„Grundlagen und Anwendung der
Genom- und Proteomforschung“
Profilmodul (Vorlesung/Seminar WS 2011/12)
Philipps-Universität Marburg
Fachbereich Biologie
Professor Dr. Egon Amann & Dr. Frank Vitzthum
Proteomics III
Frank Vitzthum
Overview
Electrophoresis
Isoelectric focussing
Disc SDS PAGE
2 Dimensional Gel Electrophoresis
Detection
Electrophoresis
-One of the most important separation techniques used today for
characterization and analysis of proteins –
Nobel Prize for Chemistry 1948:
Arne Tiselius
„for his research on electrophoresis and adsorption analysis,
especially for his discoveries concerning the complex nature of
serum proteins“
Seminal work in 1937 with the purpose to separate serum proteins
led to Tiselius‘ „moving-boundary apparatus“
Electrophoresis in In Vitro Diagnostics
- Serum-Electrophoresis -(capillary electrophoresis, agarose gels, cellulose acetate)
The overall
‚Pattern or Fingerprint‘
provides the diagnostic
information
Intensity (scan)
Gel
www.med4you.at
SELDI TOF MS Pattern / Fingerprint
- Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry Trace view
Gel view
Petricoin et al. 2002 The Lancet.
Serum-Elektrophorese
Bande
Prozent
Albumin
58,0 - 70,0
α1Globuline
α2-Globuline
β-Globuline
γ-Globuline
Hauptbestandteile
Albumin
Reduziert
Leberzirrhose,
nephrotisches Syndrom,
inflammatory bowel
disease
Erhöht
α1-Antitrypsin,
α1-Lipoprotein (HDL) Leberzirrhose
Entzündung,
nephrotisches Syndrom
5,0 - 10,0
Caeruloplasmin,
Haptoglobin
Entzündung,
nephrotisches Syndrom
8,0 - 13,0
β-Lipoprotein,
Transferrin,
Leberzirrhose typische
beta-2-Mikroglobulin, Schulterbildung in der
Plasminogen
gamma-Fraktion
nephrotisches Syndrom,
Paraproteinämie
10,0 - 19,0
Immunglobuline
(Antikörper)
chron. Krankheiten,
Plasmozytom
1,5 - 4,0
Leberzirrhose
nephrotisches Syndrom
Electrophoresis
- Molecules with a net charge migrate in an electric field –
Velocity (v) of migration (distance per time) of a molecule depends on
• Electrical field strength (E = U/d)
• Net charge of the molecule (z)
• Coefficient of friction (f)
• Size/mass and shape of molecule
• Viscosity of the medium
• Gel: molecular sieve, prevents convection, reduces diffusion
v = Ez/f
Ez = vf
Electrical force (Ez) versus friction force (vf)
Major Types of Electrophoresis
Native versus denaturing
Reducing versus non-reducing
Horizontal versus vertical
Continuous versus discontinuous
Linear versus gradient
Carrier free (free flow electrophoresis, capillary electrophoresis)
versus
Carrier (paper, cellulose acetate, polymer gels such as starch, agarose,
polyacrylamide (1959; 1975 2DGE Joachim Klose & Patrick O‘Farrell), etc.)
Isoelectric focussing (IEF)
Discontinuous sodium dodecylsulfate polyacrylamide gel electrophoresis
(disc SDS PAGE)
2D-GE (IEF/SDS-PAGE; Blue native/SDS-PAGE; CTAB/SDS-PAGE)
3D-GE (Blue native/IEF/SDS-PAGE)
Isoelectric Focussing
Separation of proteins according to their isoelectric point (IEP)
IEP: the pH-value at which the net charge is zero
E-Focussing
Diffusion
pH-gradient
Net charge
Sample application
Modification of slide of lecture: ‚Bioanalytik – Funktionsanalytik – Proteom‘ from the University of Bielefeld / Faculty Biology
Ampholytes
Modification of slide of lecture: ‚Bioanalytik – Funktionsanalytik – Proteom‘ from the University of Bielefeld / Faculty Biology
Free Carrier-Ampholytes
- Gradients difficult to reproduce, e.g. because of lot to lot variation -
Aliphatic oligoamino-oligocarbonic acids with different pI-values
Gels with 2 % carrier ampholytes almost all are charged
Start: uniform pH-value
Development of a stable pH-gradient in the electric field
Negative ampholytes migrate to anode (+); gel becomes acidic
Positive ampholytes migrate to cathode (-); gel becomes basic
→ A gradient is generated
E
Modification of slide of lecture: ‚Bioanalytik – Funktionsanalytik – Proteom‘ from the University of Bielefeld / Faculty Biology
Immobilines
- Stable gradient / higher reproducibility / higher loading capacity -
Polymers of polyanions and polycations
Stable ‘immobile’ pH-gradients polymerized within a gel matrix
(bifunctional immobilines)
Acrylamide-derivates (weak acids or basis)
Manufacturing by mixing during casting
Polymerisation on carrier slide
Desiccation
Rehydration with additives (urea, detergents, hydroxyethyldisulphide (HED))
Isoelectric Focussing (cont.)
