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