# Proteomics/Protein Identification - Mass Spectrometry

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!MALDI TOF MS

## Introduction

-   **Mass Spectrometry Overview**

Mass spectrometry is a technique in which gas phase molecules are
ionized and their mass-to-charge ratio is measured by observing
acceleration differences of ions when an electric field is applied.
Lighter ions will accelerate faster and be detected first. If the mass
is measured with precision then the composition of the molecule can be
identified. In the case of proteins, the sequence can be identified.
Most samples submitted to mass Spectrometry are a mixture of compounds.
A spectrum is acquired to give the mass-to-charge ratio of all compounds
in the sample. Mass spectrometry is also known as \'mass spec\' or MS
for short. Mass spectrometry throws light on molecular mechanisms within
cellular systems. It is used for identifying proteins, functional
interactions, and it further allows for determination of subunits. Other
molecules in cells such as lipid components can also be defined.

A mass spectrometer is composed of several different parts: a source
that ionizes the sample, the analyzer that separates the ions based on
mass-to-charge ratio, a detector that \"sees\" the ions, and a data
system to process and analyze the results. You can also measure relative
abundance of an ion using mass spectrometry. Different compounds have
differential ionization capabilities and therefore intensity of your ion
is not a direct correlation to concentration.

Mass spectrometry can be a high throughput analytical method due to the
ability for a mass spectrum to be measured rapidly and with minimal
sample handling as compared to gel methods.

It is an analytical method which has a variety of uses outside of
proteomics, such as
isotope and dating, trace gas analysis, atomic location mapping,
pollutant detection, and space exploration

-   **History of Mass Spectrometry**

The history of this technique finds its roots in the first studies of
gas excitation in a charged environment, more than 100 years ago. This
pioneering work led to the identification of two isotopes of neon
(neon-20 and neon-22) via mass to charge ration discrimination by J.J
Thomson in 1913. Over the next fifty years
the fundamental basis of the technique was further developed. After the
coupling of gas chromatography to Mass Spectroscopy in 1959 by
researches at Dow Chemical, the full potential of the technique as a
highly accurate, quantitative method for exploring compounds was
realized, spurring a wave of developments which continue to the present
day. The precision of mass spectrometry led to the discovery of
isotopes.

\
\***Implications of Mass Spectrometry for Proteomics Applications** The
technique of mass spectrometry is a valuable tool in the field of
proteomics. It can be used to identify proteins through variations of
mass spectrometry techniques. The most common first approach to
proteomics is a bottom-up approach in which the protein is digested by a
protease, such a trypsin, and the peptides are then analyzed by peptide
mass fingerprinting, collision-induced
dissociation, tandem MS,
and electron capture
dissociation. Once the
peptides masses have been determined the mass list can be sent to a
database, such as MASCOT, where the list is compared to the masses of
all known peptides. If enough peptides match that of a known protein you
can identify your protein. If the masses of your peptides do not match a
known protein you can sequence your peptide by de novo sequencing using
MS/MS methods; where you isolate your peptide and break it along the
peptide bond backbone forming y and b ions from which you can determine
the sequence. The advantages of the bottom-up approach are that the
small size of tryptic peptide ions is easy to handle biochemically than
entire protein ions because of their relatively small masses that are
easier to be determine. Beside bottom-up approach, another approach is
top-down. In top-down approach, the complete proteins are directly
analyzed by using mass spectrometer without solution digestion as
bottom-up does. The advantages of the top-down approach are that it can
sometime provide the complete coverage of the protein. But since whole
proteins are hard to handle biochemically compared to small peptide
pieces, it makes top-down approach difficult to analyze.

Another use of mass spectrometry in proteomics is protein
quantification. By labeling proteins with stable heavier isotopes you
can in turn determine the relative abundance of proteins. Companies now
produce kits, such as iTRAQ (Applied
Biosystems),
in order to do this at a high-throughput level.

