CENTRO INTEGRADO “ESCUELA DE EDUCADORES - HEZITZAILE ESKOLA” - PAMPLONA

MS involves the ionization of analytes in the ion source and their separation depending on their mass per charge (m/z) ratio in one or more mass analyzers. Subsequently, a detector registers the ion current from the analyzers, yielding the corresponding mass spectrum. During several years, a fundamental issue in the analysis of biological samples was the transference of polar and non volatile molecules into the gas phase without destroying them. The proposed solution commonly known as soft ionization techniques (MALDI and ESI) had a high impact on the peptide and protein research field. Nobel Prize awarded the latter technique in 2002 in the field of chemistry [120]. One of the most important advantages of ESI over MALDI is the possibility of on-line connection with the chromatographic eluent. In addition, while MALDI is used for relatively simple peptide mixtures, ESI-MS system is preferred for complex samples [217].

Most popular analyzers in proteomics are ion trap (IT), quadrupole (Q), time of flight (ToF) tubes, and Orbitrap cells [120, 123]. Hybrid mass spectrometers can yield additional information on analyte structure. Different fragmentation mechanisms (collision induced dissociation (CID), also known as collision activated dissociation, electron capture and electron transfer dissociation (ECD and ETD) etc.) are available [117].

48 R I INTR O DUC TI ON

Principles of ESI. ESI is a technique of ionization at atmospheric pressure where a sample

is nebulized and ionized at the end of a capillary due to the action of a strong electric field (several kV) (see Fig. I.21). After ionization and nebulization, charged droplets move forward due to the established electric field gradient and the pressure gradient (ionization takes place at atmospheric pressure while mass analyzers are at very low pressure). Moreover, the control of chamber temperature enables the evaporation of charged droplets during this transition. As a consequence of solvent evaporation, the size of droplets decreases and the charge density increases, which results in a repetitive droplet instability and explosion into finer droplets. Finally, electrostatic repulsion is sufficiently high to cause desorption of analyte ions which then pass to the MS. Ions generated by ESI usually bear multiple charges [119, 120, 122, 128].

Fig. I.21. ESI ionization overview.

In ESI, analytes compete for charge as they are extruded from spray droplets. Consequently, the main drawback of ESI is its sensitivity to easily chargeable salts and detergents [218, 219]. Recently, an additional feature in ESI named “Jet Stream” has been introduced. This technology consists of an additional sheath gas heated at high temperature that focuses the nebulizer spray and desolvates ions more efficiently, thus improving sensitivity at high LC flow rates [205].

Mass analyzers and hybrids. Key parameters of any mass analyzer are sensitivity, resolution,

49 R I INTR O DUC TI ON

quadrupole, ion trap, time of flight, and Orbitrap [217]. The schemes of all four mass analyzers are depicted on Fig. I.22. Moreover, different hybrid instruments have been developed by fusing various mass analyzers, ions optic, and fragmentation tools [131].

Fig. I.22. Schemes of Q, IT, ToF, and Orbitrap mass analyzers.

First mass analyzer introduced in the market was the quadrupole (Q). It consists of four parallel rods to which a fixed and direct current (DC) and an alternating radio frequency (RF) are applied [121] (see Fig. I.22). By applying appropriate RF/DC voltages, only a narrow m/z range can reach the detector [219]. Q is limited in mass range (until 4000 m/z) and provides low resolution. This mass analyzer can operate in single ion monitoring (SIM) or in scan modes. The SIM mode provides a significantly higher sensitivity. In the SIM mode, Q parameters are adjusted to select only one specific m/z. The time required to collect data of a particular mass is the transmission efficiency. When various m/z values are detected, the instrument works sequentially (from low to high m/z) [205]. When a high number of ions is selected, the transmission efficiency for every ion is reduced which directly decreases sensitivity. Three consecutive Q configured together (QqQ) can enhance significantly selectivity. The first and

50 R I INTR O DUC TI ON

third Q are used for scanning/filtrating ions, while the middle one is used as a collision cell. Ions are fragmented using CID, which is a low energy ‘beam type collision’ with a ground gas (e.g. nitrogen) [218]. Many MS/MS scan modes are possible in QqQ, like product ion, precursor ion, neutral loss or selected reaction monitoring (SRM or multiple reactions monitoring (MRM)). The two first modes are especially useful to identify closely related molecules or functional groups, and are out of the scope of this thesis. In the SRM mode (see Fig. I.23), Q1 and Q3 are programmed to filter just selected precursor and fragment ions, thereby increasing selectivity. When the number of monitored compounds during an analysis is too high and the transmission efficiency is very low, different time-scheduled windows with different SRM transitions and time intervals can be employed [206].

Fig. I.23. Overview of the selected reaction monitoring mode in QqQ. Source: [220].

