
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2D-gel analysis software practical-Introduction into the PDQuest software package-Demonstration of an comparative analysis of gels from two different sample types (wildtype, mutant)-Practising PDQuest analysis of the gels run during the workshop-Compare:a)control embryos vs. ethanol treated embryosb)control embryos vs. selenium treated embryos
High performance analysis of 2D-gels