Chapter 2:

Protein Separation
Learning Outcomes

• Understand the need for protein separation in a proteomics

study

• Understand the function of each step in protein separation

• Able to compare between separation techniques

• Able to highlight advantages and disadvantages of various

techniques

• Able to propose a suitable technique for separation of a

given sample
Proteome
• definition - the set of expressed proteins in a given type
of cells or an organism at a given time under defined
conditions
• the proteome is a collective of all protein isoforms since
a protein can have many isoforms based on the degree of PTM
it goes through
• proteomics – combined techniques applied for the study and
analysis of proteome
• these include:
a) proteome mapping
b) protein identification and measurement
c) protein sequencing
d) comparison of protein profiles
e) protein modeling, structure prediction
f) protein interactions study
Human serum proteome as separated by 2D gel electrophoresis

http://www.jpp.krakow.pl/journal/archive/1106_s7/articles/05_article.html
Yeast proteome as separated by 2D gel electrophoresis
Saccharomyces cerevisiae
Lower molecular weight Proteome of
Saccharomyces cerevisiae
Proteome of the mouse brain as separated by 2D gel electrophoresis

http://www.proteomefactory.com/vwr/technology/proteomeanalysis/proteomeanalysis.html
Medical Examples of Proteomics Application

*Slide from GE health Ettan DIGE

Agricultural examples of proteomics
application

Identify and
Proteins as markers for Example: Rice, tomato, corn
determine
plant responses to function of unknown
abiotic/biotic stress proteins in the
proteome

discovery of proteins
Investigate crop growth with beneficial
and quality markers activity for cosmetics,
nutraceutical and
pharmaceutical

Studies are aimed at enabling selective breeding of crop

species to increase yields and better cope with adverse
environmental conditions.
 What we need these tools for..
Large scale analysis of complex samples
• serum, urine, plasma, cell/tissue/tumour extract,culture
media…

To distinguish
• protein profiles/map/expression pattern
• identification and quantification (some sort of)
• interaction, modification

Requires high-throughput/robust analytical methods

• to deal with many samples
• in a short period of time
• to allow as many scientists to be able to perform test
• yet, maintaining consistent accuracy and minimising variation
inter/intra-samples
Common analytical tools in proteomics

Main approaches
• proteomics analysis- characterisation and
identification of proteins
• expression proteomics- expression profiles
• ‘interactome’- protein-protein interactions and
complexes

Major techniques
• Gel-based- 2D gel electrophoresis
• Mass spectrometry
• Protein arrays
• Interaction arrays
Electrophoresis for Protein separation

• one or two dimensional

• according to molecular weight (Da) and/ isoelectric point
of proteins
• most commonly used poly-acrylamide (neurotoxin) gel (PAG)
• like agarose (for DNA), acrylamide forms linkages polymerise
into long chains)
• results in protein fractionation, separation and isolation
crucial for studies
• can be prepared with a range of pore sizes (determines the
separation efficiency)
• pore size determined by Total amount of acrylamide (T) and
amount of cross-linking (C)
2D Gel Electrophoresis

Protocol for 2D gel electrophoresis for protein separation:

• sample preparation – reduction, alkylation

• isoelectric focusing (IEF) – gel rehydration
and focusing (1st dimension)
• gel electrophoresis- proteins on IEF strip (2nd
dimension)
• staining
Advantages of synthetic polyacrylamide gel is:
- thermo-stable
- transparent
- chemically inert
- withstand high voltage gradients
- can transfer proteins onto other materials e.g
membranes
- feasible for various staining
- can be digested, dried and stored
 Sample
Preparation
Rehydration and
1st dimension separation by
IEF

2nd dimension
Variable Gel Staining for Visualisation
separation by PAGE
Step 1: Sample preparation

• protein concentration of samples must be standardised for

all samples
• standard concentration assays e.g Bradford essay
• sample undergo reduction to break disulphide bonds
• this ensures proteins are in a linear form to enable
accurate migration
• addition of the right combination (based on pI IEF range)
of carrier ampholytes to assist in IEF
• ampholytes are solubles with different pI’s can form a pH
gradient
• ampholytes are already in the gel on the IPG strip/tube
• carrier ampholytes are mixed with samples to assist
proteins migrating