Pros
• high resolution (~ 70 – 100 bands)
• separation of proteins with a single charge difference
• direct determination of pI
• combination with (disc SDS)-PAGE possible
Cons
• very basic or acidic proteins are difficult to focus
• same applies to membrane proteins
• staining cumbersome
• precipitation issues
Isoelectric Focussing (cont.)
- As proteins are focused they will not elute out of the gel as is the case in PAGE -
t=0
t‘ = x
t‘ = y
Modification of slide of lecture: ‚Bioanalytik – Funktionsanalytik – Proteom‘ from the University of Bielefeld / Faculty Biology
Resolution
Ideal
‚Real‘
Efficient depletion
of one component
Depletion / Enrichment
2DGE of Plasma untreated or treated with ProteoMiner
www.bio-rad.com
To see or not to see?
Signal
100 % Purity ?
Measure, e.g. distance (mm), volume (mL), (elution) time (min), fraction (#), etc.
Signal
Measure, e.g. distance (mm), volume (mL), (elution) time (min), fraction (#), etc.
To see or not to see?
Relevance and issues
Functional proteomics (quantitation and MS protein assignment)
Presentation and documentation
(consignment notes of products, audits, publications, etc.)
Assigned purity of a substance/preparations depends on analytics
LC, SDS-PAGE, IEF, 2DGE, etc. and the respective conditions applied
Effects of substances/preparations and root cause analysis
(side reactions, impact of alterations of production processes, etc.)
Quality control
(internal and external raw materials)
Polyacrylamide Gel Electrophoresis (PAGE)
http://www.ym.edu.tw/ibm/credit/ppt/b501.pdf
Stryer, L. Biochemie. ISBN 3860253468
Denaturing SDS PAGE
Sodium dodecylsulfate (SDS); Na+ H3C-(CH2)10-CH2OSO3denatures proteins and aligns structures and negative charge per mass
Reduktionsmittel
• Dithiothreitol (DTT),
• Dithioerythrol (DTE),
• ß-Mercaptoethanol,
• Hydroxyethyldisulphide (HED)
Discontinuous (Disc) SDS PAGE
http://web.chemistry.gatech.edu/~williams/bCourse_Information/4581/techniques/gel_elect/gel.jpg
Disc SDS PAGE (cont.)
Focusing
-
increases resolution
Kathode
dE
+ Anode
Modification of slide of lecture: ‚Bioanalytik – Funktionsanalytik – Proteom‘ from the University of Bielefeld / Faculty Biology
Basic
Acidic
Deprotonated
Protonated
Positive
Charge
pK-values at 25 °C:
Negative
Charge
α-NH3+-group: 9.8
α-COOH-group: 2.4
SDS PAGE (cont.)
SigmaMarker
Wide Range
4-20 %
http://www.pagegel.com/products/gels_sds.html
Disc SDS PAGE (cont.)
O'Farrell, P. H.:
High resolution two-dimensional electrophoresis of proteins.,
J. Biol. Chem. 250, 1975, 4007-4021
IEF
SDS PAGE
9
• Application of 10µg E.coli protein (14C labelled) • Autoradiography: 825 hours
 1100 different protein spots
2 D Gelelectrophoresis
pH 6.1
40 kDa
Modification of slide of lecture: ‚Bioanalytik – Funktionsanalytik – Proteom‘ from the University of Bielefeld / Faculty Biology
2 D Gelelectrophoresis (cont.)
Pros/Characteristics
separation, display and storage of several thousands of proteins
high resolution (400 and up to 10 000 spots)
spreading of pH-range increases resolution (pH 3 – 11)
typical size range 10 – 200 kDa
detergents may be applied
detection of post-translational modifications
Cons
difficult to automate
reproducibility (position und amounts; operator dependence)
losses due to transfer from the first to the second dimension
time-consuming & labor intensive (e.g. 2 d for a single sample)
extraction of proteins for further processing
extremely large, small, hydrophobic, and basic proteins excluded/difficult to detect
low abundance proteins conceiled by high abundance proteins (insufficient
resolving power to fully separate all proteins)
quantification issues
lower peptide yield after in-gel digestion if compared to liquid phase approaches
(limited sensitivity in subsequent MS analysis)
Major Detection Methods
Visualization / Quantification of protein spots
• UV absorption
• Staining
• Computer Image Analysis
Identification of proteins
• Western Blotting
• Spot picking and Sequencing
• Mass spectrometry (MS)
• Edman sequencing
Would a simple dilution solve the issue; especially
when maximal signal intensity is reached anyhow?
Signal intensity
Measure, e.g. distance (mm), volume (mL), (elution) time (min), fraction (#), etc.
