Proteomics

1
Proteomics
Astrid Bruckmann IBL, Leiden University
Workshop Transcriptomics and Proteomics in
Zebrafish, Leiden University,13-22 March 2006
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Why Proteomics?
Genome - Transcriptome
- Proteome
from Graves and Haystead, 2002

• 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

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
understanding of biological systems at a
molecular level
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Proteomics - the classical definition
Two-dimensional gelelectrophoresis (2D-PAGE) of
cell lysates
Mass spectrometry

generates global patterns of protein expression
? annotation
?
large-scale visualization of differential
protein expression
Peptide mass fingerprinting for protein
identification

• - High resolution 2D-PAGE first developed in 1975
  (OFarrell and Klose)
• - Combination with biological mass spectrometry
  (1990s)
• - Availability of genome sequences in databases
• ? central role
  in proteomic studies

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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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Position of proteins before IEF
Position of proteins after 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

• Protein expression profiling 1000 proteins
  routinely detectable in a
• 2D-gel ? global changes in the proteome
  readily detectable

• 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

pI
MW
SYPRO Ruby stained gel
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Protein identification by peptide mass
fingerprinting
from Graves and Haystead, 2002

• 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.

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Mass spectroscopy for protein identification
MALDI-TOF spectrum
MALDI-TOF
Matrix assisted laser desorption/ionisation
time-of-flight
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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-inde
  x.html

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2D-PAGE based differential expression proteomics

• 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
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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

• Sample preparation as
• simple as possible
• Isoelectrofocusing conditions
• (patience)
• Staining fluorescent stains
• for high sensitivity and high
• linear range of detection

Currently possible to run 12 gels in parallel
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Difference in-gel 2D-PAGE system (DIGE)

• 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

from Kolkman et al. 2005
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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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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
  to
• cysteines.
• Proteins are labeled on cysteine residues with
  either the
• light or heavy form of the ICAT reagent. Protein
  samples
• are mixed and digested with a protease. Peptides
  labeled
• with the ICAT reagent can be purified using
  avidin
• chromatography.
• ICAT-labeled peptides can be analyzed by MS to
• quantitate the peak ratios and proteins can be
  identified
• by sequencing the peptides with MS/MS.

from Graves and Haystead, 2002
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2D-PAGE in functional proteomics

• Typical 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
Phenotypic differences between untreated and
treated zebrafish embryos

• Treatment startet at high oblong stage of
  development
• Samples taken from 70-90 epiboly stage

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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 Practical
3 different protein samples from a) untreated
embryos
b) ethanol treated embryos
c)
selenium treated embryos Experiment 1 7cm IPG
strips 3-9 NL Passive rehydration/loading per
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/loading per sample 2
replicates, 6 strips to be run 7cm IPG strips
7-10 Anodic cup loading to improve resolution
of basic proteins per sample 2 replicates, 6
strips to be run ?Start 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 embryos
• b)control embryos vs.
• selenium treated embryos