IPG- Immobilised pH Gradient

Step 2: Isoelectric Focusing (IEF) of proteins

• Separation of proteins based on their isoelectric point

• Proteins have either net +ve or –ve charge depends on
the surrounding pH
• the pI is the pH at which the protein has net zero charge

[+ve charge] = [-ve charge]

• if a protein is placed in a pH gradient and subjected to an

electric field, it will move until it reaches its pI
• at the pI, the protein has no charge – so it stops
moving/migrating
• difference of 1/100 pH unit is enough to separate two
proteins
• IEF enhances separation for 2D as compared to 1D (separation
by protein size only)
IEF apparatus
IPG strip post-IEF
 Step 3: 2nd dimension gel electrophoresis
• separation of proteins based on their size (molecular weight)

Each band may

contain several
proteins
 Step 4: Protein visualisation-Staining of 2D gels
• protein spots are not visible without staining
• staining has evolved from just for visualisation to a form
of direct comparison to highlight differences across samples

Three Main Types of Staining

i. Coomassie blue stains

simple and easy - cheapest
least sensitive – 5 ng protein detection limit
compatible with MS

ii. Silver nitrate stain

most sensitive – 0.1 ng - quantitative
but long process
not very compatible with MS

iii.Fluorescent stains
sensitive – almost as good as silver stain – 0.25 to 1ng
best dynamic range for quantitative
compatible with MS
 Comparison of protein visualisation
based on various staining methods

Coomassie blue

Silver stain

Fluorescent dye
Qualitative and Quantitative comparison of
Protein spots
 Matching spot-to-spot of two different samples e.g.
cancer vs normal cells
 Matching intensity of spots  concentration of protein

Example: Comparison of a
section of 2D protein profiles
from 9 different samples

Exact same spots may contain

-higher/lower [protein]
- same/different proteins
- none/with proteins

Sensitivity of dye is crucial!

Advantages and disadvantages of 2D
proteomics
Advantages
 High resolution capacity – thousands of proteins
separated at the same time
 Identification of post translational modifications
 Highly reproducible

Disadvantages
 Time consuming
 Technically difficult
 Difficult to automate
 Some proteins e.g. membrane proteins not recovered
 Not very useful in protein identification – spot
matching is difficult
Methods to Improve Protein Separation

1. Sample Fractionation

• performed to remove unwanted/high abundant proteins in order

to simplify complex protein profiles and analysis

• common techniques include:

a) chromatography- fractionation by size/charge
using normal columns separate proteins

b) removal of large abundant proteins e.g using Ab

this step allows more of the lower abundant proteins to
be loaded onto and appear clearly without being masked
by the higher abundant proteins e.g albumin. Also more
of the smaller proteins can make up the protein
concentration compared to without removal
2. Sample Enrichment
a) PTM-based enrichment of proteins of a particular type
proteins e.g using lectin columns to bind glycosylated
proteins to lectins and remove unbound proteins
(unglycosylated). Proteins that bind will be eluted
(disassociated with lectins) and collected for 2D

3. Smaller pH range IPG strip

Using a specific range of pI to focus on known proteins,
manipulation of the various IGP strips available

4. Specific gel percentage for PAGE

Using a certain percentage of gel for PAGE to separate
proteins in a desired MW range/size

*Note: sample preparation is extended (time-wise) for Options

1 & 2 and may involve additional clean-up steps (e.g salt
removal, volume reduction) before IEF
Effects of protein enrichment on protein separation
Less spots to compare, simpler analysis and clearer differences
observed
Example:

Comunale MA et al. Proteomic Analysis of Serum Associated Fucosylated Glycoproteins in the Development of
Primary Hepatocellular Carcinoma. J. Proteome Res., 2006, 5 (2), pp 308–315
Comunale MA et al. Proteomic Analysis of Serum Associated Fucosylated Glycoproteins in
the Development of Primary Hepatocellular Carcinoma. J. Proteome Res., 2006, 5 (2), pp
308–315
Isoforms in 2D profiles

