A. Cálculo principal

2.2.1

Basics of mass spectrometry

The molecular composition of samples can also be studied with mass spectrometry (ms),72, 73 which has a high sensitivity and the ability to measure a diverse array of metabolites, and is thus considered one of the main techniques for metabolic profiling complementary to nmr, discussed in §2.1. Molecules with different chemical compositional formulae including different naturally abundant isotopes will have different molecular weights. Therefore, molecular information can be extracted by measuring the molecular weight of the species, and this is what ms achieves. ms uses the property that a charged particle placed in an electric or magnetic field experiences a Lorentz force F. This force is dependent on the ion charge (q), the electric field (E), the ion velocity (v) and the magnetic field (B), according to equation 2.5 (where × represents the vector cross product).74, 75

F = q(E + v× B) (2.5)

Because the exerted force equals the product of the acceleration a and the mass m (Newton’s second law, see equation 2.6), these equations can be combined into a differential equation from which the ratio m

q can be determined, see equation 2.7. The charge q is often divided by the

elementary charge e (1.602 × 10−19_{C) to obtain the charge number z. This ratio of mass to charge} number is often referred to as mass-to-charge ratio, and represented as m/z in this thesis.

F = ma (2.6)

m

qa = E + v× B (2.7)

The first step in ms is the ionisation of molecules (to create the charge q), which can be performed in various ways. The focus in this thesis will be on a method known as matrix-assisted laser desorption/ionisation (maldi). This is an ionisation method that allows molecular ions to be formed, in contrast to other methods that can create a large number of charged fragments from each molecular ion. It is therefore known as a ‘soft’ ionisation technique and is very suitable for large biomolecules, as those are very fragile and would be comprehensively fragmented if subjected to other ionisation approaches. A matrix is applied to the sample of interest and the matrix molecules will co-crystallise with the analyte upon vaporisation of the solvents, and when a laser

0 100 200 300 400 500 600 700 800 900 1000 0 5000 10000 m/z intensity

Figure 2.6: An example mass spectrum (acquired as part of the mass spectrometry imaging study described in chapter 5), ions with m/z=50−1000 were measured.

beam is targeted to the sample plate, the matrix molecules will absorb the energy of the laser irradiation to become desorbed and ionised. Gas-phase chemistry causes the transfer of ionisation to the matrix-trapped analyte molecule [M] to create ionised analyte molecules such as [M+H]+, [M+Na]+ and [M+K]+; double or multiple charged ions can also be formed.74, 75

The ions can then be measured using time-of-flight (tof) ms, where the ions experience an electric field. The potential energy Ep based on the electric potential V is converted to a kinetic

energy that will be equivalent for equally charged molecules, see equation 2.8.

qV = Ep= Ek=

1 2mv

2 _(2.8)

The velocity v of the molecule will depend on the mass m and charge q. Thus, by measuring the time-lapse to reach the detector one can infer the mass of the molecule: the heavier particles will travel more slowly, hence reaching the detector at later times.74, 75 _{The detector monitors the} charge induced or the current produced when an ion passes by or hits a surface, and this signal is amplified and recorded for the different flight times to produce a mass spectrum. An example mass spectrum (acquired as part of the maldi imaging of the sample discussed in chapter 5) is shown in figure 2.6.

2.2.2

: mass spectrometry imaging

Rather than acquiring a mass spectrum from a biofluid sample, e.g. after separation by chromato- graphy, it is possible to directly analyse a tissue or other sample of interest using a maldi imaging approach. Whereas in conventional maldi, the sample (e.g. a protein purified from a gel) is ‘spot- ted’ on a plate, and then the matrix is applied to that in order to get a mass spectrum, one can simply use the tissue as a ‘plate’. For example, a cryo-sectioned tissue slice can be mounted on a slide, see figure 2.7 A₁ and A₂, and after the matrix is applied to the sample, figure 2.7 A₃, a mass spectrum can be generated without additional preparation, as shown in 2.7 A₄.51

Matrices used for maldi on biological substrates need to efficiently absorb the laser irradiation, have good vaporisation characteristics and be able to act as a proton source to encourage ionisation; examples of crystallising matrix molecules are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic

EXPERIMENT DATA ANALYSIS multi-way analysis two-way analysis image analysis n mass spectral profiling

B

B

B

B

A

A

A

A

Figure 2.7: (A) A sample is cryo-sectioned (step A₁) and mounted on a plate (step A₂), to which a matrix is applied (step A₃) in preparation for mass spectrometry imaging (step A₄). In mass spectrometry imaging, a mass spectrum is acquired for each position along a grid. (B) The msi data can be presented in a data tensor of sizex× y × n(step B₁) and analysed with multi-way methods such as parafac. Alternatively, the full data set can be unfolded into ax∗ y × nmatrix (step B₂) and analysed with two-way chemometric techniques. Common univariate data analysis involves the evaluation of each of thenindividual m/z values, represented as an image (step B₃), or each of thex∗ ypixels, which can be represented by a characteristic mass spectrum (step B₄).

acid) and alpha-cyano-4-hydroxycinnamic acid (αchca). The matrix molecules are dissolved in a mixture of organic and aqueous solvent to facilitate both hydrophobic and hydrophilic analyte molecules to dissolve and subsequently co-crystallise with the matrix molecules.

The acquisition of a spectrum using maldi-imaging is analogous to the approach frequently taken in metabonomics, where instead of purifying, identifying and quantifying each individual constituent, a characteristic fingerprint of the sample composition is generated with a powerful analytical approach. As a result, the information density of spectra generated in this manner is very high.

Once a spectrum has been acquired in one location, the set-up is repositioned in order to acquire a mass spectrum from a different part of the sample; using this mass spectrometry imaging (msi) approach, a ‘grid’ of mass spectra across the sample is obtained, see figure 2.7.51, 76, 77 _Thus, localised information is obtained, reporting the chemical make-up of the different regions with mass spectrometry-based molecular fingerprints. It is not hard to imagine that these distribution maps of biomolecules, e.g. shown in figure 2.7 B3, should consistently reflect properties of different tissue regions. This can, for example, aid characterisation of a tissue into different structural units, and additionally make molecular masses available that typify these segments.78 _{A plethora} of data analysis objectives and methods are available to evaluate the images and the spectroscopic data, and this will be the main topic of chapter 5: how to extract the maximum amount of useful information from an msi data set. In general, these approaches can be divided into univariate methods:

• Studying single m/z images, as shown in figure 2.7 B₃.

• Evaluation of selected mass spectra for different regions in a ‘profiling’ manner, shown in figure 2.7 B4.

and multivariate methods:

• Analysis of the complete 3-way data tensor with multi-way analysis methods such as parafac and tucker, see figure 2.7 B1.4, 79

• Unfolding the data into a two-way matrix, where the pixels are displayed in the different rows and the m/z variables in the different columns, shown in figure 2.7 B₂. Then multivariate methods such as principal component analysis and self-organising maps can be performed.

There are a large number of other ionisation, mass analysing and detection methods, which were not discussed here. It should be noted though, that some of these can also be used in an imaging set-up, for example secondary ion mass spectrometry (sims). However, maldi is the method of choice for biological matrices as it is a soft ionisation technique and therefore the compounds are not fully fragmented, and tof is the most used complementary mass analyser companion. maldi and tof are compatible through the pulse-process nature of the laser-based ionisation, moreover, tof is able to analyse a large mass range. Imaging data acquired with the maldi-tof msi experiment are discussed in chapter 5.