7 Proteomics – Applications and Techniques

Proteomics is the branch of the biological sciences which

deals with proteins. Proteins are basic constructional blocks
and functional elements of living organisms. They are
involved in all processes occurring in cells and tissues and
therefore are linked to each other by numerous interactions.
Proteomics can be viewed as the study of properties,
interactions, and functions of proteins. Proteomics can be
defined as the large-scale study of protein properties such as
expression levels, post-translational modifications and
interactions with other molecules to obtain a global view of
cellular processes at the protein level.
Proteomics can be broadly divided into two areas of research:
(i) Protein Expression Mapping - Protein expression mapping involves the quantitative study of global changes
in protein expression in cells, tissues or body fluids using 2D gel electrophoresis coupled with mass
spectrometry. The identity of proteins within spots on 2D gels can be rapidly determined by in-gel proteolysis
and peptide mass fingerprinting using mass spectrometry. The aim of protein expression mapping is to
compare the spectrum of proteins expressed in cells or tissues under different environmental conditions or
from different disease states. Furthermore, an understanding of post-translational modifications of expressed
proteins under different conditions or disease-states is sought.
a. For clinical applications, the objective of protein expression mapping is to identify proteins that are up-
or downregulated or modified in a disease-specific manner to use as diagnostic reagents or possible
therapeutic targets.
b. For basic research, the goal is to understand how the regulation of protein levels or modifications
contributes to the execution and coordination of cellular processes.
(ii) Protein Interaction Mapping - Protein-protein interaction mapping involves determining, on a proteome-
wide scale, the interaction partners for each of the encoded proteins of a cell or organism. The majority of
the proteins within a cell are thought to work in concert with other proteins via direct physical interactions
to carry out cellular processes. Therefore, creation of a protein-protein interaction map of the cell would be
of great value for understanding the biology of that cell.
SAGE – Serial Analysis of Gene Expression
Serial analysis of gene expression (SAGE) is a
transcriptomic technique used by molecular biologists to
produce a snapshot of the messenger RNA population in
a sample of interest in the form of small tags that
correspond to fragments of those transcripts. Several
variants have been developed since, most notably a more
robust version, LongSAGE, RL-SAGE and the most
recent SuperSAGE. Many of these have improved the
technique with the capture of longer tags, enabling more
confident identification of a source gene.

Isolate the mRNA and convert it to • Using biotinylation will allow you to isolate your
cDNA using reverse transcriptase cDNA fragments later on in the process.

Mix your cDNA with • These beads will bind to the biotin-cDNA complex.
streptavidin beads
Cleave the cDNA using a restriction
endonuclease enzyme, called an
anchoring enzyme

Rinse and divide in 2 different • Cleaved cDNA that is no longer bound to the
solutions beads is now removed by rinsing.

Add an oligonucleotide – either A

or B – to each solution

•This removes the cDNA from beads to create a short “tag” of around 11
Cleave the cDNA using tagging nucleotides
enzyme •Tags have sticky ends, repaired using DNA polymerase (DNAP) and gives blunt
end fragments

• Generates ditags with A and B adaptor ends.

Ligate the blunt-end tags together • This string is then amplified by PCR using A and B primers

Use Anchoring enzyme to cleave •Allows the ditags to form long chains cDNA, called cDNA
ditags to remove the A and B concatemers
oligonucleotides •Each ditag is separated by an anchoring enzymes recognition site.

Transform your concatemers into bacteria

and allow the bacteria to replicate to form
high quantities of your concatemers.

Isolate your concatemers using •Use high-throughput DNA sequencing to quantify each individual tag.
•Create a gene expression profile for your original sample of mRNA-
any extraction protocol. containing cells.
MASS SPECTROMETRY
In a mass spectrometer, operated under high vacuum, analytes are ionized, sometimes fragmented, and then
directed to a mass analyzer where they are separated according to their mass-to-charge ratios, m/z. The ion current
generated is plotted versus m / z ratios to produce a mass spectrum, which is characteristic of the original analyte
and can be used for both qualitative and quantitative analysis. Usually the z value of the ions is + 1, so the m / z
ratio actually is the same as the mass for that ion.
A mass spectrometer consists of three major parts: the ion source, the mass analyzer, and the detector. Since the
mass analyzer and the detector (and many of the ion sources) require low pressure for operation the instrument
also needs a pumping system. Moreover, another system is required to record the signal registered by the detector.