• reduction procedure in sample preparation results in the

disassociation of protein chains/subunits
• the same exact protein can also form a train/series of
spots depending on the PTM which affects their MW and pI
• e.g haptoglobin- seen as haptoglobin alpha and beta chain
• haptoglobin beta chain in ovarian cancer (below) forms a
train of 6 spots, differing in the degree of glycosylation

Saldova et al. Glycobiology (2007) 17(12):1344-56

Differential Gel Electrophoresis
(DIGE)
 Direct comparison of proteins in two samples
 Labelled using fluorescence dyes producing different
colours
 Separated together on one IPG strip during IEF and same
gel in electrophoresis
 Overlay of images allows differential expression to be
easily detected
 Eliminates the problem of spot matching
 Quantitative comparison by measuring the amount of
fluorescence
 Dyes usually used - Cy3 (green) and Cy5 (red), when
overlaid color is yellow
Cy3
Overlay- common spots in yellow

Cy5

Spot intensity measured as peak height

 DIGE sample preparation
 Proteins in samples are evenly labeled with a
fluorescence dye
 Allows for more accurate quantitative comparison of
protein abundance
 Easier spot matching
 Can label either the e amino group of lysine/ thiol
group of cysteine
DIGE with 3 dyes - 2 samples + 1 internal standard

Pooled internal standard (aliquot of all samples) as the 3rd sample

- every protein from all samples will be represented in the internal
standard and each protein can therefore be compared to itself within
the internal standard to generate a ratio of relative expression
 Having an internal standard allows us to compare results in
diff. gels
a) reduce effects of variations (either technical or gel to
gel)
b)highlight significant differences (accurate spot quantitation
and statistics) by including the same amount of standard in
each gel
 allows us to compare results from several different gels
 detect differences in abundance of < 10%
Example of 2DIGE with internal standard for statistical
significance
a Cy3 Cy5 Cy2
Control Sample 1 Control Sample 2 Internal Standard

Gel 1

Disease Sample 1 Disease Sample 2 Internal Standard

b

Gel 2

c Disease Sample 3 Disease Sample 4 Internal Standard

Gel 3

Swatton et al., Molecular Psychiatry (2004) 9, 128–143

 An internal standard is essential for accurate quantification of
protein expression.
Panels a–c show theoretical images for three gels after scanning or
Cy2, Cy3 and Cy5 fluorescence. For each gel, control or disease
samples (labelled with Cy3 or Cy5) are run alongside the internal
standard (labelled with Cy2).

The internal standard is a pool of all samples included in the

study (control samples 1 and 2 and disease samples 1–4).
 Without the internal standard, expression of protein X (circled) in
disease samples 1 and 2 would be apparently increased compared with
control samples 1 and 2.
 Inclusion of the internal standard, however, shows that protein X
expression is unchanged in disease sample 1 and actually reduced in
disease sample 2.
 If gels 1 (a) and 3 (c) are analysed without an internal standard,
protein X would be apparently missing in disease samples 3 and 4
compared with control samples 1 and 2.
 By including the internal standard, however, it is obvious that
protein X has not resolved on gel 3 (c), and is thus missing as a
result of experimental, not biological, variation.

Swatton et al., Molecular Psychiatry (2004) 9, 128–143

Mass spectrometry (MS) for Protein
Separation

 Analytical technique that measures the mass/charge ratio of the

ions formed when a molecule or atom is ionised, vaporised and
introduced into a vacuum
 may also involve breaking molecules into fragments, enabling
its structure to be determined
 identification of proteins by peptide mass fingerprinting (PMF)
 Sequence peptides by tandem MS and post source degradation
(PSD)
 Quantitative analysis using a label tag e.g. ICAT, iTRAQ
 Study post translational modification (PTM)
The Human Proteome

 ~30,000 genes
 at least 10 times as many proteins
 may exceed 1,000,000 if include modifications
 5% of the proteins make up 80% of cell weight
 the dynamic range of the protein concentrations can
vary very greatly to > 106 fold

 At any time a human cell could have ~30,000 types of

proteins
 On a 2D gel, about 1,500 spots can be seen
 i.e. only 5% of all proteins are recovered
The dynamic range of serum proteins

