Download Mass spectrometry - 123seminarsonly.com

Proteomics
1
Saurabh Sharma
Proteomics
Saurabh Sharma
M. Sc.( Honors school) 1st Year
18th January 2008
Proteomics
2
Saurabh Sharma
Table of Contents
Gel electrophoresis ....................................................................................................................................................... 4
Separation ................................................................................................................................................................ 4
Visualization ............................................................................................................................................................ 4
SDS-PAGE............................................................................................................................................................... 5
Two-dimensional gel electrophoresis ........................................................................................................................... 6
Mass spectrometry ........................................................................................................................................................ 6
Working of a Mass Spectrometer ............................................................................................................................ 8
Instrumentation ........................................................................................................................................................ 8
Ion source ............................................................................................................................................................ 8
Mass analyzer...................................................................................................................................................... 9
Types of Mass Analyzers .................................................................................................................................... 9
Sector field mass analyzer .............................................................................................................................. 9
Time-of-flight mass analyser.......................................................................................................................... 9
Quadrupole mass analyser.............................................................................................................................. 9
Figure: Quadrupole analyser ........................................................................................................................ 10
Quadrupole ion trap analyser ....................................................................................................................... 10
Fourier Transform Ion Cyclotron Resonance(FTICR) (analyser) ................................................................ 11
Orbitrap analyser .......................................................................................................................................... 11
Detector ............................................................................................................................................................. 11
Tandem Mass Spectrometry ....................................................................................................................................... 12
MALDI(Matrix assisted laser-desorption ionization) ................................................................................................ 12
Matrix .................................................................................................................................................................... 13
Laser ...................................................................................................................................................................... 14
Reproducibility and performance .......................................................................................................................... 15
AP-MALDI ................................................................................................................................................................ 15
Surface-enhanced laser desorption/ionization(SELDI) .............................................................................................. 16
Data Representations .................................................................................................................................................. 16
Mass Spectrum....................................................................................................................................................... 17
Data analysis and interpretation ................................................................................................................................. 18
Reference .................................................................................................................................................................... 18
Introduction
Large scale screening of proteins, their expression, modifications and interactions by using high-throughput
approaches. Proteins are vital parts of living organisms, as they are the main components of the physiological
metabolic pathways of cells. The term "proteomics" was coined to make an analogy with genomics, the study of
the genes. The word "proteome" is a combination of "protein" and "genome". The proteome of an organism is the
set of proteins produced by it during its life, and its genome is its set of genes.
Proteomics is often considered the next step in the study of biological systems, after genomics. It is much more
complicated than genomics, mostly because while an organism's genome is rather constant, a proteome differs
from cell to cell and constantly changes through its biochemical interactions with the genome and the environment.
One organism has radically different protein expression in different parts of its body, different stages of its life
Proteomics
3
Saurabh Sharma
cycle and different environmental conditions. Another major difficulty is the complexity of proteins relative to
nucleic acids. E.g., in human there are about 25 000 identified genes but an estimated >500 000 proteins that are
derived from these genes. This increased complexity derives from mechanisms such as alternative splicing, protein
modification (glycosylation,phosphorylation) and protein degradation.
Scientists are very interested in proteomics because it gives a much better understanding of an organism than
genomics. This is because of several reasons;
1. The level of transcription of a gene gives only a rough estimate of its level of expression into a protein. An
mRNA produced in abundance may be degraded rapidly or translated inefficiently, resulting in a small
amount of protein.
2. Many proteins experience post-translational modifications that profoundly affect their activities; for
example some proteins are not active until they become phosphorylated. Methods such as
phosphoproteomics and glycoproteomics are used to study post-translational modifications.
3. Many transcripts give rise to more than one protein, through alternative splicing or alternative posttranslational modifications.
4. Finally, many proteins form complexes with other proteins or RNA molecules, and only function in the
presence of these other molecules.
Since proteins play a central role in the life of an organism, proteomics is instrumental in discovery of biomarkers,
such as markers that indicate a particular disease.
With the completion of a rough draft of the human genome, many researchers are looking at how genes and
proteins interact to form other proteins. A surprising finding of the Human Genome Project is that there are far
fewer protein-coding genes in the human genome than proteins in the human proteome (20,000 to 25,000 genes vs.
