Introduction to Proteomics 2D gels and - PowerPoint PPT Presentation

Loading...

PPT – Introduction to Proteomics 2D gels and PowerPoint presentation | free to view - id: 1e1ca5-ZDc1Z



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Introduction to Proteomics 2D gels and

Description:

Introduction to Proteomics 2D gels and – PowerPoint PPT presentation

Number of Views:283
Avg rating:3.0/5.0
Slides: 53
Provided by: plantsciAr
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Introduction to Proteomics 2D gels and


1
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
2
Outline
  • what is proteomics?
  • why study the proteome
  • proteomic strategies
  • the 2D gel standard workflow
  • useful protein fractionation methods

3
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
4
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)

5
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)

6
Common covalent modifications of proteins
affecting activity
Biochemistry. Jeremy M Berg, John L Tymoczko,
Lubert Stryer , Neil D Clarke
7
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

8
Targeted Proteomics
Global Proteomics
identify all proteins present
  • quantitative changes abundance
  • qualitative changes PTMs
  • subcellular compartments nuclei, membranes
  • functional complexes of interacting proteins

9
There are many Proteomic Approaches using many
different technologies
Gels Proteins 1D/2D gels stains/labels
Liquid Chromatography Peptides/Proteins 1D/2D La
bels/label free
Protein Chips Protein arrays on slides
Mass Spectrometry
10
Protein chips SELDI-TOF biomarkers in fluids
plasma, CSF, urine
11
Liquid Chromatography MudPIT 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
12
Quantitative LC-MS using ICAT
Adapted from R. Aebersold, Institute for Systems
Biology, Seattle, USA
13
2D gel/MS Proteomic Workflow
Protein separation
Analysis and protein spot selection
Processing and digestion to peptides
Mass spectrometric analysis Database
interrogation
Protein identification
14
Protein separation 2-dimensional gel
electrophoresis
1st dimension Separation by charge (isoelectric
focussing)
2nd dimension Separation by molecular
weight (SDS-PAGE)
15
Example 2D gel ovarian follicle granulosa cell
proteins
pH3-10 linear IPG strip 12.5 polyacrylamide
gel Silver stained
16
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
17
2-D gel electrophoresis equipment - 2nd dimension
various lengths linear / gradient reducing /
non-reducing
Multi-gel runners higher reproducibility
18
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)
19
Comparison of gel stains
Coomassie Blue 10-50 ng/mm2
SYPRO ruby 1 ng/mm2
Silver 0.5 ng/mm2
20
2D gel/MS Proteomic Workflow
Protein separation
Analysis and protein spot selection
Processing and digestion to peptides
Mass spectrometric analysis Database
interrogation
Protein identification
21
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

22
Hereditary bovine dilated cardiomyopathy 11
proteins increased in abundance
Weekes et al (1999)Electrophoresis 20898
23
2D gel/MS Proteomic Workflow
Protein separation
Analysis and protein spot selection
Processing and digestion to peptides
Mass spectrometric analysis Database
interrogation
Protein identification
24
Gel spot excision and processing
Pick individual spots into 96-well microtitre
plates
Destain Digest (trypsin) Peptide
extraction Identify proteins using mass
spectrometry
25
Identify protein by Mass Spectrometry
MALDI-ToF
Q-ToF2 (plus capillary flow HPLC)
26
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
27
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
28
DeCyder analysis Example protein spot
Spot 1007 increased, volume ratio 3.20
29
Detection of PTMs with fluorescent gel
stains Multiplexed Proteomics Technology
Total protein SYPRO Ruby
Glycoprotein Pro-Q Emerald
Phosphoprotein Pro-Q Diamond
Image courtesy of
30
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
31
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)
32
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
33
Limitations of 2D gels
Basic / acidic Large / Small proteins Membrane
proteins Low throughput / difficult to automate
34
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
35
Zoom gels narrow range
pH 3-10









pH 3-6
pH 5-8
pH 7-10
36
Zoom gels micro range
pH 4
pH 7
245 Spots
pH 5.9
pH 4.7
479 Spots
37
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
38
Overcoming limitations - improving resolution
sample preparation
  • Fractionation/enrichment
  • sequential extraction based on solubility
  • different cellular compartments
    (nuclei/cytoplasm)
  • electrophoretic pre-fractionation
  • affinity purification (chromatography)

39
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
40
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
41
Fractionation/enrichment (II) Preparative
Electrophoresis Molecular weight based separation
Fountoulakis and Juranville (2003) Anal. Biochem.
313 267-282
42
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
43
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
44
Limitation of Proteomic Technologies
Dynamic range
45
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
46
1010 Really Is Wide Dynamic Range (Here on a
linear scale)
47
12 Proteins comprise up to 96 of the protein
mass in plasma
From Beckman Coulter
48
Immunodepletion of 6 high abundance proteins from
human serum
1 - crude serum 2 flow through fraction 3 -
bound fraction M - molecular weight standards
49
Phosphoprotein Enrichment
Western blot 1 extract 2 flow through 3 wash 4
eluate
50
Affinity purification of complexes
(Gavin, Bosche et al 2001 Nature 415141-7
Functional organization of the yeast proteome by
systematic analysis of protein complexes)
51
Vary the experimental method no one size fits
all
Directed proteomic analysis of the human
nucleolus. Andersen et al (2002) Current Biology
121-11
52
Proteomics
global screening technologies for complex
samples identifies protein targets for further
investigation after validation
About PowerShow.com