Ion Sources
In a mass spectrometer the role of the ion source is to create gas phase ions. Analyte atoms, molecules, or clusters
are transferred into gas phase and ionized either concurrently (as in electrospray ionization) or through separate
processes (as in the glow discharge).

Sr. Method Category Ion Type Application

No
1 Desorption Spray Non Volatile Direct, preparation free analysis of
electrospray Molecular Ions samples
ionization
2 Direct analysis in Discharge Non Volatile Direct, preparation free analysis of
real Molecular Ions samples
time
3 Secondary Ion (mass Particle induced Non Volatile Semiconductors; Surface analysis;
spectrometry) desorption/ Molecular Ions Imaging, Metabolite detection
ionization
4 Matrix-assisted laser Photon induced Non Volatile Soft method; Large
desorption/ desorption/ Molecular Ions molecules
ionization ionization
5 Thermal ionization Ionization by heating Atomic ions Isotope ratio, Trace analysis; Solid
samples
6 Glow discharge Plasma source Atomic ions Trace analysis
7 Electron ionization/ Electron induced Volatile Smaller molecules; GC-MS;
Chemical ionization ionization molecular ions Extensive
libraries
8 Photoionization Photoionization Volatile Smaller molecules; GC-MS
molecular ions
9 Field ionization Ionization by strong Volatile Molecular compounds
electric field Molecular ions
10 Field desorption Desorption/ionization Nonvolatile First soft method; Large molecules
by strong electric molecular
field ions

Mass Analyzers
A mass analyzer is a device that can separate species, that is, atoms, molecules, or clusters, according to their
mass. The separation should also be independent of the chemical conformation of the species. All mass analyzers
presently in use are based on electromagnetism so ions are required to obtain separation. Therefore, an ion source
has to be coupled to the analyzer. The analyzer will then separate ions coming from the source according to their
m/z. There are several types of mass analyzers used in mass spectrometric research and they can be divided into
different categories, such as magnetic or pure electric, scanning or non-scanning (pulse based), and trapping or
non-trapping analyzers.
Time-of-Flight
A time-of-flight (TOF) mass analyzer separates ions according to the time difference between a start signal and
the pulse generated when an ion hits the detector, that is, the time of flight.
Normally the sample plate is floated on a high positive or negative potential, typically 5 to 30 kV, and as the ions
enter the gas phase they are accelerated towards ground potential. When leaving the acceleration region, all intact
ions with the same charge will ideally have the same kinetic energy, but different, mass-dependent, velocities.
The ions are then allowed to drift in a field-free region towards a detector. The time difference between the start
signal and the pulse generated when the ion hits the detector is the time of flight (tTOF) and can be expressed as-

Where L = length of the field-free region,

v = ion velocity after acceleration,
m = mass of the ion,
q = charge of the ion,
Ua = accelerating electric potential difference,
z = charge state.
The faster or lighter the ion, the shorter the time of flight and the time-of-flight spectrum obtained can be
converted into a mass spectrum. There is no need to know the exact potentials and distances of the spectrometer,
as the time/mass conversion is made by calibration with ions of known masses.

Magnetic/Electric Sector
Sector analyzers are easily adaptable to continuous ion sources, such as EI, dynamic SIMS, ICP, and ESI. As in
the TOF case, the ions leaving the source in a magnetic sector mass spectrometer are accelerated to a high velocity.
In a reverse-geometry instrument, the ions then pass through a magnetic sector, in which the magnetic field is
applied perpendicularly to the direction of the ion beam. The magnetic field will not change the velocity of the
ions, but force them into a circular motion with a radius that depends on the magnetic field strength, and the mass-
to-charge ratio and velocity of the ions so that –

m is the ion mass, q the ion charge,B the magnetic field strength (or more correctly the magnetic flux density), r
the radius of the ion trajectory in the magnetic field, and Ua the potential difference over the acceleration region.
Only ions that pass through a narrow slit will be detected. A
magnetic sector will also act as a lens, and ions of the same
m/z but with slightly different velocity will not be focused at
the same point. Hence, the resolution will be limited by the
initial velocity spread of the ions. To obtain better resolution,
it is necessary to add an electric sector that focuses ions
according to their kinetic energy.
The electric sector also applies a force perpendicular to the
ion beam direction and is, therefore, also shaped as an arc. If
the electric sector is designed so that the dispersion of ions
due to their velocity spread is exactly equal and opposite to
that of the magnetic sector, the result of combining the two types of sectors is a zero net velocity dispersion, that
is, double focusing. In a forward-geometry sector instrument the electric sector is placed before the magnetic
sector. Reverse geometry means that the magnetic sector is placed before the electric sector.