Quadrupole IT consists of two parallel oval rods enabling the application of variable RF and one circular ring enabling the application of a fixed RF (see Fig. I.22). IT permits to isolate and fragmentate ions in the same space. Ions are trapped into a small volume by an oscillating electric field (RF/DC) and scanned by increasing the RF applied to the trap. IT is filled with helium gas that takes the excess of ions kinetic energy and focus them in the center of the trap. Further isolation of selected precursor ions is performed by ejecting all ions except that selected as precursor. Isolated ions are translationally excited before the collisions with helium gas. The translational energy is converted to internal energy during the collisions with helium, which leads to the ion fragmentation (so called resonance CID). Obtained daughter ions are then scanned out. The main advantage of IT is its quick shift between scanning for masses of analytes and generating fragmentation spectra of these ions [131]. IT is very sensitive since it can concentrate ions in the trapping field for different amounts of time. Additionally, IT is the only mass analyzer that can provide multiple MSn _{fragmentation and, alone, can be used to identify}

peptides. ‘Pseudo- SRM’ mode is used with IT analyzer when upon fragmentation of a precursor ion, MS/MS data are acquired on a partial mass range, which is centered on a

51 R I INTR O DUC TI ON

fragment ion [221]. The main disadvantage of IT is its low mass accuracy, partly due to the limited number of ions that can be accumulated at its center. Linear ion traps (LIT) are a recent improvement of IT, where ions are stored in a cylindric volume that is larger than a conventional IT. This feature improves IT sensitivity, resolution, and mass accuracy [131, 217, 219]. Dual pressure LIT improves sampling speed and sensitivity. In this hybrid, the first IT efficiently captures and fragments ions at relatively high pressure, whereas the second IT performs extremely fast scan at reduced pressure [131, 222].

ToF-MS is a mass analyzer where ionized molecules are accelerated by a fixed amount of kinetic energy and travel down to a flight tube (see Fig. I.22). Due to the differences in masses, ions have different velocities and reach the detector, at the far end flight tube, at different times. ToF instruments provide high mass resolution and accuracy over a broad mass range. Typically, ToF instruments can achieve very high resolutions [218, 219]. The combination of ToF with Q is other commonly applied hybrid. Q-ToF consists of one Q, one hexapole collision cell, and a ToF mass analyzer (see Fig. I.24). This configuration provides additional advantages since it is possible to select parent ions for their fragmentation and to separate fragments using high resolving power ToF [121].

Fig. I.24. Schematic diagram of a Q-ToF. Adapted from: [131].

Orbitrap is one of the newest mass analyzers. It consists of an outer electrode enclosing a central inner electrode and a ceramic ring (see Fig. I.22). In Orbitrap, moving ions are trapped into an electrostatic field. The attraction toward the central electrode is compensated by the centrifugal force that comes from the initial tangential velocity of ions (similar to satellites on orbit). This electrostatic field forces ions to move in complex spiral patterns. The axial component of ions oscillations (independent from initial energy, angles, and position) are

52 R I INTR O DUC TI ON

detected as a current in two parts of the electrode that encapsulate the core. Fourier transform turns those currents into oscillation frequencies of ions at different masses, which allows obtaining their accurate m/z values. Although it may be possible to fragment ions in the Orbitrap, it is more practical and much faster to hybrid Orbitrap with other systems. In this regards, a hybrid with sensitive and very fast LIT has been developed (LTQ-Orbitrap). LTQ- Orbitrap contains three main parts: a LIT analyzer to obtain MS and MSn spectra with very high sensitivity and mass accuracy, a C-Trap system (simplified Q) to accumulate and store ions, and an Orbitrap to analyze ions accumulated in the C-Trap (see Fig. I.25). Depending on requirements, two analyzers can be used independently or in concert. When both analyzers work simultaneously, high resolution/mass accuracy spectra are acquired by Orbitrap while fast fragmentation and MS/MS detection is carried out by LIT [223]. The next generation of LTQ Orbitrap system, termed Velos, provides even more improved sensitivity and scan speed. The most important implemented changes are: the use of a dual LIT instead of a simple LIT which accelerates the acquisition speed, improves and makes more efficient the fragmentation by a higher energy collision dissociation cell (HCD) system, etc. [222].

Fig. I.25. Schematic diagram of the LTQ Orbitrap Velos MS. Adapted from: [222].

Finally, Table I.13 groups some characterictics of the mass analyzers and hybrids described. Q-ToF and LTQ-Orbitraps are the MS equipments with the highest resolving power and mass accuracy. Regarding m/z range, Q-ToF is the system enabling to work at higher m/z values and, thus, the most suitable to work with large molecules. Last extremely important parameter is acquisition speed, being IT and LIT the systems showing the highest values [224].

