MS technologies for proteome analysis

Recently mass spectrometry (MS) has emerged as an indispensable tool to analyze protein and peptide mixtures arising from their proteolysis. Among all the available techniques commonly used for the analysis of a full proteome, MS has incrementally improved especially because it provides specificity, speed and reliability of the analytical response and it also gives the chance to handle the complexity associated to the biological system. For these reasons the employment of MS in the study of food proteomes is rapidly increasing.

For a long time MS has been restricted just for the analysis of small and thermostable compounds. The introduction of new types of mass spectrometers, which allowed the formation of ions from formation of molecular ions of intact biomolecules, as electrospray ionization (ESI),¹² and matrix assisted laser desorption/ionization (MALDI),¹³ made peptides and also polypeptides able to be analyzed by MS.

In MS technologies, ionization is fundamental, as the physics of mass spectrometry relies upon the molecule of interest being charged, resulting in the formation of either positive ions or negative ions. In this way a molecular ion species is formed and, depending on the ionization method, fragment ions may also be created. These ion species are then separated according to their mass-to-charge (m/z) ratio and the masses are assigned from the measurement of some physical parameter. Finally, the measurement of ion abundance, based on peak height or peak area, is made leading to a semi-quantitative or quantitative answer.

IoIonniizazattiionon ssoouurrcceess. As concerns ionization, electrospray ionization probably is the preferred ionization methods for proteomics.¹⁴ Here the sample is dissolved in a solvent mixture (e.g., acetonitrile-water) and then injected into a capillary held at a potential of 3-4 kV. As a result, a very fine spray of solvent droplets containing ions are formed. Multiple charged gas-phase ions are subsequently formed during the desorption process due to the evaporation of the solvent,¹⁵ which will then enter the mass analyzer (Fig 4).

Fig 4. Electrospray ionization (ESI) and ion source overview.

32

For ESI, there are several ways to deliver the sample to the mass spectrometer. In the simplest method, electrospray sources have been connected in line with LC systems that automatically purify and deliver the sample to the mass spectrometer. Examples of this method are LC and reversed phase LC (RP-LC).

The second major ionization technique, matrix-assisted laser desorption ionization (MALDI), relies on a laser which is fired at a sample plate containing a dried mixture of matrix (α-cyano-4-hydroxycinammic acid) and sample. The matrix absorbs radiation from the laser resulting in excitation of the matrix molecules. As a result, a dense plume containing both the matrix and the analyte molecules is produced. The analyte molecules interact with protons from the matrix to form mainly single charged ions that enter the mass analyzer (Fig 5). The formed ions are separated in a mass analyzer according to their mass-to-charge ratio (m/z).¹⁵

Fig 5. A schematic representation of MALDI source.

AnAnaallyyzezerrss. The analyzer is an instrument able to separate or differentiate introduced ions. Both positive and negative ions (as well as uncharged, neutral species) form in the ion source. However, only one polarity is recorded at a given moment. Basically, four types of mass analyzers have been deeply applied in the study of proteomes:

quadrupole (Q), time-of-flight (TOF), ion trap, and Fourier-transform ion cyclotron resonance (FT-ICR), which strongly differ in both the physical principles of ion separation and the analytical performance.¹⁶

Quadrupole uses oscillating electrical fields to selectively stabilize or destabilize ions.

By passing ions through a radio frequency quadrupole field, single mass/charge ratios can be measured. Only ions within a particular mass range, exhibiting oscillations of constant amplitude, could collect at the detector (Fig 6). Single quadrupole mass spectrometers require a clean matrix to avoid the interference of unwanted ions, and they exhibit very good sensitivity.

33

Fig 6. A schematic representation of the quadrupole analyzer.

In the time-of-flight analyzer, the ions enter a field-free drift range where they are not accelerated further and thus travel with a speed they have reached at the moment when passing the electrode. This speed, in turn, depends on the mass of the ions with heavier molecules having a higher moment of inertia and hence a lower velocity; ions with smaller m/z values will reach the detector first. For the resolution of the mass spectral analyses the length, L, of the field-free drift range is essential and in modern machines it measures about one meter. Further increase in resolution can be reached in the reflector mode: after having passed a distance in the drift range the ions enter another electromagnetic field and are accelerated in a nearly reversed direction towards an ion detector (Fig 7).

Fig 7. A schematic representation of the time-of-flight (TOF) analyzer.

The quadrupole ion trap,¹⁷ is the three dimensional analogue of the linear quadrupole mass filter. In this device too, ions are subjected to forces applied by a radio frequency field but the forces occur in all three, instead of just two, dimensions.

Stable motion of ions in the linear quadrupole allowed ions freedom of motion in one dimension. In the ion trap, stable motion allows no degrees of freedom. Hence, ions are trapped within the system. These features confer high sensitivity and high resolution to the system.

The first high-performance mass analyzer to employ ion trapping in an electrostatic field, the Orbitrap mass spectrometer, easily achieves ultra-high resolution (>100,000) with high mass accuracy (<1 ppm), a wide dynamic range (up to 5,000), fast scanning, and uncompromised sensitivity. Ions injected from the ion source are

34

trapped in the linear ion trap where ions of interest can be isolated and fragmented, scanned out and detected by an independent set of detectors. For high accuracy measurements, achieved with hybrid instruments such as linear trap quadrupole and Orbitrap (LTQ-Orbitrap), the ions are axially ejected from the linear trap into the C-Trap where they are captured again and 'cooled' by collisions with nitrogen gas. The ions are then squeezed into a smaller cloud within the C-Trap prior to injection into the Orbitrap. As ions enter the Orbitrap mass analyzer, the voltage on the central electrode increases, forcing the ion packets to circle around the electrode. The ions enter the Orbitrap slightly off axis and keep oscillating along the central electrode (left-right). The image current is recorded on the outer split electrodes. The signals are amplified and transformed into a frequency spectrum by fast Fourier Transformation and converted into a mass spectrum (Fig 8).¹⁸

Fig 8. A schematic representation of the hybrid LTQ- Orbitrap analyzer.

FT-ICR (Fourier Transform Ion Cyclotron Resonance) mass spectrometers are useful, high-precision analyzers. Fourier transform mass spectrometry (FTMS) detects the image current produced by ions cyclotroning in the presence of a magnetic field. The ions are injected into a static electric/magnetic ion trap, where they form part of a circuit. Detectors in the system measure the electrical signal of ions that pass near them, producing a periodic signal. The frequency of the signal can be used determine the ion's mass/charge ratio. The FTMS is highly sensitive and boasts higher precision and resolution than other methods (Fig 9).¹⁹

Fig 9. A Schematic of FT-ICR-MS showing the ion trapping, detection and signal generation.

In most cases, one or more forms of mass spectrometry, which utilize different methods of sample ionization, are used for protein identification. The first is MALDI-TOF mass spectrometry, used to perform peptide and protein mass fingerprinting; the

35

second is ESI tandem mass spectrometry (MS/MS), usually hyphenated to high performance liquid chromatography separation, used to perform peptide sequence elucidation and identification of the corresponding protein.²⁰