Quadrupole Mass Filter

The quadrupole mass filter is a relatively small device that can be set to let through ions within a very limited m/z
range only. The trajectories of the ions with either higher or lower m/z will bend and the ions will never escape
the filter. A mass spectrum is acquired by scanning through the whole m/z range of interest and detecting how
many ions pass the filter at each m/z.
The analyzer employs a combination of direct-current (DC) and radio frequency (RF) potentials. Four parallel
rods are arranged symmetrically. Opposite rods are connected electrically in pairs. The
two pairs will, at any given time, have potentials of the same magnitude, but of opposite
sign. Ions emerging from the source, typically accelerated over a potential of 5 to 20 V,
enter the analyzer region between the rods and travel parallel to the rods. At given values
of the DC and RF potentials and the RF frequency, only ions within a certain narrow
m/z range will have stable trajectories through the quadrupole. The m/z range for ions
allowed to pass through depends on the ratio between the DC and RF potentials. Ions
that do not have a stable trajectory will collide with the rods, never reaching the detector.

Quadrupole Ion Trap

In the quadrupole ion trap (QIT), ions are trapped and stored in a potential well. A mass spectrum is acquired by
ejecting the ions from the potential well in order of ascending m/z and detecting them. The trap can also be used
to selectively store ions with a particular m/z, and perform further experiments, for example, fragmentation in
several consecutive steps. The
cylindrical quadrupole ion trap is
based on the same principle as the
quadrupole mass filter, but the
geometry is different. The
cylindrical QIT, or Paul trap, was
developed almost simultaneously
with the quadrupole mass filter.
Under stable conditions, ions
moving around inside such traps
will ideally continue to do that
forever.
Orbitrap
The orbitrap is the most recently invented mass analyzer. Like with the QIT, ions are
trapped and stored in a potential well. However, instead of ejecting the ions for external
detection the frequency of the trapped oscillating ions is measured. This method
provides substantially better resolution and mass accuracy in normal operation. The
orbitrap is a pure electrostatic device operated using a logarithmic electrostatic field
between its inner and outer electrodes, as well as a quadrupolar field between its end
caps. Between the inner and outer electrodes a DC voltage is applied giving a
logarithmic potential. Ions injected (through a narrow slit in the outer electrode)
perpendicular to the wire and having an appropriate velocity will circulate in an orbit
around the wire.

Fourier Transform Ion Cyclotron Resonance

In the ion cyclotron resonance (ICR) analyzer, ions are trapped by a strong magnetic field. The magnetic field
will cause the ions to move in a circular motion with a frequency that depends on their m/z. Ions to be detected
are excited to make them move closer to the detection plates. Then a small current will be induced in the plate
each time an ion passes by. Since the ions with different m/z have different ICR frequencies, each generated
current frequency will correspond to a certain m/z value. Like the quadrupole ion trap, the ICR mass analyzers
are capable of storing ions in a cell.

Detectors
The role of the detector is to convert the energy of incoming particles into a current signal that is registered by
the electronic devices and transferred to the computer of the acquisition system of the mass spectrometer. When
an in-coming particle strikes the detector the energy from the impact causes emission of secondary particles, for
example, electrons or photons. The number of secondary particles created by an impact most often depends on
the energy and/or the velocity of the incoming ion.

Photo plate Detector

The photo plate detector is one of the oldest types of MS detectors. Photoplates were for a long time one of the
most common detectors for multicomponent detection. Even though the photoplate has advantages such as
simultaneous detection and possibilities of signal integration, compared to modern electrical devices the
disadvantages are numerous, including poor sensitivity, short linear range, off-line image processing, off-line
calibration, and shortage of commercial suppliers.

