Tandem mass spectrometry

Tandem mass spectrometry or MS/MS combines two stages of MS: First, the mass of the intact ion is determined. Next, this precursor ion is isolated and fragmented. Low-energy collision in-duced dissociation (CID) is the most common fragmentation mode applied for large biomolecules.

Therein, fragmentation is induced by collision with inert gas such as helium, argon or nitrogen. The measurement of the resulting product ions presents the second stage of the analysis.

Tandem MS can be carried out in three separate parts of the mass spectrometer (tandem in-space), i.e., selection of the desired ion, fragmentation and mass determination are performed by different components of the instruments. Since these instruments contain two mass analyzers, they are termed hybrid mass spectrometers. Quadrupole time-of-flight (Q-ToF) instruments (see below) are a prominent example of hybrid instruments that perform tandem in-space MS.

Tandem in-time instruments perform ion selection, fragmentation and mass determination in the same part of the instrument but sequentially in time. This applies to linear ion traps which can be found as stand-alone instruments. A linear ion trap is also part of most Orbitrap instruments, the second type of hybrid instrument which will be described in more detail below.

The majority of tandem mass spectrometry experiments in proteomics are carried out with data dependent acquisition (DDA) in the mass spectrometer. The instrument records a full scan (MS1), measuring the masses of all species eluting from the LC at that time point. Next, the species giving rise to the most intense signals are chosen for fragmentation. After the product ions scans (MS2, MS/MS) have been acquired, the instrument records the next set of MS1 and MS2 scans. This cycle is repeated over the entire duration of the chromatographic gradient. In DDA, low abundant species are less likely to be chosen for fragmentation, an effect that increases with sample complexity.

1.2.1.1 Quadrupole time-of-flight (Q-ToF) mass spectrometers

A Q-ToF mass spectrometer is a hybrid instrument combining a quadrupole and a time-of-flight mass analyzer (see Figure 1.3). A quadrupole is composed of four parallel metal rods that serve as electrodes. An electric field is generated by applying both direct current (DC) and radio frequency (RF) potentials to the metal rods. At a given combination of DC and RF, only ions within a narrow mass-to-charge (m/z) ratio window pass through the quadrupole. All other ions are not confined within the quadrupole and are removed by the vacuum system. If only a radio frequency is applied, ions over a wide m/z range can pass through the quadrupole. The quadrupole can scan though an m/z range by changing both DC and RF potentials while keeping their ratio constant. By detecting at which ratio ions reach a detector, a mass spectrum can be acquired.

The time-of-flight mass analyzer separates ions according to their m/z ratio in a field-free drift region. Ions with a smallm/z ratio travel faster than those with a higherm/z ratio. Important for resolution and mass accuracy is that the ions enter the flight path at the same time with the same kinetic energy. The mass spectrum is recorded by detecting at which time ions reach the detector, the time is converted to the correspondingm/z ratio. The resolution of a ToF analyzer is increased with integration of a reflectron which serves as an electrostatic mirror. Ions with higher velocity penetrate deeper into the repelling electric field of the reflectron, compensating differences in kinetic energy of ions with the same m/z ratio. In addition, the flight path is increased, also leading to higher resolution.

Figure 1.3: Schematic representation of a quadrupole time-of-flight (Q-ToF) mass spectrometer.

It contains a regular quadrupole (mass analyzer) and an RF-only quadrupole (collision cell). Ions are directed into the time-of-flight mass analyzer by the pusher. The drift region is increased by the reflectron which guides the ions toward the detector.

A simplified Q-ToF mass spectrometer is depicted in Figure 1.3. In a first scan (precursor ion scan), all ions in a wide m/z range pass through both quadrupoles. The pusher applies a short pulse of an orthogonal accelerating field to the constant ion beam passing through the second quadrupole to direct a group of ions into the field-free drift region of the ToF. This way, the MS spectrum is recorded. For fragmentation experiments, the first quadrupole serves as a mass analyzer, selecting ions of the desiredm/z ratio. These are subsequently fragmented in the second, RF only quadrupole by collision with inert gas (beam-type collision induced dissociation). The product ion scan is again recorded in the ToF mass analyzer^[22].

