A Short Review

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A Short Review
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A Short Review
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A Short Review
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A Short Review
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A Short Review
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Properties of proteins in solution
Solubility of proteins in aqueous solutions
varies enormously. The solubility of a protein
in water is determined by its free energy when
surrounded by aqueous solvent relative to the
free energy when interacting with other
molecules or within membranes.
Water molecules interact with polar side chains
to form a hydration layer containing 0.3g water
per gram of protein, about two waters for every
amino acid residue. This bound hydration layer
has different properties to bulk water. The
density is 10 higher and it has a 15 greater
heat capacity suggesting much reduced molecular
motion. Despite reduced motion, water
molecules exchange with solvent on the 10s
timescale.
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Properties of proteins in solution
Globular proteins (mostly enzymes) have a near
uniform distribution of charged and polar groups
on their surface and their solubility is governed
by interactions of polar groups with
water. Proteins that play
structural roles have polar surfaces but they
interact with other proteins more favorably than
with water.
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Properties of proteins in solution
The solubility of a globular protein is the
lowest at its isoelectric point (the pH at which
the protein has a zero net charge). Solubility
increases as the pH moves away from this
isoelectric point. For a protein, the greater
the net-charge on the protein molecule, the
greater the electrostatic repulsion between
molecules (less aggregation) and the higher the
solubility.
At extremes of pH, most proteins can undergo a
local (or global) unfolding event that would
expose nonpolar residues to the surface (and
drastically affect solubility).
Random coil
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Properties of proteins in solution
The solubilities of proteins are affected by
co-solvents (especially salts). A protein
molecule in a low ionic strength aqueous solution
is surrounded by an excess of ion charges
opposite to the net charge of the protein
molecule This screening decribed by the
Debye-Huckel theory, which at physiological
ionic strength suggests a screening distance
about 8 Å. This ion screening decreases the
electrostatic free energy of the protein and
increases its solubility. At low salt strengths,
increasing the ionic concentration increases
protein solubility (salting-in effect). At high
ionic strength, the solubility of the protein
decreases and the magnitude of this effect
depends on the nature of the salt. -salts
interact preferentially with bulk water rather
than with proteins -general electrostatic
repulsion between salt ions and the less polar
protein interior
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Effects of organic solvents on folding
Organic solvents decrease the solubility of
proteins by lowering the dielectric constant of
the solvent. Polar interactions between solvent
and the protein surface are less favorable.
This lowers the stability of the folded state
relative to the unfolded state. Organic
solvents can denature proteins. Other polymers
can decrease the solubility of proteins. Two
polymers interact unfavorably in solution
because of volume exclusion (impossible for two
molecules to occupy the same space at the same
time). The second polymer is excluded from the
solvent near the protein surface producing
preferential hydration. This volume exclusion
effect is the basis for the ability of polymers
like PEG to precipitate proteins.
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Characterization and detection of proteins
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Characterization and detection of proteins
The second most important is to know how much
protein you have.
The intrinsic spectral properties of an amino
acid side chain are barely affected by the
conformation of the polypeptide
backbone. Absorbance Max Molar Absorbance
(M-1 cm-1) Phenylalanine 257.4
nm 197 Tyrosine 274.6 nm 1420 Tryptophan 279
.8 nm 5600
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Intrinsic spectral properties of an amino acid
side chain are barely affected by conformation
of the polypeptide backbone.
Empirically, the molar extinction coefficient for
a polypeptide can be determined by measuring the
absorbance at 280 nm Molar extinction
coefficient of Tyr 1280 of Trp
5690 Molecular weight
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Characterization and detection of proteins
One can determine protein concentration by
calculating the molar extinction coefficient and
then measuring the absorbance at 280 nm. If a
protein has a molar extinction coefficient of 1,
then an absorbance of 1 at 280 nm corresponds to
a protein concentration of 1 mg/ml. Peptide
backbone absorbs light at wavelengths less than
240 nm (maxima is at 218 nm). Chose 280 nm to
measure molar extinction to avoid absorbance by
the peptide backbone.
Molar extinction coefficient of Tyr 1280
of Trp 5690 Molecular
weight Protein conc (mg/ml) Absorbance
at 280 nm extinction coeff path
length
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The Bradford Method (Coomassie Dye)
This method relies on the binding of protein to
Coomassie Brilliant Blue G-250 which causes an
absorbance shift from 465 nm to 595 nm.
The Coomassie dye binds primarily with basic and
aromatic side chains. The interaction with
arginine is very strong and less strong with
histidine, lysine, tyrosine, tryptophan, and
phenylalanine. About 1.5 to 3 molecules of dye
bind per positive charge on the protein.
Aromatic
Ionic
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Amino Acid Analysis
Amino acid analysis is a process to determine the
quantities of each individual amino acid in a
protein.
Hydrolysis A known amount of internal standard
(norleucine) is added to the sample. The tube
is placed in 6 N HCl and the protein is
hydrolyzed by the HCl. Derivatization The free
amino acids cannot be detected by HPLC unless
they have been derivatized.Derivatization by
reacting the free amino acids with
phenylisothiocyanate (PITC)
HPLC separation The PTC-amino acids are separated
on a reverse phase C18 silica column and the PTC
chromophore is detected at 254 nm. Chromatographi
c peak areas are identified and quantitated using
a data analysis system
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Exploiting chemical properties of
proteins Purification

