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Introduction to Proteomics Susan Liddell University of Nottingham [email protected]. PGT short course May 2012 UoN Graduate School Course Post-genomics and bio-informatics. Sutton Bonington Proteomics labs Division of Animal Sciences – South lab Susan Liddell, Ken Davies. - PowerPoint PPT Presentation
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Introduction to ProteomicsSusan Liddell
University of [email protected]
PGT short course May 2012UoN Graduate School Course Post-genomics and bio-informatics
Sutton Bonington Proteomics labsDivision of Animal Sciences – South lab
Susan Liddell, Ken Davies
• Supports proteomics studies & collaborative projects– gel electrophoresis (mainly 2D)– protein identification via tandem MS
• Wide variety of types of projects and organisms – including some species with unreported genome sequences
human cow horse fungi bacteria archaea plants
Dr Ken Davis
Overview
• what is proteomics?• why study the proteome• proteomic strategies
– the 2D gel standard workflow– 2D DiGE– High throughput LC-MSMS
Proteome
“the PROTein complement of a genOME”
Wasinger et al 1995 Electrophoresis: 16:1090
Proteomics
“...the identification of all the proteins encoded in the human genome....”
including modification, quantification, localisation and functional analysis for every cell type
Human Proteome Organisation (www.hupo.org)
Proteomics
study of proteins and protein function usually on a genome wide scale
Proteomics preceded genomics Human Protein Index N & L Anderson 1982
Aims of Proteomics
Global analysis of complex samples
A fundamental understanding of biological processes and mechanisms
Find changes in protein expression (biomarkers) in different biological situations (disease)
Aid in development of therapeutic agents/drugs
Why analyse the proteome? Genome considerations
Functional Annotation of the Arabidopsis Genome Using Controlled VocabulariesPlant Physiology (2004) Vol.135, p745
Arabidopsis genome annotationfunctional characterisation
26% molecular function unknown
sequence alone does not reveal biological function
Why analyse the proteome? Genome considerations
• one gene can code for more than one protein
– gene rearrangements
– RNA splicing
• unlike the genome, the proteome is highly dynamic– varies from tissue to tissue
– between different cell types
– according to developmental stage
– environment (e.g. disease)
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. 19:1720
– Anderson and Seilhamer. 1997 A comparison of selected mRNA and protein abundances in human liver. Electrophoresis 18:533
– Ingolia et al 2009 Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324(5924):218-23
• each transcript can give rise to several protein isoforms via post translational processing (>300 PTMs)
Common covalent modifications affecting protein activity
Modification Donor moleculeExample of
modified proteinProtein function
Phosphorylation ATPGlycogen
phosphorylaseGlucose homeostasis; energy transduction
Acetylation Acetyl CoA Histones DNA packing; transcription
Myristoylation Myristoyl CoA Src Signal transduction
ADP-ribosylation NAD RNA polymerase Transcription
FarnesylationFarnesyl
pyrophosphateRas Signal transduction
-Carboxylation HCO3- Thrombin Blood clotting
Sulfation3’-Phosphoadenosine-
5’-phosphosulfateFibrinogen Blood-clot formation
Ubiquitination Ubiquitin Cyclin Control of cell cycle
Biochemistry. Berg, Tymoczko, Stryer, Clarke
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
“At the end of the day, proteins, not genes, are the business end of biology.”
Targeted Proteomics
• quantitative changes : abundance
• qualitative changes : PTMs
• subcellular compartments : nuclei, membranes
• functional : complexes of interacting proteins
identify all proteins present
Global Proteomics
Gels
Proteins1D/2D gels
stains/labels
Liquid Chromatography
Peptides/Proteins1D/2D
Labels/label free
Protein Chips
Protein arrays on slides
Mass Spectrometry
There are many Proteomic Approaches using many different technologies
Electrospray ionization (ESI)
John B Fenn
Matrix-assisted laser desorption/ionization (MALDI)
Koichi Tanaka
"for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules"
The Nobel Prize in Chemistry 2002
Applications of mass spectrometry in protein analysis include
Protein identification peptide mass fingerprintingTandem MSde novo sequence
Recombinant protein evaluationconfirm identityengineered mutations, sequence changescleavages or other modificationsassess homogeneity
Identification of modificationsacetylationoxidationglycosylationphosphorylation
….anything that causes a change in mass….