1.2 Mass spectrometry in identification of biological macromolecules 7 1.2.1.2 Orbitrap mass spectrometers with linear ions traps (LTQ Orbitraps)

Figure 1.4: Schematic representation of a LTQ Orbitrap mass spectrometer. The first mass ana-lyzer is a linear ion trap with adjacent detectors. The orbitrap serves as mass anaana-lyzer and detector. From the ion trap, ions can be passed to the HCD collision cell or in-jected into the orbitrap by the C-trap. Mass spectra can be recorded in the ion trap as well as the orbitrap. Fragmentation can be performed in the ion trap (CID) or in the HCD collision cell.

A more recently developed class of hybrid mass spectrometers are LTQ Orbitraps. They contain a linear ion trap (linear trap quadrupole, LTQ) and an orbitrap mass analyzer. A simplified scheme is shown in Figure 1.4.

As implied by the name, the LTQ shares similarities to quadrupoles. It is built of four hyperbolic rods that are typically separated into three axial sections. Ions are trapped in the axial direction by applying different DC voltages to the three sections, and in the radial direction by RF potentials between opposite rods within the same section. Two of the central rods have a small slit though which ions can be ejected towards the detectors. Alternating current (AC) voltages are applied to these rods for isolation, activation, and ejection of ions.

The ion trap is held under a low helium pressure. Ions gather kinetic energy during acceleration by the ion optics between ESI source and ion trap (omitted from the simplified representation in Figure 1.4). During trapping, slow collisions with the inert gas lead to decrease of kinetic energy (cooling of ions).

In order to record a mass spectrum, the RF amplitude is increased at a constant rate from low to high voltages. This leads to successive destabilization of ions with an increasing m/z ratio. The AC is kept at constant frequency but increasing amplitude. This way, instable ions are directed through the slits towards the detector.

In order to isolate ions in a narrow m/z window, all other ions are destabilized at a constant RF amplitude by changing the AC frequency, skipping the frequency at which the ions of interest would become instable. After isolation, this AC frequency is used to increase the kinetic energy of the ion of interest. However, the AC amplitude is considerably smaller than during isolation so that the ions are not ejected. Due to the increased kinetic energy of the ions, collisions with helium lead to fragmentation (ion trap collision induced dissociation). The fragment spectrum can then be recorded as described above.

The applied potentials cannot stabilize ions with a smallm/z ratio. Thislow mass cut-off typically affects the lower third of the m/z range with respect to the uncharged precursor mass and thus prevents the detection of small fragmentation products.

The orbitrap consists of an axial central electrode and a co-axial outer electrode. The electrostatic field traps ions rotating around the central electrode and oscillating along its axis. Only the axial movement is independent of kinetic energy and spatial distribution of the ions, but it is related to the m/z ratio. The frequencies of the oscillating axial movement are detected by the current induced between the halves of the outer electrode and are converted into m/z ratios by Fourier transformation.

In a typical tandem MS experiment on LTQ Orbitraps, all ions entering the instrument are trapped in the LTQ, passed on to the C-trap and injected into the orbitrap where a high resolution precursor ion scan is recorded. Meanwhile, ions are isolated, fragmented and product ion scans are recorded in the linear ion trap (tandem in-time, see above). Since the sequencing speed of the LTQ by far exceeds that of the orbitrap, several product ion scans can be recorded in it while the precursor ion scan is acquired in the orbitrap. The CID spectra can also be recorded in the orbitrap, with the benefit of a considerably higher resolution and mass accuracy compared to the LTQ but at a significantly lower acquisition speed.