• Purification procedures attempt to maintain the
  protein in native form. Although some proteins
  can be re-natured, most cannot!
• To purify a protein from a mixture, biochemists
  exploit the ways that individual proteins differ
  from one another. They differ in
• Thermal stability

Precipitation with ammonium sulfate (salting out)
solubility
For most protein purifications, all steps are
carried out at 5C to slow down degradative
processes.
Ammonium sulfate precipitation is cheap, easy,
and accommodates large sample sizes. It is
commonly one of the first steps in a purification
scheme.
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The major problem in protein purification
Maximize yield get as many red marbles as
possible High purity take only the red
marbles. These two are opposing forces.
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Ion exchange chromatography
Anion exchange Column is postively charged
(can bind negativey charged proteins). Cation
exchange Column is negativey charged (can bind
negatively charged proteins).
Exploit the isoelectric point of a protein
to Separate it from other macromolecules.
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Size exclusion chromatography
Porous beads made of different but controlled
sizes. Smaller proteins go in and out of beads
and will be retained in the resin. Large
proteins will only go into large beads and will
be retained less. Very large proteins will not
go into any of the beads (exclusion limit). Can
be used as a preparative method or to determine
the molecular weight of a protein in solution.
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Affinity chromatography
A ligand with high affinity to the protein is
attached to a matrix. Protein of interest binds
to ligand and is retained by resin. Everything
else flows through. Can use excess of the
soluble ligand to elute the protein.
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Weighing naked proteins An introduction to
mass spectrometry

• A mass spectrometer creates charged particles
  (ions) from molecules.
• Common way is to add or take away an ions
• NaCl e- ? NaCl-
• NaCl ? NaCl e-
• It then analyzes those ions to provide
  information about the molecular
• weight of the compound and its chemical
  structure.
• There are many types of mass spectrometers and
  sample introduction
• techniques which allow a wide range of analyses.

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Mass Spectrometry

• All mass spectrometers consist of three distinct
  regions.
• Ionizer -
• Ion Analyzer -
• Detector

