Experiment-6: MALDI-TOF instrumentation and analysis of serum

protein

Theory
The term proteome describes the protein complement expressed by a genome, while
proteomics is the study of the entire compendium of proteins encoded by a genome for their
expression, localization, interaction and post-translational modifications. Mass spectrometry
is an indispensible analytical platform for proteomics studies related to protein identification
and characterization. Mass spectrometry deals with the accurate mass measurement by
producing charged molecular species in vacuum and their separation by magnetic and electric
fields based on mass to charge (m/z) ratio. Technological developments in mass spectrometry
allows the characterization of proteins to its amino acid level leading to a wide variety of
applications such as protein characterization, disease biomarker detection, drug discovery
etc.. In this module we will depict the basic principles of mass spectrometry and its
applications in proteomics using Matrix-assisted laser desorption/ionization time-of-flight-
time-of-flight (MALDI-TOF-TOF) mass spectrometry as a model. MALDI is an efficient
process for generating gas-phase ion of peptides and proteins for mass spectrometric
detection. TOF is a mass analyzer in which the flight time of the ion from the source to the
detector is correlated to the m/z of the ion. It is widely used in proteomic research as a high
through-put technique to identify proteins and their post translational modifications. It is also
applicable for detection of intact molecular weight of any analytes.
Every mass spectrometer has three basic components







Fig 1. Schematic representation of components of mass spectrometer

1. Ionizer:
Here gas phase ions of the molecules are produced. The main reason behind production of
gas phase ions is almost all the mass analyzers are capable of analyzing molecules only if
they are charged. Hence it is the most important step, success of good analysis relays on good
ionization of the molecule. Formation of ions may be due to addition of a proton or removal
of an electron. Depending upon property of analyte the resultant ion may be singly or
multiple charged. Once the molecule is ionized it is ready to be separated on mass
analyzer. Two types of soft-ionization methods namely ESI and MALDI are mostly used for
biological samples. These are referred as soft ionization techniques due to its nature of
ionization which is very helpful in ionizing and studying intact biological molecules. These
have successfully replaced old conventional physical ionization techniques which had a
drawback of being non-applicable for biological molecules.
a) Electro spray ionization (ESI):
ESI method was invented by Prof. John B. Fenn. This land mark discovery was awarded with
Noble prize in chemistry in the year of 2002. Here the neutral molecules are transformed into
ions by bombarding with high energy electrons. Ions may be singly or multipaly charged due
to loss of single of many electrons respectively. Following is the reaction occurring in the gas
phase resulting in molecular ions;
M + e

M
+
+ 2e

Where M is the atom of molecule being ionized, e

is the electron, and M
+
is the resulting
ion. In most f the case is ESI is coupled with liquid chromatography. The fraction of liquid
containing desired molecule is directly subjected to ESI source so that the molecules are
further ready for MS analysis. Depending upon application one can choose best ionization
source.








Fig 2. Electro Spray Ionization (ESI)

The ESI source acts in atmospheric pressure. De-solvation is carried out simultaneously to
get rid of liquid components present along with sample. Usually warm nitrogen gas assists in
the process of de-solvation. As the sample droplet moves further it enters a region of vacuum
where the charge on the droplet increases resulting in single or multiple charged molecules.
Further these molecules enter into mass analyzer and get separated with respect to their m/z
ratios.
b) Matrix Assisted Laser Desorption Ionization (MALDI):
MALDI is one of the most widely used ionization technique. This ionization method was
independently developed by 2 groups Koichi Tanaka and Franz Hillenkamp. Koichi Tanaka
received the noble prize for development of this soft desorption ionization methods for mass
spectrometric analyses. The chemical compound named matrix used for ionization of the
sample. Matrix molecules co-crystallized with sample molecule. Matrix molecules have a
specialized property that they can be excited to higher energy state when it encounters UV-
LASER beam. Thus while coming back to ground state they concomitantly give out energy
which in turn received by sample molecules, leads to the formation the ions of interest.
Generated ions may receive a single proton and forms [M+H]
+
. Ionized molecule enters the
mass analyzer and yields the mass spectrum. The whole process of ionization and separation
of ions takes place in high energy vacuum. There are various kinds of matrixes being used
some of them are as follows;









Fig 3. MALDI-TOF mass spectrometer basic principle

Usually these matrices are low molecular weight aromatic compounds which do not interfare
with sample molecules. Depending upon the sample to be analyzed the matrices can be
chosen. The MALDI can be utilized for wide variety of applications. MALDI can detect
proteins of mass 300000 at femto molar level.
2. Mass Analyzer:
Ions entered into the mass analyzer get separated solely on the basis of m/z ratio, irrespective
of other parameters such as chemical properties. There are various types of mass analyzers
which differ with each other with respect to the principle involved in separation. The whole
mass analyzer acts in vacuum so that the highly reactive ions do not cross react or collide
with other ions moving simultaneously in the space. This ensures the ions are separated to
give accurate results on basis of their m/z ratio. Conventionally most of the instruments
maintain pressure ranging from 10
4
to10
8
tor. The level of the vacuum maintained depends
on the mass analyzer being used. Some of the commonly used mass analyzers are as follows;
1) Quadrupole (Q)
2) Ion trap (IT)
3) Time-of-ight (TOF)
4) Fourier transform ion cyclotron resonance (FTICR)

Each of these mass analyzers has their unique advantages and disadvantages. For instance,
we will discuss bit more about TOF because this is most widely used in combination with
MALDI. As the name indicates Time Of Flight separates the ions based on the flight time
taken by ion for travel between two points. Here all the ions are accelerated at a time and it is
taken into consideration that all the released ions have same potential resulting in ions of
same kinetic energy. Depending upon the m/z ratio, lighter ions reach the target first and
heavier reach late.
3. Detector:
The process of ionization and separation in mass analyzers happens in a matter of nano
seconds hence the process of separation requires an efficient detection of the separated
molecules. The part of the mass spectrometer called detector does this task of detecting ions
after separation sent detection may be on basis of charge, mass or velocity. So main function
of the detector is to get the information from the mass analyzer and convert these to electric
signals which are subsequently captured by the electronic devise. Ultimately these signals are
read by data system to bring out mass spectrum.