Proteomic Workflow 2D gel/MS
Protein separation
Mass spectrometric analysis
Database interrogation
Analysis and protein spot selection
Processing and digestion to peptides
Protein identification
Protein separation2-dimensional gel electrophoresis
1st dimension Separation by charge(isoelectric focussing) pH 3 pH 10
pI
2nd dimension Separation by molecular weight
(SDS-PAGE) kDa
2D gel electrophoresis equipment1st dimension IEF
various lengths 5 - 24 cm
wide range pH 3-11
narrow/zoom range pH 4-5
loading methods in-gel rehydration cup, paper bridge
2-D gel electrophoresis equipment 2nd dimension SDS-PAGE
various lengths
linear / gradient
reducing / non-reducing
Multi-gel runnersincrease reproducibilityincrease throughput
Protein detection and image capture
post-gel staining
colloidal coomassie bluesilverSYPRO ruby, Deep Purple, Flamingo
pre-gel sample labelling 35S-methionineCy3, Cy5, Cy2 (DiGE)
Pro-Q Diamond – phosphoproteinsPro-Q Emerald – glycoproteinsPro-Q Amber – transmembrane proteins (1D gels)
Example 2D gelE. coli cell extract
Soo Jin Saa
100 µg
pH 4-7 IPG strip
12.5% PAGE
Silver stained
100
37
25
150
75
50
20
250
Comparison of gel stains
ColloidalCoomassie Blue
10-50 ng/mm2
SYPRO ruby
~ 1 ng/mm2
Silver
0.5 ng/mm2
Special cases
Bacteria -high nucleic acid: protein ratio -use nucleic acid removal techniques
Yeast/fungi -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/inhibitors to prevent phenolic modification
Proteomic Workflow 2D gel/MS
Protein separation
Mass spectrometric analysis
Database interrogation
Analysis and protein spot selection
Processing and digestion to peptides
Protein identification
Analysis and spot selection
Image analysis software
PDQuest (BioRad)
DeCyder (GE Healthcare)
Same Spots (Nonlinear Dynamics)
Image capture
Spot detection
Spot matching across gel set
Statistical evaluations
Find differences in spot patterns (protein expression changes) between samples using image analysis software
Weekes et al (1999)Electrophoresis 20:898
Hereditary bovine dilated cardiomyopathy:11 proteins increased in abundance
Proteomic Workflow 2D gel/MS
Protein separation
Mass spectrometric analysis
Database interrogation
Analysis and protein spot selection
Processing and digestion to peptides
Protein identification
Gel spot excision and processing
Pick individual spots
into 96-well
microtitre plates
Destain
Digest (trypsin)
Peptide extraction
Proteomic Workflow 2D gel/MS
Protein separation
Mass spectrometric analysis
Database interrogation
Protein identification
Analysis and protein spot selection
Processing and digestion to peptides
Identify proteins using Mass Spectrometry
MALDI-ToF Q-ToF2 (plus capillary/nano flow HPLC)
Investigation of proteins involved in ovarian folliculogenesis
Ken Davis, Jacqueline Cameron, Susan Liddell & Bob Webb
School of Biosciences
Waves of follicular growth and development in the cow
True dominant follicles
healthierfertilisable oocytes
early stage “dominant” follicles
oocytes with a lower fertilisation rate
1 to 2
2 to 6
15 to 20
SelectionSelectionSelectionSelection
phaseDominanceDominanceDominance
phase
9 12 15 18 0 3 6 9 12
Fol
licle
num
ber
21 Days of oestrous cycle
Ovulation
Granulosa cells surround the oocyte
granulosa cells
oocyte
GCs provide instructions directing oocyte development
Prepared GC extracts from each stage
Compare profiles
Identify proteins that differ
Proteins that
determine which follicle becomes the dominant, mature follicle that can be
fertilized
determine the quality of the oocyte
ovarian follicle granulosa cell proteins
Sets of analytical gels run
Samespots™ software indicates we needmore gels for acceptable statistics
Project ongoing .....
