Ever since the introduction of desorption based ionisation methods in the late 1970's, mass spectrometry has increasingly become the chosen method for rigorous structural characterization of biomolecules (De Hoffmann and Stroobant, 2007). However, the history of mass spectrometry starts with Sir J.J. Thomson who measured the fundamental particles-electrons e/m in 1897 (early physicists typically reported a charge to mass ratio, e/m, rather than the present MS standard of m/z). Two years later, he created an instrument that could simultaneously measure e/m and e, thus indirectly measuring the mass of the electron. Consequently, he was awarded the Nobel Prize in Physics in 1906. Then, later on, he built the first mass spectrometer to measure the masses of charged atoms. This instrument used gas discharge tubes to generate ions, which were then passed through parallel electric and magnetic fields. The ions were deflected into parabolic trajectories and then detected on a photographic plate (Jennifer Griffiths. (2008).
2.7.1 Components of Mass Spectrometry
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As illustrated in Figure 2.8, mass spectrometers are made up of the following three basic components: 1) an ionization source that converts particles into ions, 2) a mass analyzer that sorts ions according to their mass-to-charge ratio (m/z) by applying electromagnetic fields, 3) an ion detector that measures the mass-to-charge ratio (m/z) and thus calculates the abundances of each ion present (Kinter and Sherman, 2000; Hoffmann and Stroobant, 2005; Siudzak, 2006). In addition these components may include collision cells for ion activation in tandem mass spectrometer analysis. All these components are essential to create a robust instrument and their different combinations can produce unique analytical capabilities.
2.7.1(a) Ionization Source
The two most common ionization sources used in most biochemical analyses are electrospray ionization (ESI) where samples are converted to gaseous ions and matrix-assisted laser desorption/ionization (MALDI). Some ionization techniques are very energetic and can cause fragmentation during the ionization process like EI. On the other hand, there are gentler ionization techniques that produce mainly ions of the molecular species, for instance ESI and MALDI. ESI and MALDI will be described in more detail in subsequent sections.
2.7.1(b) Mass Analyser
A Mass Analyzer separates gas phase ions according to their m/z. The separation can be based on many principles, thus, there are several types of mass analyzers with different advantages and limitations. Some of the important characteristics of a mass analyzer include resolution, mass rang, scan rate and detection limit (Van Bramer, 1997). Currently, the trend in mass analyzer development is to combine different analyzer so as to increase the versatility and allow multiple experiments to be performed (De Haffmann and Stroobant, 2007).The mass analyzers that are relevant to this thesis are TOF, Quadrupole and QIT and will be discussed in more detail in the subsequent sections.
2.7.1(c) Ion Detector
Once the ions are separated by the mass analyzer, the ions will reach the ion detector whereby the mass-to-charge ratio will be measured. A mass spectrum will be generated when a signal is produced in a detector through a mass spectrometry scanning. The most commonly used detector is the electron multiplier. However, other detectors like Faraday cup, ion-to-photon, array and many others are also used (Matsuo et al., 1994; Hoffmann and Stroobant, 2005; Siudzak, 2006).
A detector usually transforms mass analyzed ions into electric currents, which are proportional to the ion abundances. A detector is selected based on its speed, dynamic range, gain and high voltage. Ion activation are techniques where mass separated/selected ions (precursor ions) are given excess energy either by collision or by irradiation to dissociate. Dissociations may either occur spontaneously (metastable ions) or can result from intentionally supplied additional activation in collision cell (Gross, 2004).Spontaneous fragmentation that occurs within the ion source is known as in-source decay, whereas outside the ion source is known as post-source decay (Harvey, 2009).
Methods for ion activation include surface-induced dissociation, infrared multiphoton dissociation, electron capture dissociation and collision-induced dissociation (CID), which is the most prominent ion collision technique. It generally involves passing an ion beam through a collision cell that contains collision gas, such as helium, argon and nitrogen, at high pressure. CID is very useful for structural elucidations of ions of low internal energy because it allows for the fragmentation of gaseous ions that are stable before the activating process (Gross, 2004).
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In tandem mass spectrometric analysis, the precursor ions are normally dissociated between the two mass spectrometric stages .There are two types of tandem mass spectrometers, those that perform tandem MS in space by the coupling of two physically distant analyzers (for example TOF/TOF and QTOF), or in time by performing a sequence of fragmentations in an ion storage device (for example QIT) (De Haffmann and Stroobant, 2007).In an MS/MS experiment, selected precursor ion are cleaved into smaller fragments which are then separated and detected. In multiple stage MS analyses (MSn), the fragments of the precursor ions can be selected and further fragmented and this process is repeated during the course of each of the activation stages.
