Introduction to Proteomics 2D gels and

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Introduction to Proteomics 2D gels and sample
preparation methods Susan Liddell University
of Nottingham susan.liddell_at_nottingham.ac.uk
PGT short course Feb 2008
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Outline

• what is proteomics?
• why study the proteome
• proteomic strategies
• the 2D gel standard workflow
• useful protein fractionation methods

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Proteome
the PROTein complement of a genOME Wasinger et
al 1995 Electrophoresis 161090 Proteomics ...t
he identification of all the proteins encoded in
the human genome... Human Proteome Organisation
(www.hupo.org) study of proteins and protein
function on a genome scale Proteomics preceded
genomics Human Protein Index. N L Anderson
1970s
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Why analyse the proteome? Genome considerations

• DNA sequence alone does not reveal biological
  function
• one gene can code for more than one protein
• gene rearrangements
• RNA splicing
• unlike the genome, the proteome is not fixed
• varies from tissue to tissue
• between different cell types
• according to developmental stage
• environment (e.g. disease)

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Why analyse the proteome? Transcript
considerations

• poor correlation between mRNA and protein
  expression levels
• Gygi et al, 1999 Correlation between protein and
  mRNA abundance in yeast. Mol.Cell.Biol. 191720
• Anderson and Seilhamer. 1997 A comparison of
  selected mRNA and protein abundances in human
  liver. Electrophoresis 18533
• each transcript can give rise to several protein
  isoforms via post translational processing (gt200
  PTMs)

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Common covalent modifications of proteins
affecting activity
Biochemistry. Jeremy M Berg, John L Tymoczko,
Lubert Stryer , Neil D Clarke
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PROTEOMICS

• proteins are the main biological effector
  molecules
• not just identifying novel genes, now
  determining the function of gene products
• analysis of protein products complements
  genomics transcriptomics

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Targeted Proteomics
Global Proteomics
identify all proteins present

• quantitative changes abundance
• qualitative changes PTMs
• subcellular compartments nuclei, membranes
• functional complexes of interacting proteins

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There are many Proteomic Approaches using many
different technologies
GelsProteins1D/2D gelsstains/labels
Liquid ChromatographyPeptides/Proteins1D/2DLa
bels/label free
Protein ChipsProtein arrays on slides
Mass Spectrometry
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Protein chips SELDI-TOFbiomarkers in fluids
plasma, CSF, urine
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Liquid ChromatographyMudPIT Multidimensional
protein identification technology
Separate complex mixtures of peptides (or
proteins) using multi-dimensional HPLC 1st
dimension strong cation exchange 2nd dimension
reversed phase Analyse using mass spectrometry
Make quantitative using labelling - heavy and
light isotopes ICAT / iTRAQ Metabolic labelling
in vivo or in vitro e.g. 15N/14N Trypsin digest
in presence of heavy water 18O/16O
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Quantitative LC-MS using ICAT
Adapted from R. Aebersold, Institute for Systems
Biology, Seattle, USA
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2D gel/MS Proteomic Workflow
Protein separation
Analysis and protein spot selection
Processing and digestion to peptides
Mass spectrometric analysis Database
interrogation
Protein identification
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Protein separation 2-dimensional gel
electrophoresis
1st dimension Separation by charge (isoelectric
focussing)
2nd dimension Separation by molecular
weight (SDS-PAGE)
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Example 2D gelovarian follicle granulosa cell
proteins
pH3-10 linear IPG strip 12.5 polyacrylamide
gel Silver stained
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2-D gel electrophoresis equipment - 1st dimension
various lengths 5 - 24 cm wide range pH
3-11 narrow/zoom range pH 4-5 loading
methods in-gel rehydration, cup, paper
bridge
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2-D gel electrophoresis equipment - 2nd dimension
various lengths linear / gradient reducing /
non-reducing
Multi-gel runners higher reproducibility
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Protein detection and image capture
pre-gel sample labelling 35S-methionine Cy3,
Cy5, Cy2 (DiGE) ALEXA fluors post-gel
staining colloidal coomassie blue silver SYPRO
ruby / Deep Purple / Flamingo Pro-Q Diamond
phosphoproteins Pro-Q Emerald
glycoproteins Pro-Q Amber transmembrane
proteins (1D gels)
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Comparison of gel stains
Coomassie Blue 10-50 ng/mm2
SYPRO ruby 1 ng/mm2
Silver 0.5 ng/mm2
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2D gel/MS Proteomic Workflow
Protein separation
Analysis and protein spot selection
Processing and digestion to peptides
Mass spectrometric analysis Database
interrogation
Protein identification
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Analysis and spot selection

• Image analysis software
• PDQuest (BioRad)
• DeCyder (GE Healthcare)
• Progenesis (nonlinear dynamics)
• Image capture
• Spot detection and matching
• Identify variant protein spots
• Statistical evaluations

