Principles of separation of

Biomolecules
 Principle

 Proteins move in the electric field. Their relative speed depends on the
charge, size, and shape of the protein.
Electrophoresis
Electrophoresis is the migration of charged molecules, particles or ion in a
liquid medium under the influence of an electric field
There are several varieties of migration techniques defined by the type of
support used
1. Paper chromatography– amino acids, small peptides
2. Polyacrylamide gel electrophoresis– Proteins, small DNA/RNA (<500bp)
3. Agarose gel electrophoresis – DNA/RNA
GEL ELECTROPHORESIS

What is a gel?
Gel is a cross linked polymer whose composition and porosity is
chosen based on the specific weight and porosity of the target
molecules.

Types of Gel:
 Agarose gel.
 Polyacrylamide gel.
TWO-DIMENSIONAL ELECTROPHORESIS
 This technique combines the technique IEF (first dimension), which
separates proteins in a mixture according to charge (PI), with the size
separation technique of SDS-PAGE second dimension).

 The combination of these two technique to give two-dimension(2-

D)PAGE provides a highly sophisticated analytical method for
analysing protein mixtures.
Isoelectric focusing (IEF)
• This separates proteins based on isoelectric point
• The isoelectric point is the pH at which the protein has no net charge.
• pH gradients may be large 2-10 or small 6-7
• Typically this is done with an immobilized pH gradient gel strip or
with a tube gel containing a low concentration of polyacrylamide.
• Ampholytes are added to create a pH gradient in an electric field and
the proteins are loaded.
• The IEF gel is placed in an electrophoresis system for up to 24 hours
and the proteins form tight bands at their isoelectric point.
• The IEF gels are now ready for the second method.
Figure is from “Principles of Biochemistry” Lehninger, Fourth Edition
SDS Polyacrylamide Gel Electrophoresis
• The second dimension separates the proteins based on size.
• There are two parts, the stacking gel which concentrates the sample and
the running gel that is used to separate the proteins.
• The IEF gel is soaked in a solution containing chemical to denature the
proteins including sodium dodecyl sulfate a detergent which gives the
proteins a net negative charge. This means that all proteins will move in
one direction.
• The IEF gel is then put in the one long well in the stacking gel, sealed in
place with agarose, and the proteins subjected to an electric field to
separate.
• The larger proteins are found at the top and the smaller ones are found at
the bottom of the gel.
2-D Gel Electrophoresis
2-Dimensional Gel Electrophoresis
• In a 2D gel the proteins appear as spots on the gel rather than bands.
These spots can then be further processed or used for mass spectrometry
directly.
• Further processing usually includes spot excision, trypsin digestion, and
mass spectrometry.
• Analysis may also include differential 2D gel electrophoresis
• In this case a control and sample are separately labeled with a fluorescent
molecule.
• The samples are mixed and electrophoresed in the same gels.
• A laser scanner is used to identify each spot and a program puts the two images
together.
2-Dimensional Gel Electrophoresis

 Using this method one can routinely resolve between 1000 and 3000 proteins from a
cell or tissue extract and in some cases the separation of between 5000 and 10000
proteins also possible.

 The result of this is a gel with proteins spread out on its surface. These proteins can
then be detected by a variety of means, but the most commonly used stains are silver
and coomasie staining.
Visualization of proteins
Coomassie blue staining
Detect 36-47ng

Silver staining
Detect 0.5-1.2ng

Fluorescent staining
Detect 1-2 ng

From Jefferies, et al., Images from

http://www.kendricklabs.com/2d+CoomassieBlue.htm
http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html#Section%201 http://www.unil.ch/dbcm/page48211_fr.html
Advantages of 2D-gel analysis

1) Very sensitive
2) High resolution
>10,000 different proteins
3) Unbiased search