> 500,000 proteins). The human body may even contain more than 2 million proteins, each having different
functions. The protein diversity is thought to be due to alternative splicing and post-translational modification of
proteins. The discrepancy implies that protein diversity cannot be fully characterized by gene expression analysis,
thus proteomics is useful for characterizing cells and tissues.
Gel electrophoresis
Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid, ribonucleic acid, or protein
molecules using an electric current applied to a gel matrix. It is usually performed for analytical purposes, but may
be used as a preparative technique prior to use of other methods such as mass spectrometry, PCR, cloning, DNA
sequencing, or Southern blotting for further characterization.
Separation
The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most
cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and
composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or
oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker,
producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a
few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix.
"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the
gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move
through the matrix at different rates, usually based on size, toward the negative cathode if positively charged or
toward the positive anode if negatively charged.
Proteomics
4
Saurabh Sharma
Figure:
Gel
electrophoresis
apparatus - An agarose gel is
placed in this buffer-filled box and
electrical current is applied
Visualization
After the electrophoresis is
complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie
blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's
components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the
gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility,
an autoradiogram can be recorded of the gel.
If several mixtures have initially been injected next to each other, they will run parallel in individual lanes.
Depending on the number of different molecules, each lane shows separation of the components from the original
mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead
to overlapping bands, or to indistinguishable smears representing multiple unresolved components.
Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel
with the same speed, which usually means they are approximately the same size. There are molecular weight size
markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the
gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to
determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the
size of the molecule.
SDS-PAGE
Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into
the gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are
usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate
(SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the
size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall
negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods
instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is
relative only to its size and not its charge or shape.
Proteomics
5
Saurabh Sharma
Proteins are usually analyzed by sodium
dodecyl
sulfate
polyacrylamide
gel
electrophoresis (SDS-PAGE), by native gel
electrophoresis, by quantitative preparative
native
continuous
polyacrylamide
gel
electrophoresis (QPNC-PAGE), or by 2-D
electrophoresis.
Figure: SDS-PAGE autoradiography - The indicated
proteins are present in different concentrations in the two
samples.
Two-dimensional gel
electrophoresis
The two dimensions that proteins are separated into using this technique are isoelectric point and mass. It was
invented by O' Ferrel and Klose independently in 1975.
This is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions
oriented at right angles to one another. This allows the sample to separate over a larger area, increasing the
resolution of each component.
The model is based on two crucial observations:
1. A molecule placed between two oppositely charged poles will migrate between them at a speed
inversely proportional to its mass-to-charge ratio (molecular weight / net charge).
2. Placed in an appropriate medium, a protein will migrate within a gradient of increasing pH
until it reaches a spot where the net charge of all its side-chains is 0. This point
the isoelectric point of the protein in the given medium.
The polyacrylamide gel provides a supporting matrix through which proteins can migrate. This gel has a pH
gradient from top to bottom - the top is more acidic than the bottom.
A complex protein mixture is loaded in the middle of the left side, where the pH is neutral, and a voltage is applied
across the gel. The proteins then migrate through the gel until they reach their isoelectric point (the point at which
their charge is the same as the surrounding pH).
This technique is called isoelectric focussing.
The gel is then soaked in a denaturing solution
(which causes the proteins to unfold) containing
the detergent sodium dodecylsulphate (SDS).
This molecule has a very strong negative charge
and binds to all proteins, effectively making
them all the same charge.
A voltage is then applied across the gel from side
to side with the anode (positive terminal) at the
right. The proteins all move towards the anode,
but smaller proteins move faster through the gel
than larger ones, thus separating the proteins
Proteomics
6
Saurabh Sharma
according to their size.
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is used to separate mixtures of proteins, and is
particularly useful for comparing related samples such as healthy and diseased tissue. The first step is to load the
proteins onto the gel, which has a pH gradient from top to bottom.
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of ions. The underlying
principle of all mass spectrometers is that the paths of gas phase ions in electric and magnetic fields are dependent
on their mass-to-charge ratios which is used by the analyzer to distinguish the ions from one another. The mass
spectrum is measured by a mass spectrometer.