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Mass Spectrometry (MS)
1. Ionizer
Electron ionization
Ionization is the act of placing a charge on a
neutral molecule.
The sample must be delivered as a gas which is
usually accomplished by heating the sample to
vaporize it off of the probe. Once in the gas
phase, the compound passes into an electron
ionization region where it interacts with a beam
of electrons of nearly homogeneous energy (70
electron volts), typically causing electron
ejection and some degree of fragmentation.
Electron ionization is most useful for compounds
below a molecular weight of 400
Daltons Electron ionization is principally used
as a detector for gas chromatography
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Interpretation of Data
EI ionization introduces a great deal of energy
into molecules. It is known as a "hard"
ionization method. This is very good
for producing fragments which generate
information about the structure
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Mass Spectrometry (MS)
1. Ionizer
Electrospray
Ionization is the act of placing a charge on a
neutral molecule.
Electrospray ionization generates ions directly
from solution by creating a fine spray of
highly charged droplets in the presence of a
strong electric field.
As the droplet decreases in size, the electric
charge density on its surface increases. The
mutual repulsion between like charges on this
surface becomes so great that it exceeds the
forces of surface tension, and ions begin to
leave the droplet through what is known as a
"Taylor cone". The ions are then
electrostatically directed into the mass
analyzer. Vaporization of these charged droplets
results in the production of singly or
multiply-charged gaseous ions.
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Electrospray Ionization Mass Spec
During standard electrospray ionisation, the
sample is dissolved in a polar, volatile solvent
and pumped through a narrow, stainless steel
capillary at a flow rate of between 1 µL/min and
1 mL/min. A high voltage of 3 or 4kV is applied
to the tip of the capillary, and as a consequence
of this strong electric field, the sample
emerging from the tip is dispersed into an
aerosol of highly charged droplets.
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Electrospray Ionization Mass Spec
The charged droplets diminish in size by solvent
evaporation, assisted by a warm flow of nitrogen
known as the drying gas which passes across the
front of the ionization source. Eventually
charged sample ions, free from solvent, are
released from the droplets, some of which pass
through a sampling cone or orifice into an
intermediate vacuum region, and from there
through a small aperture into the analyzer of
the mass spectrometer, which is held under high
vacuum. The lens voltages are optimized
individually for each sample.
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Mass Spectrometry (MS)
1. Ionizer
MALDI
Ionization is the act of placing a charge on a
neutral molecule.
In MALDI analysis, the analyte is first
co-crystallized with a large molar excess of a
matrix compound, after which pulse UV laser
radiation of this analyte-matrix mixture results
in the vaporization of the matrix which carries
the analyte with it. MALDI has had its biggest
impact on the field of protein research. The
ability to generate MALDI-MS data on whole
proteins and proteolytic fragments is extremely
useful for protein identification and
characterization.
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Matrix Associated Laser Desorption/Ionization
MALDI is based on the bombardment of sample
molecules with a laser light to bring about
sample ionisation. The sample is pre-mixed with
a highly absorbing matrix compound for the most
consistent and reliable results. The matrix
transforms the laser energy into excitation
energy for the sample, which leads to sputtering
of analyte and matrix ions from the surface of
the mixture. In this way energy transfer is
efficient and also the analyte molecules are
spared excessive direct energy that may
otherwise cause decomposition.
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Matrix Associated Laser Desorption/Ionization
MALDI is a soft ionisation method and so
results predominantly in the generation of singly
charged molecular-related ions regardless of the
molecular weight, hence the spectra are
relatively easy to interpret. In positive
ionisation mode the protonated molecular ions
(MH) are usually the dominant species.
Positive ionisation is used in general for
protein and peptide analyses. In negative
ionisation mode the deprotonated molecular ions
(M-H-) are usually the most abundant species.
Negative ionisation can be used for the analysis
of oligonucleotides and oligosaccharides.
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Mass Spectrometry (MS)
2. Analyzer
Immediately following ionization, gas phase ions
enter a region of the mass Spec. known as the
mass analyzer.
The mass analyzer is used to separate ions within
a selected range of mass-to-charge (m/z) ratios.
The analyzer is an important part of the
instrument because of the role it plays in the
instrument's accuracy and mass range. Ions are
typically separated by magnetic fields, electric
fields, or by measuring the time it takes an ion
to travel a fixed distance.
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Mass Spectrometry (MS)
2. Analyzer - Magnetic Analysis
Immediately following ionization, gas phase ions
enter a region of the mass Spec. known as the
mass analyzer.
In magnetic analysis, the ions are accelerated
(using an electric field) and are passed into a
magnetic field. A charged particle traveling at
high speed through a magnetic field will
experience a force, and travel in a circular
motion with a radius depending upon the m/z and
speed of the ion. A magnetic analyzer separates
ions according to their radii of curvature, and
therefore only ions of a given m/z will be able
to reach a point detector at any given magnetic
field. A primary limitation of typical magnetic
analyzers is their low resolution.
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Mass Spectrometry (MS)
2. Analyzer - Double sector magnetic/electrostatic
Immediately following ionization, gas phase ions
enter a region of the mass Spec. known as the
mass analyzer.
Combines the magnetic mass analyzer with an
electrostatic analyzer. The electric sector
acts as a kinetic energy filter allowing only
ions of a particular kinetic energy to pass
through its field, irrespective of their
mass-to-charge ratio. Given a radius of
curvature, R, and a field, E, applied between two
curved plates, the equation R  2V/E allows one
to determine that only ions of energy V will be
allowed to pass. Thus, the addition of an
electric sector allows only ions of uniform
kinetic energy to reach the detector, thereby
increasing the resolution of the two-sector
instrument to 100,000.
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Mass Spectrometry (MS)
2. Analyzer - Tandem mass spectrometry
In contrast to electron ionization (EI) which
produces many fragment ions, the new ionization