Establish proteome maps of GC proteins
CALREGULIN
GRP94
ATPase
ALBUMIN
HSP70
PROTEIN DISULPHIDE ISOMERASES
TUBULIN
HAEMOGLOBIN
HSP60
UBIQUITIN
ENOLASE
ACTINS
GLUCOSIDASE II
PHOSPHOGLYCERATE MUTASE
ANNEXIN
PEROREXOXIN
ALDEHYDE REDUCTASE
Reference profile to support differential analyses projects
So far identifying proteins of a fairly abundant “housekeeping” nature
How to find regulator proteins – of lower abundance?
Limitation of Proteomic Technologies
Dynamic range
Don’t see the lower abundance proteins in complex mixtures
Anderson NL, Anderson NG The human plasma proteome: history, character, and diagnostic prospects
Molecular and Cellular Proteomics 2002 1:845-867
Proteins measured clinically in plasma span > 10 orders of magnitude in abundance
1010 Really Is Wide Dynamic Range(Here on a linear scale)
2D gels only ~2-3 orders of proteins detected only the most abundant proteins
Mass spectrometers detection range of ~ 3 (to 5) orders
How to overcome the dynamic range and detect proteins of lower abundance?
Reduce the complexity and dynamic range
Fractionation techniques include remove abundant proteinsdifferent cellular compartmentsdifferential protein solubilitysequential chromatography (2D, 3D)affinity purification
Fractionation : Remove abundant proteins12 proteins in plasma comprise ~ 96% of the protein mass
Figure courtesy of Beckman Coulter
Immunodepletion of 6 high abundance proteins from human serum
1 - crude serum
2 – flow through fractiondepleted of high
abundance proteins
3 - bound fraction
M 1 2 3
Figure courtesy Agilent Technologies
RuBiSCo immunodepletion
• the biggest single obstacle in plant proteomics
• ribulose-1,5-bisphosphate carboxylase oxygenase• enzyme catalyses the first major step of carbon fixation
– the energy supplied by photosynthesis is used to convert carbon dioxide into available food
• ~ 40%-60% of the total protein in green plant tissues
• the most abundant protein in plants
• the most abundant protein on Earth
RuBiSCo immunodepletion tool
IgY antibodies raised against purified RuBiSCo from spinachcross species
Petunia leafTotal Depleted
Arabidopsis leaf
Large Sub-Unit
Small Sub-Unit
Total Depleted
100
75
150
50
37
20
25
15/10
250
Susan Liddell, unpublished
Reduce the complexity and dynamic range
Fractionation techniques includeremove abundant proteinsdifferent cellular compartmentsdifferential protein solubilitysequential chromatography (2D, 3D)affinity purification
Fractionation: Different cellular compartments example of a subproteome experiment
Investigation of proteins involved in nuclear transfer
Jacqueline Cameron, Susan Liddell & Keith Campbell
School of Biosciences
Nuclear transfer
MII oocyte
spindle
Inject new, somatic nucleus
Enucleation
DC electric pulse
Development
Gestation Transfer tosurrogate
The meiotic spindle is removed from oocyte
Does this also deplete associated proteins that are required for subsequent development and cell cycle control?