Signal intensity
Dynamic range
Limit of detection
Measure, e.g. distance (mm), volume (mL), (elution) time (min), fraction (#), etc.
Increase dynamic range by appropriate detection
Signal intensity
Dynamic range
Measure, e.g. distance (mm), volume (mL), (elution) time (min), fraction (#), etc.
Protein Staining in Gels
Sensitivity Dynamic
(ng/5 mm band
range
or spot)
Silver
1 - 30
Eosin Y.
10
Fluor. Dyes (SYPRO)
1 - 50
Stains-all
100-200
Coomassie R250
200-400
Coomassie G250
25
Nitroblue Tetrazolium
200 - 400
Na-Acetat
1000-3000
KCI
2000-4000
Radio-Labeling
<1
(ord. magn.)
1
3-4
2
Time
1-2h
30 min
1h
3-4 days
2-4 h
2-4 h
20 min
20 min
10-40 min
Variability
high
moderate
low
low
low
low
Blotting
no
yes*
yes*
yes*
yes*
yes*
yes*
4-5
* Before blotting the dyed gels need to be treated, e.g. by elusion of salts or incubation in
0.1% SDS-solution (Perides et al. 1986)
Multiplexing
- Overlay of multiple pictures of a single gel of different dyes or labels detected
separately by a respective scanner or detection system and representing different
properties of proteins Miscolored Multiplexing
Protein quantity
(e.g. fluorescent dyes, silver staining, etc.)
Protein synthesis
(autoradiogram of radioactively,
pulse labeled proteins)
Protein phosphorylation (ProQ-Diamond dye)
Protein glycosylation (Emerald ProQ dye)
Different samples
(Differential in Gel Electrophoresis; DIGE)#
Enzymes
(fluorescent suicide inhibitors)
http://de.wikipedia.org/wiki/2D-Gelelektrophorese
2GE-Glycoproteomics
1. Pro-Q Emerald 300
staining & detection
of glycoproteins
2. SYPRO Ruby
staining & detection
of proteins
Lysates from rat liver tumor cells
Two different gels
Lysates from rat normal liver cells
http://probes.invitrogen.com/handbook/print/0901.html
The same single gel
Overlaying of Images of Different Gels and Correct
Matching of Proteins is Difficult
Gel-to-gel variations (reproducibility) can mask or mimic
biological variation between samples.
The same/similar sample(s) in different gels show(s) different
•
spatial resolution
•
spot intensities
because of
•
variations in protein uptake by isoelectric focusing strips
•
incomplete protein transfer from 1.D to 2.D
•
local inconsistencies in
• gel composition,
• field strength or
• pH gradients
Image Software
• Background reduction
• Artefact reduction
• Image warping
→
Corrected spot pattern for
quantitative analyses
Image Warping by Respective Software
- Account for different migration (warping) when two different gels are overlayed -
Corresponding spots at different positions
Corresponding spots at the same position
Similarly expressed spots appear black
Differentially expressed spots appear in the
respective colour
http://de.wikipedia.org/wiki/2D-Gelelektrophorese
Differential in Gel Electrophoresis (DIGE)
480/530 nm
540/590 nm
620/680 nm
Three different samples
labelled separately
before separation
Run and analysed/illuminated successively
on the same single gel
Differential in Gel Electrophoresis (DIGE)
Excision / Digestion / Liquid Chromatography / Mass Spectrometry / Identification
Gert Van den Bergh & Lutgarde Arckens, 2005, Expert Rev. Proteomics 2(2)
DIGE Considerations
• Cyanine dyes need to guarantee co-migration of proteins
• Dyes are charge-matched and molecular mass-matched
• Dyes induced (mass) shifts need to be minimal
• N-hydroxy succinimidyl ester derivatives, are covalently tagged to the
ε-amino group of lysine residue of proteins, and replace the ε-amino
group positive charge with the positive charge of the dye introduce
small but matched increases in molecular weight
• Minimal labeling to prevent precipitation of proteins (dyes are
hydrophobic)
• Saturation dyes saturate cysteine residues instead of minimally labeling lysine
residues
• No labeling when respective amino acid is missing (lysine / cysteine)
• Large number of pairwise comparisons
Pooled sample with equal amounts of all sampes as reference to normalize
all spots across all gels
DIGE Pros & Cons
Pros
• No need to run multiple gels – up to three different samples per gel
• Co-migration on the same gel reduces running differences between samples
• Samples subjected to the same environment & procedures
• Experimental variation is reduced in this way
• No need to process a gel for visualization after electrophoresis
• Low sensitivity (minimal and saturation labeling: 1 and 0.1 ng/spot)
• Up to 4 orders of magnitude dynamic range
• Detection of approximately 1500 protein spots from less than 6.6 µg protein
material
Cons
• Costs (chemicals, equipment)
• Sample preparation
• „Preferential“ labeling/precipitation with saturation labeling
Gert Van den Bergh & Lutgarde Arckens, 2005, Expert Rev. Proteomics 2(2)