Introduction to ProteomicsSusan Liddell

University of Nottinghamsusan.liddell@nottingham.ac.uk

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