Identification of such proteins would provide deeper understanding of the process and allow controlled
replenishment to improve the outcome of NT
spindle
DIfference Gel Electrophoresis (DIGE)
Figure adapted from Cambridge Centre for Proteomics
Sample 2 Cy5Sample 1 Cy3Label samples
Analyse
Scan gelcapture Cy3, then Cy 5overlay the images
Mix samplesrun on ONE gel
Cy3/Cy5
Unlu M, Morgan ME, Minden JS Electrophoresis 1997Difference gel electrophoresis: a single gel method for detecting changes in protein extracts
DeCyder analysis – Example protein spot
Spot 1007 increased, volume ratio 3.20
Oocytes & SpindlesSaturation DiGE labelling
50 Spindles Cy5 / Red50 Enucleated Oocytes Cy3 / Green
Detection of PTMs with fluorescent gel stains“Multiplexed Proteomics Technology”
GlycoproteinPro-Q Emerald
PhosphoproteinPro-Q Diamond
Total proteinSYPRO Ruby
Image courtesy of
Trains of glycosylated forms
Phosphorylatedproteins
Phosphorylatedprotein
Limitations of 2D gels
Some classes of proteins are difficult to obtain on 2D gels
basic / acidic proteinslarge / small proteinsmembrane proteins
Low throughput / difficult to automate
Another approach : high throughput LC-MSMS
Proteomic Workflow high throughput LC-MSMS
Digestion of complex protein sample
Mass spectrometric analysis
Database interrogation
Protein identification
Quantitationtagging, non-tagging approaches
Peptide separationHigh resolution HPLC
(often multidimensional)
High throughput LC-MSMSExample of an off-gel shotgun approach
Analysis of the articular cartilage secretome
Ali Mobasheri, Abigail Clutterbuck, Kirsty Hillier, Adam WilliamsSchool of Veterinary Medicine & Science
Susan LiddellSchool of Biosciences,
Julia SmithBruker UK Ltd
Mechanically unique connective tissue acts to– withstand and distribute load– act as an elastic shock absorber– provide a wear resistant surface to articulating
joints
Extra-cellular matrix (ECM), only 1 cell type
Created by chondrocytes, composed of collagens and aggregating proteoglycans
Is continually remodelled, but cannot repair
Inflammation causes disruption of the ECM
Articular Cartilage
Articular Cartilage and Osteoarthritis (OA)
OA is the most common form of arthritis in humans and animals
Major cause of pain and disability
Chronic disease characterised by progressive destruction of articular cartilage and subchondral bone
Obtain basic proteome profile of the tissueUse a controlled pathological insult, pro-inflammatory cytokines (IL-1β) to stimulate inflammation
Monitor the protein response
Identifying biomarkers of early stage of OA may provide molecular insights into disease onset and discover diagnostic and therapeutic targets
Investigate effects of anti-inflammatory drugs and plant derived compounds
Aims
Explant Cartilage Culture
Chondrocytes remain phenotypically stable in their native matrix
Secretome – degeneration during OA is enhanced/mediated by disruption of normal protein secretion levels
Serum free culture
Suitable for proteomic work
0 10 20 30 40 50 Time [min]0.0
0.5
1.0
1.5
9x10Intens.
22Apr2010 sample 11_C1_01_772.d: TIC +All MS 22Apr2010 sample 11_C1_01_772.d: TIC +All MSn
496.23
626.68
744.16
851.32
646.61 858.44418.09
+MS, 15.9min #1405
0.0
0.5
1.0
1.5
2.0
2.5
6x10Intens.