Faraday Detector
Unlike the photoplate, the Faraday detector (or Faraday cup) is still very much in use today. The main reasons for
its lasting popularity are accuracy, reliability, and rugged construction. The simplest form of Faraday detector is
a metal (conductive) cup that collects charged particles and is electrically connected to an instrument that
measures the produced current. An important application for Faraday detectors is precise measurements of ratios
of stable isotopes

Electron Multipliers
An electron multiplier (EM) amplifies a weak current of incoming particles by using a series of secondary
emission electrodes or dynodes to produce a considerably higher current at the anode. When a particle impinges
on the dynode, energy is transferred directly to the electrons in the dynode material and a number of secondary
electrons are emitted. The dynode often consists of an alloy of an alkali or alkali earth metal with a more noble
metal. Thus, a thin insulating film of oxidized alkaline metal is formed on a conducting support. A good dynode
material should emit many secondary electrons per primary incoming particle, have linear gain for high currents,
and have low thermionic emission, that is, low noise.
Scintillation Detector
The scintillation detector makes use of the fact that certain materials, when struck by a particle, emit a small flash
of light, so-called scintillation. Generally the detector consists of a scintillating material that is optically coupled
to an amplifying device such as a photomultiplier. When an ion passes through the scintillator, it excites the atoms
and molecules, causing light to be emitted. The light is transmitted to the photomultiplier, where it is converted
into a weak current of photoelectrons, which is further amplified by an electron-multiplier system.

Cryogenic Detector
Cryogenic or energy-sensitive calorimetric detectors measure the heat generated by a particle impacting on a
superconducting thin film. Cryogenic detectors measure low energy solid-state excitations (below 5
millielectronvolts) and must therefore be operated at temperatures typically below 2 K to avoid excessive thermal
excitations. Compared to ionization-based detectors, which rely on electron volt energies needed to produce
secondary electrons or electronic excitations, cryogenic detectors are more sensitive to slow-moving (large) ions.
An advantage of cryogenic detectors is that they are able to distinguish the charge state of the ion. Another
advantage is that, contrary to EM detectors such as the MCP, for example, the detection efficiency of cryogenic
detectors is independent of the velocity of the ions.