Mass
spectrometers consist of three basic parts: an ion source, a mass analyzer, and a detector system. The stages within
the mass spectrometer are:
1. Production of ions from the sample
2. Separation of ions with different masses
3. Detection of the number of ions of each mass produced
4. Collection of data to generate the mass spectrum
Proteomics
7
Saurabh Sharma
Working of a Mass Spectrometer
Figure:
Schematic
diagram of
working of
a Mass
spectromet
er
Instr
umen
tation
Ion
source
The ion
source is
the part
of
the
mass
spectrom
eter that
ionizes
the
material under analysis (the analyte). The ions are then transported by magnetic or electric fields to the mass
analyzer.
Techniques for ionization have been key to determining what types of samples can be analyzed by mass
spectrometry. Electron ionization and chemical ionization are used for gases and vapors. In chemical ionization
sources, the analyte is ionized by chemical ion-molecule reactions during collisions in the source.
The gas phase reaction producing electron ionization is
where M is the atom of molecule being
+ °°
ionized, e − is the electron, and M
is the resulting ion. Two techniques often used with liquid and solid
biological samples include matrix-assisted laser desorption/ionization (MALDI, by F. Hillenkamp) and
electrospray ionization (by John Fenn )
Mass analyzer
Mass analyzers separate the ions according to their mass-to-charge ratio. All mass spectrometers are based on
dynamics of charged particles in electric and magnetic fields in vacuum where the following two laws apply:
F = q(E+v X B) (Lorentz force law)
F = ma (Newton's second law of motion)
where F is the force applied to the ion, m is the mass of the ion, a is the acceleration, q is the ionic charge, E is the
electric field, and v x B is the vector cross product of the ion velocity and the magnetic field
Equating the above expressions for the force applied to the ion yields:
Proteomics
8
Saurabh Sharma
(m/q)a = E + v X B
This equation reveals that two particles with the same physical quantity m/q behave exactly the same. Together
with the particle's initial conditions it completely determines the particle's motion in space and time and therefore
is the basis of every mass spectrometer.
There are many types of mass analyzers, using either static or dynamic fields, and magnetic or
electric fields, but all operate according to this same law. Each analyzer type has its strengths and weaknesses.
Many mass spectrometers use two or more mass analyzers for tandem mass spectrometry (MS/MS).
Types of Mass Analyzers
Sector field mass analyzer
A sector field mass analyzer uses an electric and/or magnetic field to affect the path and/or velocity of the charged
particles in some way. As shown in the figure, sector instruments change the direction of ions that are accelerated
through the mass analyzer. The ions enter a magnetic or electric field which bends the ion paths depending on their
mass-to-charge ratios, deflecting the more charged and faster-moving, lighter ions more. The ions eventually reach
the detector and their relative abundances are measured. The analyzer can be used to select a narrow range of m/q
or to scan through a range of m/q to catalog the ions present.
Time-of-flight mass analyser
It uses an electric field to accelerate the ions through the same potential, and then measures the time they take to
reach the detector. If the particles all have the same charge, the kinetic energies will be identical, and their
velocities will depend only on their masses. Lighter ions will reach the detector first.
Quadrupole mass analyser
The quadrupole mass analyzer is one type of mass analyzer used in mass spectrometry. In a quadrupole mass
spectrometer the quadrupole mass analyzer is the component of the instrument responsible for filtering sample
ions, based on their mass-to-charge ratio (m/z). A quadrupole mass analyzer is essentially a mass filter that is
capable of transmitting only the ion of choice. A mass spectrum is obtained by scanning through the mass range of
interest over time.
Figure:
Quadrupole
analyser
The
quadrupol
e consists
of four
parallel
metal
rods.
Each
opposing
rod pair is
connected together electrically and a radio frequency voltage is applied between one pair of rods, and the other. A
direct current voltage is then superimposed on the R.F. voltage. Ions travel down the quadrupole in between the
rods. Only ions of a certain m/z will reach the detector for a given ratio of voltages: other ions have unstable
trajectories and will collide with the rods. This allows selection of a particular ion, or scanning by varying the
voltages.