2.8 The Mass Spectrometric Analysis of Glycan
The mass spectrometric analysis of glycan started about 40 years ago with the analyses of monosaccharides and hydrolysed small oligosaccharides using GC-MS, (Sweet et al., 1974; Weber and Carlson, 1982). Since then, tremendous developments and achievements have been made on both analytical methods and instrumentation.
Mass Spectrometry is a powerful analytical technique that is used for the identification of unknown compounds, the quantification of known compounds and the elucidation of the structure and chemical properties of molecules (ASMS, 2001; Gross, 2004). Currently, Mass Spectrometry is the preferred method in glycomic and glycoproteomic research. This method has been at the forefront of this area of research for over 20 years (Haslam et al., 2006). Currently, other than MALDI-TOF/TOF and ESI-QTOF, LC-ESI-MS is another widely used technique (Karlsson et al., 2004; Wada et al., 2007).
Developments in Mass Spectrometry can be exploited using various types of instrumentation, for example,
Quadruple time of light (Q-TOF)
Electrospray-Quadrupole-time of light (ESI-QTOF)
MALDI- quadruple ion trap-TOF (MALDI-QIT-TOF)
MALDI is frequently coupled to time-of-flight (TOF) mass analyzers while the electrospray ionization (ESI) is frequently coupled to an ion-trap, ion-cyclotron resonance (ICR), Orbitrap, quadrupole, triple quadrupole, or a hybrid mass analyzer. While in terms of physical principles, the two techniques are very different, both are capable of introducing large biomolecules into the gas phase with minimal degradation of their parent structures. An increase in understanding of mass spectrometry has contributed to the development of several fragmentation techniques which allow researchers to quickly obtain structural information about a broad range of important biomolecules. These developments have contributed significantly to science and their developers were awarded the Nobel Prize in Chemistry in 2002.
Currently, mass spectrometry has progressed from the analysis of elements and small volatile molecules to large biological macromolecules, particularly with no mass limitations (El-Aneed et al., 2009) as a result of refinements made to every component of mass spectrometers. This has led to outstanding improvements as well as hybrid instruments which were realized by combinations of the same or different analyzers (De Hoffmann and Stroobant, 2007). Thus a whole range of mass spectrometers has enabled researchers to better understand and conduct glycomic and glycoproteomic research.
In this regard, it is important to note that the complete structural characterization of oligosaccharides is more challenging to obtain than that of proteins or oligonucleotides because of the existence of additional specific characteristics such as isomeric states, linkage positions and branching capabilities. However, the determination of all this structural information for a comprehensive characterization can be obtained by mass spectrometry, although sometimes it may require more than a single mass spectrometric technique. Various sample treatments prior to analysis might also be crucial for a successful experiment. The next section discusses some of the important strategies for glycan mass spectrometric analysis.
2.9 Strategies for Glycan Mass Spectrometric Analysis.
Electron ionization (EI),
Electron ionization (EI) was previously called electron impact. It is the original mass spectrometry ionization technique introduced in 1918 by A.J. Dempster. Since then there have been tremendous improvements in mass spectrometers, as well as the invention and optimization of ways to introduce and separate samples in mass spectrometers. For instance, GC was first coupled to a mass spectrometer in 1956 by F.W.McLafferty (McLafferty, 1957) and R.S.Gohlke(Gohlke, 1959). About 30 years later, another important addition to mass spectrometry repertoire was the discovery of MALDI-MS by two separate groups, that is, Tanaka et al. and Hillenkamp et al. (Karas et al., 1987; Tanaka, 1988).
2.9 b) Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF)
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MALDI-MS was first introduced by Tanaka, Karas and Hillenkamp in 1988 (Kinter and Sherman, 2000; Hoffmann and Stroobant, 2005; Siudzak, 2006). Although MALDI-MS was originally developed for analysis of large peptides and proteins (Karas and Hillenkamp, 1988; Mock and Cottrell, 1992), it was soon applied to oligosaccharide analysis too (Harvey, 1996).
The MALDI strategy involves two procedures. Firstly, the compound to be analyzed is mixed in solvent containing small organic molecules in solution which is known as the matrix and the matrix will have a strong absorption at the laser wavelength. This is followed by the ablation of bulk portions of this dried mixture by intense pulses of UV laser, which then vaporize the matrix compound and produce a plume that carries the protonated peptide or protein into the gas phase (Matsuo et al., 1994; Splenger, 1997). Once ions are formed in the gas phase, the desorbed charged molecules can be directed electrostatically from the MALDI ionization source to the mass analyzer. As illustrated in (Figure 4) where is the figure?) ionization on MALDI occurs by protonation in the acidic environments produced by the acidity of matrix compounds and by the addition of appropriate volume of dilute acid usually with a trace of trifluoroacetic acid to the samples. Because the laser desorption generates ions in discrete packets, MALDI is usually associated with time-of-flight (TOF) mass analysis (Spengler, 1997; Kinter and Sherman, 2000; Desiderio and Nibbering, 2001; Siudzak, 2006).