In addition to ion trap CID, LTQ Orbitraps offer a second fragmentation mode corresponding to beam-type CID on Q-ToF instruments, termedhigher-energy collision dissociation(HCD). Ions are again collected in the linear ion trap, the desired ion is isolated and passed to the HCD collision cell (multipole). There, the ions are fragmented by collisions with nitrogen molecules. The product ions are ejected into the C-trap and transferred into the orbitrap where the fragment spectrum is recorded^{[22, 25]}. HCD is slower compared to MS/MS in the ion trap but does not exhibit a low mass cut-off. In addition, HCD fragmentation corresponds to beam-type CID and is beneficial for some applications in comparison to ion trap CID (see 1.3.3.4).

1.2.1.3 Fragmentation of peptides

Figure 1.5: Nomenclature of peptide fragments resulting from backbone cleavage. Cleavage of the alkyl carbonyl bond produces a- and x-ions, cleavage of the amide bond b- and y-ions, and cleavage of the amino alkyl bond leads to c- and z-ions. a-, b-, and c-ions contain the peptide N-terminus while the corresponding C-terminal ions are called x, y, and z^{[26, 27]}.

Mass spectrometric analysis of peptides is usually carried out from acidic solutions in positive ion mode. Fragmentation of peptides is mostly charge-directed, i.e., a proton at the cleavage site is required. On the protonated peptide ions, charges are preferentially located on basic residues

1.2 Mass spectrometry in identification of biological macromolecules 9 (arginine, lysine, histidine) and at the peptide N-terminus. The energy transferred to the peptide upon collision with inert gas atoms or molecules can initiate redistribution of protons (mobile proton model) prior to fragmentation^[28]. However, fragmentation of protonated peptides is highly complex and follows many different pathways^[29]. Therefore, reliable prediction of observable fragments and especially their relative intensities is still not possible to the extent that this information could be the basis for automated peptide identification. Instead, all ions within a series are assumed to occur with the same probability and intensities are disregarded.

Figure 1.5 shows the three possible sites of fragmentation on the peptide backbone. Cleavage of the amide bond occurs most frequently, especially under CID conditions. The resulting spectra therefore contain b- and y-ion series as well as a-ions that are formed after loss of CO from b-ions. Within one series, the distance between two neighboring signals equals the mass of the corresponding amino acid (in its chain form without the water lost during amide bond formation). Therefore, amino acid sequences can be derived from calculated mass differences of fragment ions.

1.2.1.4 Fragmentation of RNA

Figure 1.6: Nomenclature of RNA fragments. Bases are simplified as gray spheres. In analogy to the nomenclature of peptide sequence ions, fragments resulting from cleavage of the phosphate backbone are termed a, b, c, or d for fragments containing the 5’ end or w, x, y, or z if the charge is retained on the 3’ end. Loss of a base is denoted as -Bn(X), where n is the position of the base counting from the 5’ end and X is the one letter code of the base^[30].

In contrast to the widespread application of mass spectrometry techniques in proteomics, MS is much less frequently applied in investigation of DNA or RNA. For most questions, (DNA) sequencing techniques are preferred as they can handle longer oligonucleotide segments, are less expensive and provide greater multiplexing capabilities.

Mass spectrometric analysis of oligonucleotides is usually carried out from basic solutions in neg-ative ion mode. The nomenclature follows rules similar to those of peptide fragments (see Figure

1.6). Upon CID fragmentation, the N-glycosidic bond is often cleaved to release the nucleic acid base, either as neutral loss or as a base anion. Additionally, backbone fragmentation predominantly leads to the formation of c- and y-ions. For DNA oligonucleotides, loss of the base is more dom-inant and backbone fragmentation leads to the formation of a- and (w-B)-ions. Fragmentation of protonated DNA in positive ion mode leads to similar product ion types. It has been proposed that the abundance of protonated bases after fragmentation correlates with the proton affinity, with C∼G > A » T ^[31].