53 R I INTR O DUC TI ON

Table I.13. Overview of some selected commercially available mass spectrometers with their technical

specifications provided. Source: [224].

The selection of a suitable MS system obviously depends on the requirements of the analysis in terms of resolution, mass accuracy, and acquisition speed. QqQ provides good linear dynamic range, high precision, and less matrix interferences for product ion measurements. QqQ is perfectly adapted for targeted analysis and quantitative applications. In fact, SRM using QqQ is a golden standard for LC-MS quantification. IT and LIT are fast, sensitive, and able to perform multi-stage fragmentation. They are perfect for both targeted and non-targeted analysis coupled to fast and highly efficient LC systems. Q-ToF instrument can acquire data over a wide mass range with high mass accuracy, resolving power, and speed. Therefore, it is particularly well suited for non-targeted analysis and, in some cases, for targeted [224]. On the other hand, Orbitrap instruments offer extremely high resolving power. However, such high quality measurements sacrifice the time of analysis proportionally with requested resolution [223]. LTQ-Orbitrap shows low sensitivity and, due to the slower data acquisition rate, it requires slower chromatography. Latest generation of this instrument has improved significantly this inconvenient by the introduction of the dual-LIT. Therefore, LTQ-Orbitrap instruments are especially important in non-targeted analysis of complex samples.

Application of tandem MS to the sequencing of peptides. Tandem MS enables to obtain the

primary structure of peptides. In the first stage, the peptide ion is isolated and fragmented and the MS/MS spectrum of peptide fragments is generated. Fragment ions produced by tandem MS can be separated into two classes. One class retains the charge on the N-terminal while the

5 _{Defined as m/z value of particular peak divided by the peak full width at half maximum. Resolving Power is} defined for a particular m/z value.

6 _{Defined as the inverse of Resolving Power expressed as Δm/z for a given m/z value.}

7 _{Defined as the relative difference between the experimental m/z value related to its theoretical value including} the sign (+ or -) and expressed in ppm.

8 _{Defined as the limits of m/z over which the mass analyzer can measure ions.}

Mass analyzer type Resolving Power5 (defined at m/z) Resolution6 (Δm/z) Mass accuracy (ppm) using internal calibration7 m/z range8 Acquisition speed (Hz) QqQ 7,500 (m/z 508) 0.07 5 10-3,000 5 IT - 0.1 - 50-6,000 52 LIT - 0.05 - 15-4,000 66 Q-ToF 42,000 (m/z 922) 0.02 <1 50-10,000 50 LTQ-Orbitrap 240,000 (m/z 400) 0.002 <1 50-4,000 8 (at RP= 15,000)

54 R I INTR O DUC TI ON

cleavage is observed in the C-terminal. This fragmentation can occur at three different positions, each of which is sequence designated as types an, bn, and cn (see Fig. I.26.). The

second class of fragment ions generated from the N-terminal retains the charge on the C- terminal, while cleavage is observed from the N-terminal. Like the first class, this fragmentation occurs at three different positions, types xn, yn, and zn (see Fig. I. 26).

Fig. I.26. Peptide fragmentation nomenclature.

MS/MS fragmentation can be performed by various fragmentation methods. CID is the most widely applied where peptide ions undergo dissociation at amide bonds generating b- and y- type fragment ions. In contrast, ECD and ETD lead to the cleavage of N-Cα backbone bonds

generating c- and z- type fragment ions [225]. While the differences among spectra obtained with CID and ECD/ETD are obvious, interestingly differences have also been observed among CID types. Indeed, the energy applied to a peptide in IT (resonance CID) and Q (beam type CID) analyzers is different. Obtained spectra differences have been associated mainly to gas conditions and kinetic energy. Lately, introduced HCD fragmentation type is other beam CID that has shown to generate spectra similar to Q fragmentation [226].

On the other hand, peptide fragmentation information can sometimes be incomplete or some peaks can belong to other peptide series, which result in complex MS/MS spectra analysis. Indeed, peptides obtained by enzymes, which do not cut at basic residues, do not possess charge at C- or N-terminal of peptides. In this case, the abundance of b- and y- ions series can be reduced and some abundant internal ions are generated. This fact complicates spectrum interpretation and peptide identification. Peptides with basic residues at C- or N-terminus, like tryptic peptides, cleave easily in MS/MS obtaining fragments that deliver richer sequence

55 R I INTR O DUC TI ON

information on b- and y- ion series and, consequently, are much easier to interpret. Obtained data can be treated using database search or de novo sequencing. De novo sequencing is influenced by the quality of data, in terms of mass accuracy and resolution, as well as the information obtained from the MS/MS spectrum. Database search is easier since the number of possible peptide amino acids sequences that occur in nature is limited [120].