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Hereditary bovine dilated cardiomyopathy11
proteins increased in abundance
Weekes et al (1999)Electrophoresis 20898
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2D gel/MS Proteomic Workflow
Protein separation
Analysis and protein spot selection
Processing and digestion to peptides
Mass spectrometric analysis Database
interrogation
Protein identification
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Gel spot excision and processing
Pick individual spots into 96-well microtitre
plates
Destain Digest (trypsin) Peptide
extraction Identify proteins using mass
spectrometry
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Identify protein by Mass Spectrometry
MALDI-ToF
Q-ToF2 (plus capillary flow HPLC)
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DIGE (DIfference Gel Electrophoresis)
Unlu M, Morgan ME, Minden JS Electrophoresis
1997 18(11)2071-7 Difference gel
electrophoresis a single gel method for
detecting changes in protein extracts
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DIGE method outline
Sample 2 Cy5
Sample 1 Cy3
Label samples
Mix samples, run on one gel
Scan gel capture Cy3, then Cy 5
merge/overlay the images
Cy3/Cy5
Analyse
Adapted from Cambridge Centre for Proteomics,
University of Cambridge
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DeCyder analysis Example protein spot
Spot 1007 increased, volume ratio 3.20
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Detection of PTMs with fluorescent gel
stainsMultiplexed Proteomics Technology
Total protein SYPRO Ruby
Glycoprotein Pro-Q Emerald
Phosphoprotein Pro-Q Diamond
Image courtesy of
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Sample Buffer for 1st dimension key ingredients
Urea/Thiourea denaturing and solubilising Deterge
nt solubilising non-ionic e.g.
CHAPS Ampholytes uniform conductivity,
solubilising Reducing agent DTT, TBP, HED
(destreak) Dye Bromophenol blue
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1st dimension what to avoid
NaCl lt 10 mM SDS lt 0.25 Tris lt 50
mM phosphates nucleic acids lipids phenolics in
soluble material Heat (always lt 30C when urea
present)
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Special cases
Bacteria -high nucleic acid protein ratio
-use nucleic acid removal techniques Yeast/fun
gi -tough cell walls require vigorous disruption
to lyse -protease activity high Cultured
cells -salt (especially phosphate ions) from
medium -wash in salt free buffer /
osmoticum Plant tissues -dilute source of
protein -precipitation is usually used
-protease activity is high -reductants/inhib
itors to prevent phenolic modification
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Limitations of 2D gels
Basic / acidic Large / Small proteins Membrane
proteins Low throughput / difficult to automate
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Overcoming limitations -improving resolution
experimental design
Cup-loading / in-gel rehydration Zoom IPG strips
and Large format 2nd D gels - increase gel area
for greater separation - higher sample load
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Zoom gels narrow range
pH 3-10









pH 3-6
pH 5-8
pH 7-10
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Zoom gels micro range
pH 4
pH 7
245 Spots
pH 5.9
pH 4.7
479 Spots
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Overcoming limitations - improving resolution
sample preparation
Improve solubilisation clean up to remove
interfering contaminants chaotropes e.g.
thiourea detergents e.g ASB-14 reducing agents
e.g. Destreak
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Overcoming limitations - improving resolution
sample preparation

• Fractionation/enrichment
• sequential extraction based on solubility
• different cellular compartments
  (nuclei/cytoplasm)
• electrophoretic pre-fractionation
• affinity purification (chromatography)

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Fractionation/enrichment (I) Sequential protein
extraction
based on solubility in a series of buffers
Cell lysate
SOLUTION 1 Tris
water soluble proteins
pellet 1
SOLUTION 2 Urea/ CHAPS
moderately insoluble proteins
pellet 2
SOLUTION 3 stronger solubilisers (thiourea,
strong detergents)
enriched in hydrophobic membrane proteins
pellet 3
detergent/chaotrope resistant fraction
e.g.cytoskeletal proteins
Figures courtesy BioRad
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Fractionation/enrichment (II)Preparative
Electrophoresis - Liquid phase isoelectric
focusing
Cell lysate separated into pH fractions Fractions
run on (zoom) 2D gels Higher loading
Figures courtesy Invitrogen
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Fractionation/enrichment (II) Preparative
Electrophoresis Molecular weight based separation
Fountoulakis and Juranville (2003) Anal. Biochem.
313 267-282
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Fractionation/enrichment (III)Chromatography/affi
nity purification
1) Size gel filtration/size exclusion 2)
Charge ion-exchange cation (basic proteins) and
anion (acidic proteins) 3) Hydrophobic/polar
reversed phase, hydrophobic interaction,
hydrophilic interaction
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Fractionation/enrichment (III)Chromatography/affi
nity purification
4) Affinity specific interaction with a ligand
bound to column - general ligand e.g. chemical
group - immobilised metal -Histidine containing
proteins - highly specific ligand e.g.antibody
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Limitation of Proteomic Technologies
Dynamic range
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Proteins measured clinically in plasma span gt 10
orders of magnitude in abundance
Anderson NL, Anderson NG The human plasma
proteome history, character, and diagnostic
prospects Molecular and Cellular Proteomics 2002
1845-867
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1010 Really Is Wide Dynamic Range(Here on a
linear scale)
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12 Proteins comprise up to 96 of the protein
mass in plasma
From Beckman Coulter
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Immunodepletion of 6 high abundance proteins from
human serum
1 - crude serum 2 flow through fraction 3 -
bound fraction M - molecular weight standards
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Phosphoprotein Enrichment
Western blot 1 extract 2 flow through 3 wash 4
eluate
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Affinity purification of complexes
(Gavin, Bosche et al 2001 Nature 415141-7
Functional organization of the yeast proteome by
systematic analysis of protein complexes)
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Vary the experimental method no one size fits
all
Directed proteomic analysis of the human
nucleolus. Andersen et al (2002) Current Biology
121-11
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Proteomics
global screening technologies for complex
samples identifies protein targets for further
investigation after validation