From Andersson, L (2002) Mol. Cell. Proteomics 1, 845-867

Three basic components of MS

Ion source Mass Detector

Analyzer

Ion formation Ion separation Detection

MALDI – matrix Quadrupole Electron multiplier

assisted laser
TOF – time of
desorption
flight
ionisation
Ion Trap
ESI – electrospray
ionisation Fourier
transform
What is involved in MS?

 measures the mass-to-charge ratio (m/z) of

individual ions
 molecules converted to ions in the ionisation stage
 possess net charge (- or +)
 separated and detected based to m/z ratio
m/z ratio (m=mass, z= charge)

Example of peptide fragment/sequence

QAEVALRCAV mass = 1059 Dalton

QAEVALRCAV-H+ mass = 1060 Da

m/z = 1060/1
= 1060

H+- QAEVALRCAV-H+ mass = 1061 Da

m/z = 1061/2
= 530.5
MS in proteomics

After 2D gel
Cut protein
spot

MALDI –TOF for

PMF

Tandem MS/MS
for sequencing
 different proteins will give different PMF
 If the gene sequence is known, the PMF of a protein can
be derived using software
Important parameters in MS

 Resolution – ability to distinguish between

peptides , the smallest difference in m/z that can be
detected – down to 1 Da
 Sensitivity – ability to detect even the lowest
amount of peptide, the minimum amount of
molecule require for detection – down to
femtomole level
 Accuracy – correct ID of peptides
Sample preparation for MS-
Protein fragmentation

 Purified protein or spot from 2D gel is first fragmented to

smaller peptides
 e.g. by digestion with trypsin
 cleaves on the C-side of Arg and Lys (peptides will have R
or K at the C-termini)
 other proteases can be used e.g. clostripain, endopeptidase
 Digested sample is then ionized to generate charged ions in
a gas phase
1. Protein spot excision from 2D gels

•Automated/ manual removal of spots containing proteins of interest

•Spots transferred directly into eppendorfs or 96-well plates for
trypsin digestion
• Must be careful to avoid contamination skin/nail/hair proteins e.g
keratin

Automated spot
cutter
2. Protein digestion and extraction

Trypsin digestion- cleaves peptide bonds for MS analysis

In-gel- proteins need not be extracted until after digest

Protocol involves:
-Destaining –removal of dye from gel spot (interference)
* silver stained spots are not favourable
- reduction/alkylation- breaking disulphide bonds
- proteolytic cleavage
- extraction of digested peptides by incubation with extraction buffer
and supernatant collected for next step ie MS

Trypsin cleaves at the carboxyl end of basic amino acids arginine

(R) and lysine (K)
Endoglycosidase digestion (optional)

• if a protein is known/expected to be N- glycosylated and there

is a need to analyse/sequence the N-glycan, it must be cleaved
of before the protein is trypsin digested
• removal can be done using endoglycosidases which either
cleaves of the glycan completely from the Asn or leaves two
GlcNAcs still attached to Asn
• important to remember before performing MS analysis as the
masses of peptides will be affected by the GlcNAc presence
Methods of Sample Ionisation
1. MALDI
 matrix assisted laser desorption ionisation
 sample is co-crystallised with a matrix (low MM organic
matrix)
 a laser beam is used to excite the matrix (absorbs laser
wavelength (UV or IR) causing matrix-protein to expand into
gas-phase
 peptides are ionised by protonization
(addition of H+), using energy from
the laser (excited state proton transfer-
between photonized organic matrix and
a sample molecule)
Good quality matrix should:
a) have the same solubility as the analyte in specific solvents
b) stable in the vacuum environment
c) prevents cluster formation
d) absorbs a desired wavelength
e) can cocrystalize the sample
f) promotes analyte ionization

Dried-droplet method: drop of aqueos matrix solution mixed

with sample,left to dry, stable for storage
Requires removal of salt and contaminants prior to sample
measurement, high content of additives such as detergents e.g
SDS can suppress ionization, resulting in no/poor spectra
acquisition
2. SELDI-MS
(Surface Enhanced Laser Desorption/Ionisation MS)
Step 1 Protein (mix) spotted on a surface (chemically modified)

Step 2 Some proteins bind to the surface, others are removed by

washing
Step 3 After washing, matrix (e.g EAM) applied to the surface,
allowed to crystallize with the sample peptides
Step 4 Analyse by TOF-MS

