Proteomics
description
Transcript of Proteomics
Proteomics
Astrid BruckmannIBL, Leiden University
Workshop Transcriptomics and Proteomics in Zebrafish,Leiden University,13-22 March 2006
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
Types of proteomics and their application to biology
from: Graves and Haystead, 2002
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
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
First dimension: Isoelectrofocusing (IEF)
strip containing a pH gradient immobilized on a gel matrix (Garfin et al. 2000)
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
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)
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
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
Mass spectroscopy for protein identification
MALDI-TOFMatrix assisted laser desorption/ionisation time-of-flight
MALDI-TOF spectrum
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
• 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
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
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
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)
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
- 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
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)
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
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
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
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
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
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