Mass Spectrometry

A mass spectrometer can be seen as a small scale determining the mass of molecular analytes.

Contrary to macroscopic objects, the mass of biomolecules like proteins and peptides cannot be measured as a response to gravity. In fact, mass spectrometers assess the influence of electromagnetic forces on ions with differing mass¹³⁷. Therefore, the biomolecules need to be ionized, which makes them susceptible to electric and magnetic fields, guiding the path through the mass analyzer and enabling measurement of mass to charge (m/z) ratios of these ions at the detector. This results in spectra with increasing m/z-values plotted against the intensity of these

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ions. In bottom up proteomics, peptides and peptide fragments analyzed by mass spectrometry can be used for the identification of proteins.

Electrospray ionization

Earlier ‘hard’ ionization methods lead to physical destruction of biomolecules, hampering identification of proteins and peptides by mass spectrometry. The development of ‘soft’ ionization techniques (awarded with the Nobel Prize in 2002) was a huge step, which made the routine mass spectrometric analysis of large polar organic molecules like proteins and peptides possible¹³⁸. The two used methods nowadays are matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI).

In MALDI, peptides are co-crystallized with suitable matrix molecules and subsequently hit by a laser. Upon contact with the laser, the matrix layer is heated, expands, and desorbs in the vacuum.

In this process, ions are generated and transferred first to matrix molecules and eventually to the analyte^{139, 140}. MALDI commonly leads to singly charged species.

Figure 7: Electrospray ionization. a) Schematic overview of the electrospray ionization process. Surface tension and electric force on the droplet lead to formation of a Taylor cone eventually resulting in droplet fission. b) Three models explain the transfer of a charge onto the analyte (modified from ¹⁴¹)

ESI is more suited for online coupling of liquid chromatography and mass spectrometry^{142, 143}. For the projects described in this thesis, a nano-ESI source was used. It is suited for flow rates down to

<10 nL/min, leads to less sample consumption with increased sensitivity and enhanced ionization efficiency¹⁴⁴. ESI-ion sources operate at atmospheric pressure¹⁴⁵. The mobile phase carrying the peptides for that chromatographic fraction is exiting the analytical column of the LC through a spray capillary. An electric potential is applied to this capillary resulting in a charged liquid. In positive ion mode, electrons are conducted towards the anode and positive ions accumulate at the capillary tip forming a droplet because of surface tension. With increasing voltage in the electric field, the electric force on the droplet reaches the amount of surface tension leading to the formation of a cone-shaped stream (Taylor Cone)¹⁴⁶. After reaching a certain voltage threshold, a jet of liquid is emitted from the cone. These small droplets are subject to rapid solvent evaporation leading to high charge density. At the Rayleigh limit¹⁴⁷, surface tension and Coulomb repulsion are in balance.

Repeated fission and/or evaporation events ultimately result in highly charged nanodroplets^{148, 149} (Figure 7a). Three models describe how charged peptides can then be generated¹⁴¹. Low molecular weight analytes likely follow the ion evaporation model (IEM). Charge repulsion on the droplet surface leads to an active ion generation process called ion evaporation (Figure 7b)¹⁵⁰. The charged

17 residue model (CRM) is a passive process, where the charge ends up on the analyte due to subsequent solvent evaporation. This model might apply to large globular species¹⁵¹. Another mechanism called chain ejection model (CEM) has been described for unfolded proteins. Briefly, polymer chains of a protein are subsequently emitted from the droplet¹⁵². The charged peptides can then enter the mass analyzer. Electrospray ionization leads to multiply charged ions, facilitating the detection of large molecules with mass spectrometers of limited m/z range and enabling ion dissociation for tandem MS measurements¹⁴². Recently, it has been shown that addition of low percentages of dimethylsulfoxide (DMSO) to LC solvents enhances ionization of peptides. The lower surface tension of DMSO might help faster and more efficient generation of charged nanodroplets.

This increases the ESI response and consequently improves sensitivity of proteomic measurements¹⁵³.

Mass analyzers - LTQ-Orbitrap Elite

The charged peptide ions generated by ESI then enter the mass spectrometer. Electric fields direct the peptides towards the mass analyzer where they are separated depending on their m/z ratio.

