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Mass spectrometry analysis deals with ionized molecules because ions are easier to manipulate than neutral molecules. Hence, the first step of a MS experiment consists in the production of gas phase ions from the sample. Then, the ionized molecules are separated by their mass-to-charge ratio (m/z) in a mass analyzer, and finally, the different ions are measured and the signal generated is amplified and recorded as a mass spectrum, where the relative ion intensity (ordinate) is plotted against the m/z value (abscissa) [197, 198]. The unit of mass used is the unified atomic mass (u), defined as 1/12 of the mass of one 12C atom, normally represented by the term Dalton (Da) [199].

The analysis of the charged atoms or molecules is performed with a mass spectrometer, in different but sequential parts, as depicted in Figure I.10. First, the sample inlet introduces the sample into the ion source. Depending on the type of sample and ionization method, the sample can be introduced directly into the ionization chamber, as in MALDI-TOF, or via a chromatographic interface, like the LC-MS [197, 199]. Once inside the ion source the sample is ionized, generally by electron ejection or electron capture, protonation or deprotonation, or by adduct formation [200]. The first ionization methods, electron ionization (EI) and chemical ionization (CI), were developed mainly for the analysis of organic compounds. These very energetic techniques cause broad molecular fragmentation, and are only suitable for the ionization of volatile and thermostable samples [201]. The development of new

35 technologies, like electrospray and atmospheric pressure ionization, which are used to analyze liquid samples, or plasma desorption and matrix-assisted laser desorption/ionization that are mostly used to ionize solid samples into the gas phase, permitted the analysis of proteins, peptides and polymers. Nowadays, several methods for sample ionization are easily accessible and their use depends on both the type of sample, and the mass spectrometer available [202]. An extensive description of these methods can be found in the books of Hoffman and Stroobant [200], J. Gross [198], and references cited therein. The most important ionization methods used in proteomics are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), which are described in sections I.3.2.1 and I.3.2.2, respectively.

Figure I.10: Basic diagram of a mass spectrometer. (adapted from Gross, J. H. [198])

After sample ionization, the ions are directed into the mass analyzer, where they are separated according to their m/z ratio using appropriate electric fields, magnetic fields, or both [203]. The principle of separation varies with the type of mass analyzer and the ions can be distinguished by the differences on their momentum, velocity and kinetic energy [197, 203]. Several mass analyzers are currently available, each one with different features, advantages and limitations. The better known are the quadrupoles (Q) and magnetic sectors (B), time-of-flight (TOF), ion cyclotron resonance (ICR), ion traps (IT) and more recently the orbitrap (OT) [197, 198, 200]. The choice of the mass analyzer depends not only on the objective of the work, but also on their intrinsic characteristics, such as: (i) mass range, i.e. the maximum m/z value detected; (ii) resolution, which is the ability to distinguish between ions with a small m/z difference; (iii) mass accuracy, which represents the difference between the measured m/z and the theoretical m/z value, usually expressed in parts per million (ppm); (iv) ion transmission, i.e. the ratio between the number of ions that reach the detector and the number of ions entering the analyzer; and (v) scanning speed, which is the time needed by the mass analyzer to scan a particular mass range and produce a mass spectrum [200, 203]. An overview of the most important features and differences between the referred mass analyzers is given in Table I.3.

Another important aspect in the choice of the mass analyzer is the ability to perform tandem mass spectrometry (MS/MS). In MS/MS the precursor ion is selected and fragmented into characteristic secondary ions, known as product ions, which can be used for structural or sequencing studies [204, 205]. There are different ways to obtain ion fragmentation: (i) metastable or spontaneous fragmentation; (ii) collision induced dissociation (CID), which occurs when the parent ion collides

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with neutral gas molecules; (iii) electron-capture dissociation (ECD), which involves the capture of a low energy electron by a protonated ion and subsequent fragmentation; and (iv) electron-transfer dissociation (ETD), in which free radical anions are used to fragment molecular ions, by electron transference between multiple charged species [206-208]. MS/MS experiments can be performed by (i) coupling two or more mass analyzers of the same type, as the triple quadrupole (QqQ) or TOF/TOF systems; (ii) by coupling different mass analyzers creating hybrid systems, such as the quadrupole- time-of-flight (Qq-TOF); or (iii) by doing the appropriate temporal sequence of events in the same device, normally ion-traps (IT), as the quadrupole ion-trap (QIT) or linear ion-trap (LIT) [197, 209].

Table I.3: Overview of the main characteristics of different mass analyzers available. (adapted from

Gross, J. H. [198] and Hoffmann et al. [200])

a

Varies with reflectron and linear modes (see section I.3.2.2. for details)

The last part of the mass spectrometer is the detector that records and amplifies the ion current of the mass resolved ions. The type of detector used depends on the design of the mass analyzer, but generally they can be divided in two groups: (i) focal-point detectors, which only count ions of a single mass at a time; and (ii) focal-plane array detectors, that monitor all ions all the time, resulting in improved detection and sensitivity [210, 211]. Among the several types of detectors developed over the years, the Faraday cup detector is the simplest, consisting only in a metal box with a collector electrode at the bottom to measure the ion current [210, 212]. Currently, the most common ion detector in MS is the electron multiplier (EM), also known as secondary-electron multiplier detector (SEM), which relies on the emission of secondary electrons produced when the accelerated ion beam strikes the conversion dynode. This event generates an electron cascade that hits other dynodes to produce even more electrons, resulting in a gain of at least 106 electrons for each ion [197]. There are also other examples of detectors, such as photographic plates, photomultipliers and multichannel plate

TOF Q B IT ICR OT Mass Range (Da) Unlimited 4000 20 000 6000 > 10 000 6000 Resolution 15 000a 2000 100 000 4000 500 000 100 000 Accuracy (PPM) 5-50 a 100 <10 100 <5 <5 Sampling

mode Pulsed Continuous Continuous Pulsed Pulsed Pulsed Dynamic

Range 10

2_-106 ₁₀7 ₁₀9 ₁₀2_-105 ₁₀2_-105 ₁₀2_-105

MS/MS Great Great Excellent Excellent Great Excellent

37 detectors, which are described in detail in dedicated bibliography [197, 210]. Ideally, a MS detector should have the following characteristics: (i) wide mass-range and mass independent response; (ii) low noise level; (iii) simultaneous detection; (iv) short recovery time and fast response; and also (v) high saturation level and a wide dynamic range [210].

Finally, the ion source, the analyzer and the detector, operate under high vacuum conditions during mass analysis. The vacuum system allows ions to move through different parts of the mass spectrometer without colliding with air molecules. Normally, the pressure at the ion source is maintained between 10-4 and 10-8 torr, although atmospheric pressure can also be used. At the mass analyzer the pressure is usually lower than 10-8 torr [197, 198].