Currently, in oligosaccharide analysis, MALDI-TOF/TOF-MS is the most powerful mass spectrometric method for mass fingerprinting because of its sensitivity of detection and ability to analyze glycan from complex mixtures derived from a variety of organisms and cell lines (Haslam et al., 2006; Parry et al., 2007).This instrument consists of a MALDI source, a short linear TOF, a collision chamber for CID, a second TOF with a reflectron and tow MCP detectors, one each for the linear and reflection modes as illustrated in Figure 2.9
Soft ionization methods, such as MALDI and ESI, permit the use of mass spectrometers to analyze very large biological molecules such as nucleotides, proteins and glycoconjugates. Ionisation in a MALDI source requires biomolecules to be mixed with a low molecular weight ultraviolet-absorbing organic compound, known as the matrix. Then the dried crystallized matrix compound is irradiated with a laser beam, which causes evaporation and ionisation, and the ions are directed towards the TOF analyzer as illustrated in (Figure2.10) Where is the figure?
While the ionisation process is still not completely understood, it is widely believed that the matrix allows the energy from the laser to be dissipated and assists the formation of ions by proton transfer and chemical processes (Dell et al., 2008). Apart from having an absorption frequency compatible with MALDI laser, it is important for a matrix to have sample solubility, reactivity and volatility and suitable desorption properties (Hossain and Limbach, 2009). Most matrices employed for analyzing substances in the positive ion mode are acidic, for example, Î±-cyano-4-hydroxy cinammic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), which help the ionization of biomolecules (Kinter and Sherman, 2000).
The MALDI ion source is very suitable for accurate overall glycan profiling because it produces mainly singly charged molecular ions with minimal fragmentation (Dell et al., 2008). In addition, ionisation in MALDI produces a pulsed sample ion current, which is ideally suited to the TOF mass analyzer. In-source metastable fragmentation was found to be prominent in early MALDI studies of glycans. Although this allows PSD of oligosaccharides in MALDI-TOF instrument it also complicates MS profiling. This shortcoming was overcome by increasing the pressure in the MALDI source and by establishing glycan molecules via Derivatisation (Zaia, 2004).
In a MALDI-TOF-MS analysis, excited ions from the ion source are attracted to the TOF analyzer where ions of different m/z are dispersed in time during their flight along a field-free drift linear path of known-length. The lighter ions arrive earlier at the detector than the heavier ones (Gross, 2004). Ions generated by hundreds of laser shots are accumulated from different points of laser irradiation. This makes the MALDI-TOF an excellent technique in mass spectrum reproducibility (Wada et al., 2007).
TOF analysis was originally designed for GC (Gohlke, 1995). Currently, though TOF is very commonly coupled to MALDI with the capability to produce mass spectra of proteins of at least 100,000 Da. Over time, various modifications have been made to instruments with TOF analyzers, for example, incorporation of delayed pulsed extraction and reflections to improve the mass resolution by correcting the energy dispersion of ions with the same m/z but with different kinetic energy so that they arrive at the detector at the same time. A time-of-flight (TOF) mass analyzer is well suited with the MALDI source because of the pulsed nature of the MALDI process. TOF analyzers were first described in 1949 (Pfenninger et al., 1999), and commercialized in 1955 (Reilly and Colby, 1996). TOF analyzers intrinsically demonstrate high transmission unlike the scanning-type mass analyzers where most of the ions are lost during scanning.
In delayed pulsed extraction, ions are initially allowed to separate according to their kinetic energy in a field-free region before an extraction pulse. This allows the less energetic ions to receive more kinetic energy and join the more energetic ions at the detector. The reflection, meanwhile, creates a retarding field at the end of the TOF tube and acts by deflecting the ions back through the flight tube. Ions with higher kinetic energy and hence with more velocity will penetrate the reflection much deeper than ions with less kinetic energy. Thus the high energy ions will spend more time in the reflection (De Haffmann and Stroobant, 2007). Although TOF has almost unlimited mass rang the reflection mode is restricted to analyses below masses of about 10,000 Da (Dell et al., 2008).