The mass analyzer should (i) determine the true mass of the analyte with high accuracy, (ii) be able to separate two similar masses (mass resolution) and (iii) detect even low amount of ions (sensitivity). Furthermore, the analyzers can only detect the (iv) abundance of ions in a certain range (dynamic range), are (v) limited in the m/z range they can acquire and (vi) vary in scan speeds.

Table 1 gives an overview of the four most commonly used analyzers for proteomic experiments today.

Table 1: Properties of mass analyzers used for proteomics (adapted from ^{154, 155}).

Analyzer Mass accuracy

Mass resolution

Sensitivity m/z range scan speed dynamic range Orbitrap <2 ppm >200,000 +++ 200-4000 moderate 10⁴

2D Ion Trap 0.1-0.5 Da 2000 ++ 50-2000 moderate 10⁴

Quadrupole 0.1-1 Da 1000 +++ 200-4000 moderate 10⁵

TOF <5 ppm >20,000 ++ >50,000 fast 10³

Time of flight (TOF) mass analyzers measure the time an ion needs to travel a certain distance within a vacuum. At the beginning, the ions all have the same kinetic energy, for analytes with equal charge, the flight time is dependent of the mass and therefore directly proportional to the square root of m/z¹⁵⁶.

Quadrupoles possess four parallel rods around a common axis. Two opposing rods are always electrically paired, resulting in helicoidal motion of ions through the quadrupole. With changing radio frequency applied to the rods only ions with a defined (range of) m/z value(s) have stable trajectories through the quadrupole and therefore make their way to a detector or further analysis, other ions will fall out along the way. It is mostly used as mass filter in hybrid instruments^{137, 157}. By adding two additional electrodes on both ends, ions can be trapped inside the quadrupole, also called linear (2D) ion trap (LTQ). Ions can spread out axially which leads to high ion capacity inside the linear trap. Besides radial confinement, main radio frequency (RF) now induces ‘secular’ ion motion, which is proportional to the main RF amplitude and the mass. Smaller ions move more than larger ones. Whether an ion can be kept in the trap or falls out, hence, depends on its m/z–ratio at a given main RF amplitude. Ions can then be ejected depending on their m/z-ratio by applying an

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additional ‘resonance ejection’ current at the exit rods. During a scan, the main RF is constantly increased and different ions can be measured. For isolation of an ion of interest, the exit rod current is superimposed (isolation waveform) so that all ions, except the desired one are ejected.

Quadrupoles and ion traps determine m/z-ratios in relation to ion stability in an electromagnetic field^{137, 158}.

A further development is the Orbitrap. Here, ions are radially trapped by oscillation around a spindle shaped electrode. This electrode is covered by an outer barrel-like electrode. Electromagnetic forces induce ion oscillation along the z-axis as well as radial movement around the central spindle electrode (Figure 8a, b). Dependent on their m/z ratios, ions differ in their oscillation frequency independent of energy and spatial spread. During a scan, the frequency of the whole ion population is assessed simultaneously. Fast Fourier Transformation of recorded transients then de-convolutes the time domain oscillation data and enables the generation of mass spectra. The longer ions are allowed to move around the central spindle, the higher is the resolution of the masses^159-162.

Figure 8: Tandem mass spectrometry. a) Schematic of a LTQ-Orbitrap Elite mass spectrometer ¹⁶³. Novel elements compared with previous versions of LTQ-Orbitraps are highlighted in red. Improvements in the ion optics introduced a rotated square quadrupole with a beam blocker to prevent neutral and low charged ions from transferring further. The dual ion trap can now cover higher dynamic ranges and the high-field Orbitrap has increased resolving power. b) Orbitrap mass analyzer¹³⁷. c) Peptide fragment nomenclature after Roepstorff-Fohlman¹⁶⁴.