techniques are relatively gentle and do not
produce a significant amount of fragment ions.
Tandem mass spectrometry allows one to induce
fragmentation and mass analyze the fragment
ions. This is accomplished by collisionally
generating fragments from a selected ion and
then mass analyzing the fragment ions.
Fragmentation can be achieved by inducing
ion/molecule collisions by a process known as
collision-induced dissociation (CID).
Collision-induced dissociation is accomplished by
selecting an ion of interest with a mass
analyzer and introducing that ion into a
collision cell. The selected ion then collides
with a collision gas resulting in fragmentation.
The fragments are then analyzed to obtain a
fragment ion spectrum.
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Mass Spectrometry (MS)
2. Analyzer - Quadrupole
Quadrupoles are four precisely parallel rods with
a direct current (DC) voltage and a superimposed
radio-frequency (RF) potential. And by scanning
a pre-selected radio-frequency field one
effectively scans a mass range. Quadrupole mass
analyzers have been used in conjunction with
electron ionization sources since the 1950s and
are the most common mass spectrometers in
existence today. Quadrupoles have three primary
advantages. First, they are tolerant of
relatively poor vacuums, which make them
well-suited to electrospray ionization since the
ions are produced under atmospheric pressure
conditions. Secondly, quadrupoles are now
capable of routinely analyzing up to a m/z of
3000, which is useful because electrospray
ionization of proteins and other biomolecules
commonly produces a charge distribution below m/z
3000. Finally, the relatively low cost of
quadrupole mass spectrometers makes them
attractive as electrospray analyzers.
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Mass Spectrometry (MS)
2. Analyzer - Quadrupole Ion Trap
In an ion trap the ions are trapped in a radio
frequency quadrupole field. One method of using
an ion trap for mass spectrometry is to generate
ions externally with electrospray which are then
injected into the trapping volume. The ions are
then ejected and detected as the radio frequency
field is scanned. Further, it is also possible
to isolate one ion species by ejecting all others
from the trap. The isolated ions can
subsequently be fragmented by collisional
activation and the fragments detected to
generate a fragmentation spectrum.
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Mass Spectrometry (MS)
3. Detectors
Once the ion passes through the mass analyzer it
is then detected by the ion detector, the final
element of the mass spectrometer. The detector
allows a mass spectrometer to generate a signal
current from incident ions by generating
secondary electrons, which are further amplified.
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Mass Spectrometry (MS)
3. Detectors- Faraday Cup
A Faraday cup operates on the basic principle
that a change in charge on a metal plate results
in a flow of electrons and therefore creates a
current. One ion striking the surface of the
Faraday cup induces several secondary electrons
to be ejected and temporarily displaced. This
temporary emission of electrons induces a current
in the cup and provides for a small
amplification of signal when an ion strikes the
cup. This detector is relatively insensitive,
yet robust and simple in design.
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Mass Spectrometry (MS)
3. Detectors- Electron multiplier
Whereas a Faraday cup uses one surface, an
electron multiplier is made up of a series of
surfaces maintained at ever increasing
potentials. Ions strike the surface, resulting
in the emission of electrons. These secondary
electrons are then attracted to the next surface
where more secondary electrons are generated,
ultimately resulting in a cascade of electrons.
Typical amplification or current gain of an
electron multiplier is one million.
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Mass Spectrometry
3. Detectors- Photomultiplier dynode
With the photomultiplier conversion detector,
electrons strike a phosphorus screen. The
phosphorus screen, much like the screen on a
television set, releases photons once an
electron strikes. These photons are then
detected by a photomultiplier, which operates
with a cascading action much like an electron
multiplier. The primary advantage of the
conversion dynode setup is that the
photomultiplier tube is sealed in a vacuum
unexposed to the internal environment of the
mass spectrometer.
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Applications
Atomic mass The discovery of isotopes with the
first mass spectrometer answered the question
about the integer value of atoms (e.g. carbon-12,
nitrogen-14 and oxygen-16). Geochemistry The
early studies of the radioactive decay of uranium
and thorium into lead caused the British
physicist Ernest Rutherford to suggest that this
process could be used to determine the age of
rocks and, consequently, of the Earth by
observing the amount of helium retained by a
rock relative to its uranium and thorium
contents. Organic Chemistry With a
high-resolution mass spectrometer it is
possible to carry out mass measurements on the
molecular ion (or any other ion in the spectrum)
to an accuracy of approximately one part in one
million. This mass provides the best index for
determining ionic formulas.
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Applications
Combinatorial chemistry Combinatorial chemistry
is used to create large populations of
molecules, or libraries, whereby the generation
of huge numbers of compounds increases the
probability that they will find novel compounds
of significant therapeutic or commercial
value. BiochemistryThe ability to analyze
complex mixtures has made electrospray and MALDI
very useful for the examination of proteolytic
digests, an application otherwise known as
protein mass mapping. Through the application
of sequence specific proteases, protein mass
mapping allows for the identification of protein
primary structure. Performing mass analysis on
the resulting proteolytic fragments thus
yields information on fragment masses with
accuracy approaching 5 ppm, or 0.005 Da for a
1,000 Da peptide. The protease fragmentation
pattern is then compared with the patterns
predicted for all proteins within a database and
matches are statistically evaluated.
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Applications
BiochemistryProtein mass mapping has also been
used for studying higher order protein structure
by combining limited proteolytic digestion, mass
analysis, and computer-facilitated data analysis.
In the analysis of protein structure, enzymes
are used to initially cleave surface accessible
regions of the protein or protein complex.
These initial cleavage sites are then
identified using accurate mass measurements
combined with the protein's known structure and
the known specificity of the enzyme.
Computer-based sequence searching programs
allow for the identification of each proteolytic
fragment, which in turn can be used to map the
protein's structure.