400 600 800 1000 1200 1400 1600 m/z
Digest the secreted proteins with trypsin
Peptides separated by RP-HPLC
Peptides analysed in a Bruker amaZon ETD ion trap
Generate lists of proteins identified
Search databases using Mascot software
Incubate explants
Experimental workflow
Some of the proteins identified in the canine cartilage secretome
Most of the proteins identified are secreted proteins
Many of the identified proteins are arranged into large multi-molecular assemblies in articular cartilage
Some proteins identified have regulatory rather than structural roles
Validation with western blottingApplying quantitative analysis of the MS data
Now refining the approach to allow detection of additional regulatory molecules
Conclusions of Canine Cartilage Secretome Study
uses unbiased global screening technologies to analyse complex samples
a proteome is dynamic and can vary depending on physical conditions, cell cycle, environment,
health/disease etc
a proteome is a “snapshot “of protein expression/modification by specific cells/tissues, under
particular conditions
identifies protein targets for further investigation after validation
Proteomics
11TH EAST MIDLANDS PROTEOMICS WORKSHOPLoughborough University
Wednesday 28th November 2012http://www.empw.org.uk
extra slides
Sample Buffer for 1st dimension – key ingredients
Urea/Thiourea denaturing and solubilising
Detergent solubilising
non-ionic e.g. CHAPS
Ampholytes uniform conductivity, solubilising
Reducing agent DTT, TBP, HED (destreak)
Dye Bromophenol blue
1st dimension – what to avoid
NaCl < 10 mM SDS < 0.25%
Tris < 50 mM phosphates
nucleic acids lipids
phenolics insoluble material
Heat (always < 30°C when urea present)
Overcoming limitationsexperimental design
Zoom IPG strips and Large format 2nd D gels
- increase gel area for greater separation
- higher sample load
Zoom gels: narrow range
pH 3-6 pH 5-8 pH 7-10
pH 3-10
Zoom gels: micro rangepH 4 pH 7
pH 4.7 pH 5.9
245 Spots
479 Spots
Overcoming limitationssample preparation
Improve solubilisation
clean up to remove interfering contaminants
chaotropes e.g. thiourea
detergents e.g ASB-14
reducing agents e.g. Destreak
Overcoming limitations - improving resolution: sample preparation
Fractionation/enrichment
sequential extraction based on solubility
different cellular compartments (nuclei/cytoplasm)
electrophoretic pre-fractionation
affinity purification (chromatography)
Cell lysate
pellet 1
SOLUTION 1 Tris
water soluble proteins
pellet 2
SOLUTION 2Urea/ CHAPS
pellet 3
SOLUTION 3stronger solubilisers
(thiourea, strong detergents)
moderately insoluble proteins
enriched in hydrophobic & membrane proteins
detergent/chaotrope resistant fraction e.g.cytoskeletal proteins
Fractionation/enrichment (I)Sequential protein extractionbased on solubility in a series of buffers
Figures courtesy BioRad
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
Fractionation/enrichment (III) Preparative Electrophoresis
Molecular weight based separation
Fountoulakis and Juranville (2003) Anal. Biochem. 313: 267-282
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
Fractionation/enrichment (IV)Chromatography/affinity 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
Fractionation/enrichment (IV)Chromatography/affinity purification
Affinity Enrichment - Phosphoproteins
Western blot1 Untreated extract2 Unbound3 Wash4 Eluate (bound)
Affinity purification of complexes
(Gavin, Bosche et al 2001 Nature 415:141-7 Functional organization of the yeast proteome by systematic analysis of protein complexes)
Directed proteomic analysis of the human nucleolus.Andersen et al (2002) Current Biology 12:1-11
Vary the experimental method –no “one size fits all”
Protein chips : SELDI-TOFbiomarkers in fluids – plasma, CSF, urine
Maldi MS imaging
A. Histological image of the tissue sectionB. Selected mass m/z 14,836 – correlates with non-cancerous area C. Selected mass m/z 13,777 – correlates with cancerous area
Analyse the correlating masses to identify the proteins
invasive ductal pancreatic cancer tissue by MALDI-IMS
MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology
Walch et al. Histochem Cell Biol (2008) 130:421–434
Liquid ChromatographyMudPIT Multi-dimensional protein identification technology
Separate complex mixtures of peptides using
multi-dimensional HPLC
1st dimension – strong cation exchange
2nd dimension – reversed phase
Analyse using mass spectrometry
Use labels to make quantitative - 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
Quantitative LC-MS using ICAT
Adapted from R. Aebersold, Institute for Systems Biology, Seattle, USA