One of the most powerful ways to identify a biological molecule is to
determine its molecular mass together with the masses of its component
building blocks after fragmentation. There are two dominant methods for
doing this. The first is electrospray ionization (ESI), in which the
ions of interest are formed from solution by applying a high electric
field. This is done by applying a high electric field to the tip of a
capillary, from which the solution will pass through. The sample will be
sprayed into the electric field along with a flow of nitrogen to promote
desolvation. Droplets will form and will evaporate in a vacuumed area.
This causes an increase in charge on the droplets and the ions are now
said to be multiply charged. These multiply charged ions can now enter
the analyzer. ESI is a method of choice because of the following
properties: (1)The \"softness\" of the phase conversion process allows
very fragile molecules to be ionized intact and even in some
non-covalent interactions to be preserved for MS analysis. (2)The
eluting fractions through liquid chromatography can then be sprayed into
the mass spectrometer, allowing for the further analysis of mixtures.
(3)The production of multiply charged ions allow for the measurement of
high-mass biopolymers. Multiple charges on the molecule will reduce its
mass to charge ratio when compared to a single charged molecule.
Multiple charges on a molecule also allows for improved fragmentation
which in turn allows for a better determination of structure. The second
is matrix-assisted laser desorption/ionization (MALDI) in which the
molecular ions of interest are formed by pulses of laser light impacting
on the sample isolated within an excess of matrix molecules. This
enables the determination of masses of large biomolecules and synthetic
polymers greater than 200,000 Daltons without degradation of the
molecule of interest. The advantages of MALDI are its robustness, high
speed, and relative immunity to contaminants and biochemical buffers.

A type of mass spectrometer often used with MALD is TOF or Time of
Flight mass spectrometry. This enables fast and accurate molar mass
determination along with sequencing repeated units and recognizing
polymer additives and impurities. This technique is based on an
ultraviolet absorbing matrix where the matrix and polymer are mixed
together along with excess matrix and a solvent to prevent aggregation
of the polymer. This mixture is then placed on the tip of a probe; then
the solvent is removed while under vacuum conditions. This creates
co-crystallized polymer molecules that are dispersed homogeneously
within the matrix. A pulsing laser beam is set to an appropriate
frequency and energy is shot to the matrix, which becomes partially
vaporized. In turn the homogeneously dispersed polymer within the matrix
is carried into the vapor phase and becomes charged. To obtain a superb
signal-to-noise ratio, multiple laser shots are executed. The shapes of
the peaks are improved and the molar masses determined are more
accurate. Fianlly, in the TOF analyzer the molecules from a sample are
imparted identical translational kinetic energies because of the
electrical potential energy difference. These ionic molecules travel
down an evacuated tube with no electrical field and of the same
distance. The smallest ions arrive first at the detector, which produces
a signal for each ion. The cumulative data from multiple laser shots
yield a TOF mass spectrum, which translates the detector signal into a
function of time, which in turn can be used to calculate the mass of the
ion.

In addition to these ionization techniques, highly powerful mass
analyzers have been developed. These analyzers measure the mass/charge
ratio of intact ionized biomolecules, as well as their fragmentation
spectra, with high accuracy and high speed. The measurement of
fragmentation spectra is called tandem MS or MS/MS. In conjunction with
single stage MS (with intact precursor ions) tandem MS can be utilized
to help elucidate a protein since the problem of elucidation will reduce
to assembling the puzzle pieces of the fragmented protein.

## References

1.  American Society for Mass Spectrometry - What is MS?,
    <http://www.asms.org/whatisms/p4.html>
2.  Mass Spectrometry in the Postgenomic Era
3.  Annual Review of Biochemistry Vol. 80: 239-246 (Volume publication
    date July 2011) DOI: 10.1146/annurev-biochem-110810-095744
    <https://ted.ucsd.edu/webapps/portal/frameset.jsp?tab_tab_group_id=_2_1&url=%2Fwebapps%2Fblackboard%2Fexecute%2Flauncher%3Ftype%3DCourse%26id%3D_767_1%26url%3D>
4.  University of Illinois at Urbana-Champaign School of Chemical
    Sciences <http://scs.illinois.edu/massSpec/ion/esi.php>
5.  University of Southern Mississippi School of Polymers and High
    Performance Materials <http://www.psrc.usm.edu/mauritz/maldi.html>

fr:Chimie organique/Spectrométrie de
masse