Proteomics
9
Saurabh Sharma
Quadrupole ion trap analyser
The quadrupole ion trap works on the same physical principles as the QMS, but the ions are trapped and
sequentially ejected. Ions are created and trapped in a mainly quadrupole RF potential and separated by m/q, nondestructively or destructively.
Figure: Scheme of a Quadrupole ion trap of classical setup with a
particle of positive charge (red), surrounded by a cloud of similarly
charged particles (red). The electric field E (blue) is generated by a
quadrupole of endcaps (a, positive) and a ring electrode (b). Picture
1 and 2 show two states during an AC cycle.
There are many mass/charge separation and isolation
methods but most commonly used is the mass instability
mode in which the RF potential is ramped so that the orbit of ions with a mass a > b are stable while ions with
mass b become unstable and are ejected on the z-axis onto a detector.
Ions may also be ejected by the resonance excitation method, whereby a supplemental oscillatory excitation
voltage is applied to the endcap electrodes, and the trapping voltage amplitude and/or excitation voltage frequency
is varied to bring ions into a resonance condition in order of their mass/charge ratio.
The cylindrical ion trap mass spectrometer is a derivative of the quadrupole ion trap mass spectrometer.
Fourier Transform Ion Cyclotron Resonance(FTICR) (analyser)
Fourier
transform
mass
spectrometry, or more precisely
Fourier transform ion cyclotron
resonance MS, measures mass by
detecting the image current
produced by ions cyclotroning in
the presence of a magnetic field.
Instead of measuring the deflection
of ions with a detector such as an
electron multiplier, the ions are
injected into a Penning trap (a static
electric/magnetic ion trap) where
they effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of ions
which pass near them over time producing cyclical signal. Since the frequency of an ion's cycling is determined by
its mass to charge ratio, this can be deconvoluted by performing a Fourier transform on the signal.
FTMS has the advantage of high sensitivity (since each ion is 'counted' more than once) and much high resolution
and thus precision.
Orbitrap analyser
In the Orbitrap, ions are electrostatically trapped in an orbit
around a central, spindle-shaped electrode. The electrode
confines the ions so that they orbit around the central
electrode and oscillate back and forth along the central
electrode's long axis. This oscillation generates an image
current in the detector plates which is recorded by the
instrument. The frequencies of these image currents depend on
the mass to charge ratios of the ions in the Orbitrap.
Similar to Fourier transform ion cyclotron resonance mass
Proteomics
10
Saurabh Sharma
spectrometers, Orbitraps have a high mass accuracy, high sensitivity and a good dynamic range
Detector
The final element of the mass spectrometer is the detector. The
detector records the charge induced or current produced when
an ion passes by or hits a surface.
Figure: Continuous dynode electron multiplier detector
Typically, some type of electron multiplier is used, though other
detectors including Faraday cups and ion-to-photon detectors
are also used. Because the number of ions leaving the mass
analyzer at a particular instant is typically quite small,
significant amplification is often necessary to get a signal. Microchannel Plate Detectors are commonly used in
modern commercial instruments. In FTMS and Orbitraps, the detector consists of a pair of metal surfaces within
the mass analyzer/ion trap region which the ions only pass near as they oscillate. No DC current is produced, only
a weak AC image current is produced in a circuit between the electrodes. Other inductive detectors have also been
used.
Tandem Mass Spectrometry
Multiple stages of mass analysis separation can be accomplished with individual mass spectrometer elements
separated in space(i.e. they are separated physically) or in a single mass spectrometer with the MS steps separated
in time(i.e. done at different time). In tandem mass spectrometry in space, the separation elements are physically
separated and distinct, although there is a connection between the elements to maintain high vacuum. These
elements can be sectors, transmission quadrupole, or time-of-flight. In a tandem mass spectrometry in time
instrument, the separation is accomplished with ions trapped in the same place, with multiple separation steps
taking place over time. A quadrupole ion trap or FTMS instrument can be used for such an analysis. Trapping
instruments can perform multiple steps of analysis, which is sometimes referred to as MSn (MS to the n). Often the
number of steps, n, is not indicated, but occasionally the value is specified; for example MS3 indicates three stages
of separation.