• Binding to the SELDI surface acts as a separation step and the subset of
proteins that bind to the surface are easier to analyze
• Common surfaces include CM10 (weak-positive ion exchange), H50
(hydrophobic surface, similar to C6-C12 RP chromatography, IMAC30 (metal-
binding surface), and Q10 (strong anion exchanger)
• Surfaces can also be functionalized with antibodies, other proteins, or DNA
PRINCIPLE OF SELDI-TOF CHIP ASSAYS

Source: urology.jhu.edu
Advantages of SELDI:

• does not require protein electrophoresis

• only a small amount of sample (fluid, tissue) required
• robust and can be automated (HTP)
• high detection limit (femtomolar)
• allows analysis of hydrophobic proteins (membrane bound)

Example: rapid discovery of differentially expressed proteins

using femtomolar quantities of crude protein derived from
biopsy material (Lin et al. Modern Pathology, 2004)

Disadvantages:
• results are biased towards peptides and smaller proteins
(proteins <30 kDa)
• sensitivity and resolution of the TOF analyser falls off
markedly above 30kDa.
3. Electrospray ionisation (ESI)

 Sample added to a solvent

 An electrical field is applied
 Produces positively charged ions in gas phase
oxidation reduction

Taylor
cone
From right to left:
Delivery needle with Taylor cone  droplet formation, Coulomb
fission
(droplet explodes) with droplet evaporation  gas-phase ion
formation in transfer capillary

Taylor cone- positively charged, enriched with +ve ions due to

oxidation on the inner walls of the electroconductive
delivery needle

Nanoelectrospray
- Efficiency contributed by initial diameter of droplet formed and flow
rate
- Compared to ESI- higher sensitivity (1fmol vs 10fmol)
- smaller sprayer diameter (1-25µm vs 50-200µm)
- flow rate (1-1000nL/min vs 1-500µL/min)
- requires less voltage due to the positioning of the
delivery needle much closer to the transfer capillary
Methods of Ion Separation (Mass Analysers)
1. TOF (Time-of-flight) Separator

 ions are accelerated in an electrical field

 flies to the detector, passing through a ‘reflectron’
 the ions are all given the same amount of energy
 thus the time-of-flight (TOF) depends on the mass
 the m/z ratio can then be calculated from the TOF
 a peptide mass fingerprint is generated
 matched against theoretically predicted tryptic peptides of all
known proteins
 excessive internal energy of ions themselves or collisions
with free gas cause dissociation immediately after the ions
generated by laser illumination exit the high speed field
region
 MALDI is often used with a TOF separator
http://antoine.frostburg.edu/che...d017.htm
MALDI-TOF-TOF

Also many other hybrid machines e.g. Q-TOF, Ion

trap-TOF etc.
2. Quadrupole mass analyser

 Consists of 4 circular rods

 perfectly parallel to each other
 an direct current voltage and a radio frequency voltage
are applied across the rods
 only ions of a certain m/z can travel thru the centre of the
rods at a given ratio of voltage
 other ions has unstable trajectories and will collide with
the rods
 by adjusting the voltage ratio
 an entire m/z spectrum can be scanned  PMF
 or ions with a particular m/z value can be selected
2D LC-MS for high throughput proteomics
 aka shotgun proteomics
 2D gels are a bit tedious (imagine having to cut 1000
spots and do MS one by one)
 Automation  separate protein sample using
2Dimensional -HPLC
 i.e. use two types of columns to separate a complex
protein sample
 (same principle as 2D gel – separate using two
different methods e.g. charge and size)
 Each fraction is then fed sequentially into an MS
 can analyse the protein content of an entire tissue
1st dimension – separate by
charge on ion exchange
column

2nd dimension – separate by

hydrophobicity on reverse
phase column
 SUMMARY OF PROTEIN SEPARATION TECHNIQUES

COMPLEX PROTEIN
SINGLE MASS SPECTROMETRY

GEL ELECTROPHORESIS no exact

sequence
id based on
matches

TANDEM MASS
SPECTROMETRY
Exact amino acid
WESTERN sequence, correct
BLOTTING protein ID
No sequence
Specific binding