Mass spectrometers employing a combination of the above-mentioned mass analyzers are very common. One popular combination is employed in the LTQ-Orbitrap Elite (Thermo Scientific, Figure 8a) and was mainly used for this study. It is the latest addition to an instrument family of Orbitrap mass analyzer with a preceding ion trap. Ions are transferred via improved ion optics to the ion trap. The ion optics employ a bent transfer quadrupole with a neutral blocker to prevent transfer of uncharged species. In the ion trap, ions are trapped until a certain amount of charges is collected (standard = 10⁶). Those are then further moved to the C-trap where ions are focused and transmitted to the improved high-field Orbitrap and subsequently analyzed163, 165, 166. This

19 arrangement combines the high sensitivity of ion traps with very high resolution and mass accuracy of the orbitrap¹⁶⁷.

Another successful combination features a quadrupole in front of the Orbitrap¹⁶⁸. With an ultra-high-field Orbitrap, this type of mass spectrometer is capable of deep proteome analysis in shorter time than the LTQ-Orbitrap combination¹⁶⁹.

Tandem mass spectrometry for peptide sequencing

Edman degradation was long the only method to determine the amino acid sequences in peptides and proteins^{170, 171}. With the introduction of soft ionization techniques and routine measurements of peptides and proteins in mass spectrometry, it is nowadays also possible to define a peptide sequence by mass spectrometry. This is realized in tandem mass spectrometry approaches. After obtaining a MS1 spectrum, single precursor ions are selected and subjected to a collision cell where peptides are fragmented. Then, fragments are again transferred to the mass analyzer and their m/z-ratio is again determined (MS/MS or MS2)^172-174. Ideally, the peptide breaks in a way that fragments differ by the mass of one amino acid. According to Roepstorff and Fohlman, resulting fragments containing the N-terminus of the peptide are called a, b or c, depending on the position of bond breakage. Respective C-terminal fragments are referred to as x, y or z-ions (Figure 8c)¹⁶⁴. There are different techniques to obtain fragment ions. In collision induced dissociation (CID), isolated ions are fragmented with neutral gas molecules (helium, argon or nitrogen)^{173, 175}. This can take place in an ion trap where the isolated ions are brought into resonance, but not ejected. They will then collide with inert helium-atoms, leading to vibrational excitation and finally breakage.

Unfortunately, MS2 spectra readout in ion traps lacks resolution and mass accuracy and suffers from a cutoff of small fragments. Higher energy trap dissociation (HCD) was developed in the C-trap leading to a hardware addition of an octopole collision cell (HCD-cell) in the rear end of the LTQ-Orbitrap devices. In this cell, ions are accelerated by current offsets (beam type activation), are then collided with nitrogen-atoms (or other gases) and are subsequently readout in the Orbitrap.

This generates tandem mass spectra with better mass accuracy and resolution and improves the detection of peptide modifications¹⁶⁷. In CID and HCD, the breakage mainly occurs at the peptide bond, generating y- and b- ions. Another method (not used in this thesis) is electron transfer dissociation (ETD). Here, fragments are generated by the influence of electrons. The transfer of an electron onto the positively charged peptide leads to an unstable positive radical ion and eventually fragmentation of the peptide at the N-Cα-bond. ETD mainly generates c- and z- ions (Figure 8c)¹⁷⁶. The LTQ Orbitrap Elite allows two ways of obtaining MS/MS spectra. Ions are collected in the ion trap, which now acts as a mass filter and only keeps the desired m/z-ratio stable. They can now either be fragmented in the ion trap by CID and readout by the detectors (multipliers) of the ion trap (high-low) or the filtered ions can be transferred to the HCD collision cell, where they are colliding with nitrogen atoms. Fragments are then subsequently read out in the Orbitrap (high-high)¹⁶⁶.

The possibility to measure fragment ions led to the development of different acquisition approaches. In classical DDA, the mass spectrometer switches between scanning peptide ions (MS1) and sequencing of peptide derived fragment ions (MS2). Peptides of the MS1 scan are selected for MS2 often dependent on their intensity¹⁷⁷. This process is limited by reproducibility, sensitivity and speed by which the mass spectrometer can acquire these spectra^{178, 179}. In DIA approaches, fragment ions for MS2 spectra are grouped into m/z-dependent sections and subsequently measured. Spectra are then compared to a spectral library for identification¹⁸⁰.

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