Proteomics
11
Saurabh Sharma
[EI: Electron Ionization, ESI: Electron spray Ionization]
MALDI(Matrix assisted laser-desorption ionization)
Traditional Mass Spectometry fails with peptides, proteins, and DNA. Molecules of that size and charge are not
volatile. Furthermore, under high energy electron bombardment, they would disintigrate into countless
components.
However high molecular weight ions can be made to jump into the vacuum with the help of MALDI, developed by
Franz Hillenkamp at the end of 1980's.
The proteins are first incorporated in the crystals of UV-adsorbant material. The proteins are first incorporated in
the crystals of UV-adsorbant material. The protein doped crystals are then pushed in the high vacuum of the MS
and irradiated with UV laser pulse. This explosively releases releases the UV-adsorbent molecules and with them
the built-in protein ions. Molecule with such properties of co-crystal formation, proton transfer, UV adsorption are
called matrix.
The proteins(+ protons) enter the gas phase without any hydrate and counter ions such as Na+ or Cl-. It is the single
polypeptide chains that appear in the gas phase. Quarternary proteins disintigrate into their subunits in the acidic,
denaturing matrix solution itself.
Proteomics
12
Saurabh Sharma
Figure: Matrix-assisted laser desorption ionization depicted with matrix in blue and analyte in red
Matrix
The matrix consists of crystallized molecules, of which the three most commonly used are 3,5-dimethoxy-4hydroxycinnamic acid (sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5dihydroxybenzoic acid (DHB)
Matrix compounds is selected to some extent by trial and error, but they are based on some specific molecular
design considerations:

They are of a fairly low molecular weight (to allow facile vaporization), but are large enough (with a low
enough vapor pressure) not to evaporate during sample preparation or while standing in the spectrometer.

They are acidic, therefore act as a proton source to encourage ionization of the analyte.

They have a strong optical absorption in the UV, so that they rapidly and efficiently absorb the laser
irradiation.

They are functionalized with polar groups, allowing their use in aqueous solutions.
The matrix solution is mixed with the analyte (e.g. protein-sample). The organic solvent allows hydrophobic
molecules to dissolve into the solution, while the water allows for water-soluble (hydrophilic) molecules to do the
same. This solution is spotted onto a MALDI plate (see figure). The solvents vaporize, leaving only the
recrystallized matrix, but now with analyte molecules spread throughout the crystals. The matrix and the analyte
are said to be co-crystallized in a MALDI spot.
Proteomics
13
Saurabh Sharma
Figure: MALDI plate
Laser
The laser is fired at the crystals in the MALDI spot. The matrix absorbs the laser energy and it is thought that
primarily the matrix is ionized by this event. The matrix is then thought to transfer part of its charge to the analyte
molecules (e.g. protein), thus ionizing them while still protecting them from the disruptive energy of the laser. Ions
observed after this process consist of a neutral molecule [M] and an added or removed ion. Together, they form a
quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na]+in the case of an added sodium
ion, or [M-H]- in the case of a removed proton. MALDI generally produces singly-charged ions, but multiply
charged ions ([M+nH]n+) can also be observed, usually as a function of the matrix, the laser intensity and/or the
voltage used. Note that these are all even-electron species. Ion signals of radical cations can be observed eg. in case
of matrix molecules and other stable molecules.
Lasers Used for MALDI
Laser
Wavelength (nm)
Nitrogen laser
337
Nd:YAG(neodymium-doped
yttrium aluminium garnet;
Nd:Y3Al5O12)
355, 266
Er:YAG (erbium-doped yttrium
aluminium garnet (Er:Y3Al5O12)
2940
CO2
10,600
Reproducibility and performance
The sample preparation for MALDI is important for the result. Inorganic salts which are also part of protein
extracts interfere with the ionization process. The salts are removed by solid phase extraction or washing the final
target spots with water. Both methods can also remove other substances from the sample. The matrix-protein
mixture is not homogenous because the polarity difference leads to a separation of the two substances during
crystallization. The spot diameter of the target is much larger than that of the laser, which makes it necessary to do
several laser shots at different places of the target within the target spot. The matrix composition, the addition of
trifluoroacetic acid and formic acid, delay between laser pulses, delay time of the acceleration power, laser
Proteomics
14
Saurabh Sharma
wavelength, energy density of the laser and the impact angle of the laser on the target are among others the critical
values for the quality and reproducibility of the method.