Solid-State Detector
Solid-state detectors (SSD) consist of silicon or germanium that emits electrons in response to ionizing radiation.
SSDs are mainly used to measure the energy of a particle as a compliment to an m/z determination. The addition
of an SSD to determine the residual energy of the ion allows the unambiguous determination of mass, charge, and
energy independently, instead of obtaining just ratios of energy to charge or mass to charge.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Detectors
The actual separation of each component in the sample is carried inside a column; however this separation needs
to be "collected" for us to be able to see it. The detectors are used for this purpose. The separated components are
monitored and expressed electronically. There is no universal detector that can monitor all compounds and there
are many detectors used for LC analysis. Some are listed below.
1. UV, VIS, and PDA Detectors
The UV, VIS, and PDA detectors are categorized as absorbance detectors. They provide good sensitivity for
light-absorbing compounds at ~pg level. They are easy to operate and provide good stability. UV detector is
a very commonly used detector for HPLC analysis. During the analysis, sample goes through a clear color-
less glass cell, called flow cell. When UV light is irradiated on the flow cell, sample absorbs a part of UV
light. Thus, the intensity of UV light observed for the mobile phase (without sample) and the eluent
containing sample will differ. By measuring this difference, the amount of sample can be determined. Since
the UV absorbance also differs depend on what wavelength is used, it is important to choose an appropriate
wavelength based on the type of analyte. A standard UV detector allows user to choose wavelength between
195 to 370 nm. Most commonly used is 254 nm. Compared to a UV detector, a VIS detector uses longer
wavelength (400 to 700 nm). There are detectors that provide wider wavelength selection, covering both UV
and VIS ranges (195 to 700 nm) called UV/VIS detector.
PDA detects an entire spectrum simultaneously. UV and VIS detectors visualize the obtained result in two
dimensions (light intensity and time), but PDA adds the third dimension (wavelength). This is convenient to
determine the most suitable wavelength without repeating analyses.
2. Refractive-Index Detector
RI detector measures change in reflex index. A glass cell is divided into two chambers (cells). The effluent
from LC column flow through the "sample cell", while other cell called "reference cell" is filled with only
mobile phase. When the effluent going through the sample cell does not contain any analyte, the solvent inside
both cells are the same. When a beam is irradiate on the cells, the observed beam will be straight in this case.
However, in a case the effluent contains any components other than mobile phase; bending of the incident
beam occurs due to the reflex index difference between the two solvents. By measuring this change, the
 presence of components can be observed.
RI detector has lower sensitivity compared to UV detector, and that's the main reason why RI is not as
commonly used as UV. However there are some advantages over UV detector.
 It is suitable for detecting all components. For an example, samples which do not have UV absorption,
such as sugar, alcohol, or inorganic ions obviously cannot be measured by a UV detector. In contrast,
change in reflective index occurs for all analyte, thus a RI detector can be used to measure all analyte.
 It is applicable for the use with solvent that has UV absorbance. A UV detector cannot be used with
solvent which has UV absorbance. Sometimes the organic solvent used for GPC analysis absorbs UV,
and thus UV detector cannot be used.
 It provides a direct relationship between the intensity and analyte concentration. The amount of UV
absorbed depends on each analyte, thus the intensity of UV detector peak does not provide information
on the analyte concentration. While intensity observed by a RI detector is comparable to the
concentration of analyte.
Because of those advantages, RI is often used for the detection of sugars and for SEC analysis.
3. Evaporative Light Scattering Detector
ELSD provides good sensitivity for non-volatile analytes at ng level. The column effluent is nebulized and
then evaporated to make it form fine particles. The analyte is then radiated with a laser beam and the
scattered radiation is detected. The target sample includes lipids, sugar, and high molecular weight analytes.
It is used in the similar way as a RI detector, but can provide more sensitive detection with stable base line.
Another advantage is that ELSD can be used for the gradient method whereas RI cannot.
4. Multi-Angle Light Scattering Detector
For the SEC analysis, MW of analyte is estimated from the calibration curve drown using a set of known
standards. However, by using a MALS, MW can be determined directly without the need of calibration
curve. Also MALS can provide an absolute MW of the analyte with very low detection limit.
5. Mass Spectrometer
The analytes are detected based on their MW. The obtained information is especially useful for compound
structure identification. However, its use is not limited to structure identification and can be used to quantify
very low detection limit of elemental and molecular components.
6. Conductivity Detector
Solutions containing ionic components will conduct electricity. Conductivity detector measures electronic
resistance and measured value is directly proportional to the concentration of ions present in the solution.
Thus it is generally used for ion chromatography.
7. Fluorescence Detector
The advantage of fluorescence method is its high sensitivity for selective groups of compounds at ~fg level.
By using a specific wavelength, analyte atoms are excited and then emit light signal (fluorescence). The
intensity of this emitted light is monitored to quantify the analyte concentration. Most pharmaceuticals,
natural products, clinical samples, and petroleum products have fluorescent absorbance. For some
compounds which do not have fluorescence absorbance or low absorbance, they can be treated with
fluorescence derivatives such as dansylchloride. The system is easy to operate and relatively stable.
8. Chemiluminescence Detector
Similar to FL, but instead of using a light source to excite the analyte atoms, the excitation is initiated by
chemical reaction. Since it is not relied on the external excitation source, the noise is small, results in high
signal to noise ratio, i.e. it provides even higher sensitivity than FL.
9. Optical Rotation Detector
Specific for the optical isomer measurement. The column can separate R- and L- type optical isomers, but the
general detectors (e.g., UV) cannot distinguish which is R nor does L. OR detector provide this information.
10. Electro Chemical Detector
There are several different types of ECs. The detection is based on amperometry, polarography, coulometry,
and conductometry. They offer high sensitivity, simplicity, convenience, and wide-spread applicability. It is
especially suitable for the use with semi-micro or capillary type system.
PROTEIN–PROTEIN INTERACTIONS
In general, proteins have to interact with each other to carry out biochemical functions. Thus, mapping out
protein–protein interactions is another important aspect of proteomics. Inter-protein interactions include strong
interactions that allow formation of stable complexes and weaker ones that exist transiently. Proteins involved in
forming complexes are generally more tightly co-regulated in expression than those involved in transient
interactions. Protein–protein interaction analysis at the proteome level helps reveal the function of previously
uncharacterized proteins on the basis of the “guilt by-association” rule.

Protein Protein Interaction Mapping

Experimental Determination Protein Interaction Prediction

Classical Rosetta Stone

Yeast Two Method Use of Gene Interlogs (
Large-scale affinity Linkage (Based Based on Phylogenetic Hybrid
Hybrid (Based on on Gene Sequence
Methods ( purification technique, Domain
Profiles Methods
attaching fusion tags to Neighbors) Homology)
Bait and Prey) proteins and purifying
Fusion)
the associated protein
complexes, Detection by
Gel Electrophoresis and
MS