In the last 15 years, mass spectrometry (MS) has revolutionized the

identification of proteins. Complex mixtures of proteins are digested, separated

and then identified by MS. By the combination of these powerful analytical tools,

novel proteins and peptides, post-translational modifications, disease markers

and molecular interactions have been discovered.

          Mass spectrometers measure mass-to-charge ratios (m/z) of ionized

molecules. The instrumentation consists of three main components:

A. Ion source

B. Mass analyzer

C. Detector

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A. Ion sources

           Since m/z measurements are carried out in gas phase, analytes need to be

transferred into the gas phase and ionized in order to be detected. It was not until

the development of soft ionization methods that mass spectrometry became

fully available for proteomics. Soft ionization refers to methods by which labile molecules

like proteins and peptides are not destroyed during the ionization process. These

are electrospray ionization and matrix-assisted laser desorption/ionization.

          MALDI uses short pulses of laser light to ionize biomolecules. The sample is

mixed with small organic molecules called matrix, dried on a plate and inserted into

the mass spectrometer for analysis. The matrix molecules absorb light at the wavelength

emitted by the laser. When hitting the sample/matrix mixture, the laser beam expels

into the gas phase matrix molecules (M), sample neutrals (A) and matrix ions, (MH)⁺

and (MH)^-.

           The (MH)⁺ ion transfers the proton to the sample neutrals forming (A)⁺ ions

from which the m/z ratio is measured by the mass analyzer. The most commonly

utilized matrices in proteomics are α-cyano-4-hydroxy-cinnamic acid (for peptides,

abbreviated CHCA) and 3,5-dimethoxy-4-hydroxy-cinnamic acid (for proteins,

called sinapinic acid).

B. Mass analyzers

            In proteome analysis there are four main mass analyzers: time-of-flight,

fourier transform, quadrupole and ion traps. The following describes time-of-flight

and quadrupoles mass analyzers.

     The combination of high m/z range, sensitivity and compatibility with pulsed

ionization methods has made TOF the most commonly used mass analyzer in

MALDI. As the term TOF implies, these mass analyzers measure the time from

ions are produced/accelerated until they reach the detector. Ions formed at

the same time (and place) are accelerated by a fixed electrical potential.

After the acceleration step, ions enter into a field free region where they freely

“fly” towards the detector. As all ions with the same charge obtain the same kinetic

energy after acceleration, ions with lower m/z ratios achieve higher velocities than

those with higher m/z ratios. Ion velocities are inversely related to the square root

of m/z by the relation: m/z = 2 t² K / L², where m is the mass, z is the charge of

the ion, t is the drift time, K is the kinetic energy and L the drift length. Since in

these systems K and L are constant, m/z values are obtained by measuring the

drift times.

            These analyzers uses the stability of the trajectories in oscillating

electric fields to separate ions according to their m/z ratio. Quadrupoles are

made up of four parallel rods. A group of negatively charged ions entering the space

between the rods will be attracted towards the positively charged rod. If the potential

changes sign before the ions reach the rod, the ions will change direction. If the change

of polarity is made at a certain frequency -and intensity- only ions of a particular

m/z will pass all-the-way through the rods for detection. Mass-to-charge spectra

are generated by scanning different frequencies in short period of time (usually

faster than one full scan in less than 1 ms)

            Quadrupoles are low-resolution instruments but combined with TOF

mass analyzers they have become excellent tools in polypeptide analysis. 

            The most common detector in MALDI-TOF-MS is the multichannel plate

detector. It consists of a large flat detection area containing “holes” or channels.

The inlet and outlet of each channel are kept at an electric potential which leads

to the creation of a cascade of electrons, from ions hitting the walls of the channels.

The electrons in turn hit a scintillator plate and a photomultiplier tube detects the

photons generated. The signals are finally sent to a microprocessor that calculates

drift times and m/z values.

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