AP-MALDI
Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an ionization technique (ion
source) that in contrast to vacuum MALDI operates at normal atmospheric environment.The main difference
between vacuum MALDI and AP-MALDI is the pressure in which the ions are created. In vacuum MALDI, ions
are typically produced at 10 mTorr or less while in AP-MALDI ions are formed in atmospheric pressure.
Disadvantage of the AP MALDI source is the limited sensitivity observed and the limited mass range.
AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from proteomics to drug
discovery fields. Popular topics that are addressed by AP-MALDI mass spectrometry include: proteomics,
DNA/RNA/PNA, lipids, oligosaccharides, phosphopeptides, bacteria, small molecules and synthetic polymers,
similar applications as available also for vacuum MALDI instruments.
The AP-MALDI ion source is easily coupled to an ion trap mass spectrometeror any other MS system equipped
with ESI (electrospray ionization) or nanoESI source.
Surface-enhanced laser desorption/ionization(SELDI)
Surface-enhanced laser desorption/ionization (SELDI) is an ionization method in mass spectrometry that is used
for the analysis of protein mixtures.SELDI is typically used with time-of-flight mass spectrometers and is used to
detect proteins in tissue samples, blood, urine, or other clinical samples. Comparison of protein levels between
patients with and without a disease can be used for biomarker discovery.
SELDI-TOF-MS is a variation of matrix-assisted laser desorption/ionization (MALDI) that uses a
target modified to achieve biochemical affinity with the analyte compound. In MALDI, a protein or peptide sample
is mixed with the matrix molecule in solution and small amounts of the mixture are deposited on a surface and
allowed to
dry. The
sample
and matrix
cocrystallize
as
the
solvent
evaporates
.
In
SELDI the
protein
mixture is
spotted on
a surface
modified
with
a
chemical
functionali
ty. Some
proteins in
the sample bind to the surface, while the others are removed by washing. After washing the spotted sample, the
Proteomics
15
Saurabh Sharma
matrix is applied to the surface and allowed to crystallize with the sample peptides. 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 reverse phase
chromatography), IMAC30 (metal-binding surface), and Q10 (strong anion exchanger). Surfaces can also be
functionalized with antibodies, other proteins, or DNA.
Spectrometric Data
Data Representations
Most common type of data produced by a Mass spectrophotometer is a Mass Spectrum.
However some MS's may also produce a Contour map ( The kind of map used to represent topology of a
geographical area, points of same intensity joined to each other). Hence we get a 3D data representation.
Mass Spectrum
A mass spectrum is an intesity vs. m/z(mass to charge ratio) plot representing a chemical analysis. The x-axis of a
mass spectrum represents a relationship between the mass of a given ion and the number of elementary charges that
it carries. This is written as the IUPAC* standard m/z to denote the quantity formed by dividing the mass of an ion
by the unified atomic mass unit and by its charge number (positive absolute value). This has been referred to as a
mass-to-charge ratio.
The y-axis of a mass spectrum represents signal intensity of the ions. The signal intensity may
represent Counts per second(cps) or power of the signal sine wave, etc depending on the kind of Mass spectrometer
and hence the detector being used;
When using counting detectors the intensity is often measured in counts per second (cps). When using analog
detection electronics the intensity is typically measured in volts. In FTICR(Fourier transform ion cyclotron
resonance) and Orbitraps the frequency domain signal (the y-axis) is related to the power (~amplitude squared) of
the signal sine wave (often reduced to an rms power); however, the axis is usually not labeled as such since in most
forms of mass spectrometry, the intensity of ion current measured by the spectrometer does not accurately
represent relative abundance, but correlates loosely with it. Therefore it is common to label the y-axis with
"arbitrary units".