Limitations of 2D-gel analysis

1) Lack of resolution of all proteins present
2) Irreproducibility of results
Typical steps in 2D-gel analysis
1) Isolate sample
2) Separate proteins by 2DGE
3) Visualize proteins and excise spots of interest
4) Digest proteins with trypsin
5) Use MALDI-MS to measure molecular mass
6) Use LC-MS/MS or MALDI-MS/MS to obtain sequence information
Mass Spectrometry
• Separates ions based on mass to charge ratio. Charges are placed on
the protein or the peptide by ionization.
• Two most common types of ionization are:
Matrix-Assisted Laser Desorption Ionization (MALDI)
• MALDI causes fragmentation of the protein during ionization. Can be used to
get more information about the fragments. Easier to do than ESI.
Electrospray ionization (ESI)
• ESI can give whole protein masses as well as complex masses. If the proteins
is first separated by reverse phase HPLC before injection only the subunits
masses will be known.
Matrix-Assisted Laser Desorption Ionization (MALDI)

• MALDI is soft ionization technique used in mass spectrometry, allowing the

analysis of biomolecules such as DNA, proteins, peptides & sugar or
polymers such as dendrimers and macromolecules
• MALDI causes fragmentation of the protein during ionization. Can be used
to get more information about the fragments. Easier to do than ESI.

It is three steps method.

I. The sample is mixed with a suitable matrix & applied to a metal plate.
II. A pulsed laser irradiate a sample triggering desorption of matrix material.
III. Ionization of analyte molecules
Essential Features of all MS

• Production of ions in the gas phase

• Acceleration of the ions to a specific velocity in an electric field
• Separation of ions in a mass analyzer
• Detection of each species of particular m/z ratio
MALDI TOF SCHEMATIC
REPRESENATION

Sample Linear
plate Extraction Reflector detector
grids Timed ion detector
selector Reflector
Laser

Camera
Pumping Pumping
MALDI Matrix
• Matrix consists of crystallized molecule of which the most 3
commonly used are 3,5dimthoxy-4-hydroxy cinnamic acid
(sinapinic acid), alpha-cyano-4 hydroxycinnamic acid (CHCA/alpha-
cyano/alpha-matrix), 2,5-dihydroxybenzoic acid (DHB).
• A solution of one of these molecules is made, often in a mixture of
highly purified water & an organic solvent ACN i. e. acetonitril.
• Trifluoroacetic acid may also be added .
• A good example of matrix solution would be 20ug/ml sinapinic acid
in ACN:water:TFA (50:50:0.1).
Ionization Mechanism

• It involve absorption of light by the

matrix
• Transfer of this energy to the analyte
• which then ionizes into the gas phase as
a result of the relatively large amount of
energy absorbed.
• To accelerate the resulting ions into a
flight-tube in the mass spectrometer
they are subjected to a high electrical
field .
Time Mass Detectors
• The typical detector used with MALDI is the time of flight mass
detector (TO-FMS)
• TOF is a method where the ions are accelerated by an electric field,
resulting in ions of the same strength to have the same kinetic
energy .
• Time it takes for each ion to traverse the flight tube and arrive at the
detector is based on its mass-to-charge ratio.
• Therefore heavier ions of the same charge reach with lower speeds
and ions with higher charge will increase its velocity compared with
ions of same mass.
MALDI Advantages

• Gentle Ionization technique

• High molecular weight analyte can be ionized
• Molecule need not be volatile
• Sub-picomole sensitivity easy to obtain
• Wide array of matrices

see reference 8
TOF Advantages
• All ions detected at once
• High mass accuracy and resolving power possible
• Reasonable performance for cost
• High mass and low charge ions not a problem
 APPLICATIONS
1. Proteomics
• To identify, verify, and
quantitate: metabolites,
recombinant proteins, proteins
isolated from natural source,
peptides & their amino acid
sequences
2. Pharmaceutical Analysis
• Bioavailability studies
• Drug metabolism studies,
pharmacokinetics
• Characterization of potential
drugs
• Drug degradation product
analysis
• Screening of drug candidates
• Identifying drug targets

3. Microbiology
• It is used for the identification of
microorganisms.
• Species diagnosis by this
procedure is much faster, more
accurate & cheaper than other
procedures based on
biochemical tests.
Continued…
4. Forensic analysis
5. Environmental analysis
• Pesticides on foods
• Soil and groundwater
contamination