*
International Union of Pure and Applied Chemistry
Proteomics
16
Saurabh Sharma
Signal intensity may be dependent on many factors, especially the nature of the molecules being
analyzed and how they ionize. The efficiency of ionization varies from molecule to molecule and from ion source
to ion source. For example, in electrospray sources in positive ion mode a quaternary amine will ionize
exceptionally well whereas a large hydrophobic alcohol will most likely not be seen no matter how concentrated.
In an EI source these molecules will behave very differently. Additionally there may be factors that affect ion
transmission disproportionally between ionization and detection.
On the detection side there are many factors that can also affect signal intensity in a non-proportional way.
The size of the ion will affect the velocity of impact and with certain detectors the velocity is proportional to the
signal output. In other detection systems, such as FTICR, the number of charges on the ion are more important to
signal intensity. In Fourier transform ion cyclotron resonance and Orbitrap type mass spectrometers the signal
intensity (Y-axis) is related to the amplitude of the free induction decay signal. This is fundamentally a power
relationship (amplitude squared) but often computed as an [rms]. For decaying signals the rms is not equal to the
average amplitude. Additionally the damping constant (decay rate of the signal in the fid) is not the same for all
ions. In order to make conclusions about relative intensity a great deal of knowledge and care is required.
A common way to get more quantitative information out of a mass spectrum is to create a standard curve to
compare the sample to. This requires knowing what is to be quantitated ahead of time, having a standard available
and designing the experiment specifically for this purpose. A more advanced variation on this the use of an internal
standard which behaves very similarly to the analyte. This is often an isotopically labeled version of the analyte.
There are forms of mass spectrometry, such as accelerator mass spectrometry that are designed from the bottom up
to be quantitative.
Data analysis and interpretation
Mass Spectrometric data analysis depends on the kind of experiment producing the data. There are several factors
that should be kept in mind while analysing and interpreting the data provided by a Mass Spectrometer or a
MALDI apparatus:
1. Different kinds of ion sources behave differently from each other and produce different kinds of data. For
example, electron ionization source produces many fragments, Tandem mass spectrometry purposly
produces fragment ions post source and has a great impact on the kind of Data achieved by an experiment.
2. Thee kind of sample being analysed also effects the data interpretation. For example if the sample is a
relatively crude preparation of a biological sample, the sample likely contains a certain amount of salt that
may form adducts with the analyte molecules in certain analyses.
3. How and from where a sample is prepared and how it is used/ introduced also has considerable effect on the
result. For example, the kind of matrix used for MALDI.
4. Most importantly, one must understand the goal of the project (and collect the right kind of data in the first
place) while interpreting a spectrometric data. One may be interested in knowing the masses of the
molecules or the purity of the sample or the structure of the molecule or all the above.
Reference
1. http://www.iupac.org/goldbook/T06250.pdf
2. Atmospheric pressure matrix-assisted laser desorption/ionization(AP MALDI) on a quadrupole ion trap
mass spectrometer. Susanne C. Moyer, Lisa A. Marzilli, Amina S. Woods, Victor V. Laikod, Vladimir M.
Doroshenkod, Robert J. Cotter
Proteomics
17
Saurabh Sharma
3. Proteinchip surface enhanced laser desorption/ionization (SELDI) mass spectrometry: a novel protein
biochip technology for detection of prostate cancer biomarkers in complex protein mixtures. GL Wright Jr,
LH Cazares, S-M Leung, S Nasim, B-L Adam, T-T Yip, PF Schellhammer, L Gong & A Vlahou
4. Development of a Three-Dimensional Topographic Map Display for Capillary Electrophoresis/Mass
Spectrometry with an Ion Trap/Reflectron Time-of-Flight Mass Spectrometer Detector: Applications to
Tryptic Digests of Isoforms of Myelin Basic Protein. Michael X. Li, Jing-Tao Wu, Steve Parus and David
M. Lubman
5. Preparative Native Continuous Polyacrylamide Gel Electrophoresis (PNC-PAGE): An Efficient Method for
Isolating Cadmium Cofactors in Biological Systems. Bernd Kastenholz
6. 2D Gel Electrophoresis - An Overview. Louigi Addario-Berry;
http://www.mcb.mcgill.ca/~hallett/GEP/PLecture3
7. http://genome.wellcome.ac.uk