Each component of the MALDI-TOF mass fingerprint can be rigorously characterized by subjecting each molecular ion to collision activation in MS/MS experiments. The combination of a short linear TOF and a reflectron TOF analyzer (TOF/TOF) separated by an ion selector and a collision cell as illustrated in Figure 2.11(figure) enhances tandem mass spectrometric analysis in MALDI-MS. At present, this is the leading type of mass analyzer for MALDI instrument. In MALDI-TOF/TOF-MS/MS, excited ions from the MALDI source are separated in the first TOF analyzer and selected with a timed ions selector (TIS) or mass "gate" based on their arrival time at the TIS gate. The TIS is used to isolate specific molecules for fragmentation based on their m/z before they enter a collision cell which contains the collision gas. The selected ions are then fragmented in the cell before being accelerated by a second source into the second TOF analyzer with the reflectron capability. Ions and fragmented ions are resolved according to their m/z before arriving at the detector. This technique has greatly enhanced the sensitivity and resolution of tandem mass spectrometric data and results in exceptional determination of glycan compositions and sequences.
In MALDI-MS analysis, glycans are mixed with an excess amount of a matrix compound. The matrix absorbs most of the laser energy and then transfers the energy directly to analytes. Analytes are typically ionized as alkali metal adducts, such as [M + Na] +, or protonated ions ([M + H] +). In positive ion mode, glycans lacking in proton affinity usually form sodium adducts. However, in negative ion mode, acidic glycans are easy to observe whereas neutral glycans do not ionize so well. A diversity of matrixes can be used for glycan analysis, therefore, it is important to choose the right matrix and sample preparation to obtain a good MS spectrum of glycans (Harvey, 1999). For example, sinapinic acid (SA) is a common matrix used for protein analysis; alpha-cyano-4 hydroxycinnamic acid (CHCA) for peptide analysis and 2,5-dihydroxy benzoic acid (DHB) is frequently used for small protein analysis. Apart from the tolerance of sample contamination, a matrix also serves to minimize sample damage from the laser pulse by absorbing most of the energy and increases the efficiency of energy transfer from laser to the analyte.
In the case of neutral glycans, 3-amino-4-hydroxybenzoic acid was the first matrix developed (Mock, Davey, and Cottrell, 1991). However, currently, 2,5-dihydroxybenzic acid (DHB) is a more popular matrix in carbohydrate analysis (Stahl et al., 1991). DHB mainly produces [M + Na]+ ions, apart from other minor weak ions such as [M + Ka]+. The performance of DHB matrix has been further improved by adding other substituted benzoic acids (Strupat and Hillenkamp, 1991). This matrix is commonly called "super-DHB" and has enhanced sensitivity and MS resolution because it causes the disordered crystallization of matrix and analyte mixture.
Other matrixes, such as hydroxyisoquinoline88, arabinosazone89, have also been introduced for the analysis of neutral glycans. In the case of acidic glycans, like sialic acids and sulfated glycans, different matrixes must be used because acidic N-glycans produce poor MALDI spectra when ionized with DHB matrix alone. Some of the different matrices that have been used for MALDI analysis of acidic glycans include 6-Aza-2-thiothymine (Papac and Jones, 1996), 2, 4, 6-trihydroxyacetophenone (Papac and Jones, 1996), spermine with DHB as a co-matrix (Mechref and Novotny, 1998) and 5-chloro-2-mercaptobenzothiazol (Pfenninger et al., 1999).
While MALDI/TOF-MS offers a highly sensitive approach for molecular-weight profiling of all glycans released from isolated glycoproteins, however, MALDI/TOF-MS profiles on their own provide limited glycan compositional data. This is due to the ubiquitous isomerism of glycan structures (Mechref, and Novotny, 2002). Thus in order to obtain a more complete structural characterization of glycans in terms of branching, linkage position and a monomer anomericity it is necessary to use other methodologies (Mechref and Novotny, 1998; Mechref, and Novotny, 2002).
Generally, glycans are cleaved in two ways. Glycosidic cleavages result from breaking the bond linking two sugar residues while cross-ring cleavages result from breaking any two bonds on the same sugar residue. Glycosidic cleavages provide the sequence and branching information, while cross-ring cleavages reveal some additional details on a linkage. In this regard, the Domon-Costello nomenclature has been widely adopted (see Figure 2.12?) to describe glycan fragmentations (Domon, and Costello, 1988).
In order to obtain a more detailed structural characterization of glycans, tandem MS (MS/MS) techniques may be needed. Although fragmentation occurs in the drift region (a field-free region) after extraction from the source (called postsource decay or PSD), molecular ions are mainly detected. PSD spectra of glycans provide mainly glycosidic cleavages and very weak cross-ring fragmentation because such fragmentation requires high energy (Spengler et al., 1994). In this context, collision induced dissociation (CID) can provide the necessary high energy to promote a glycan fragmentation process and provide more detailed information on cross-ring fragments (Harvey et al., 1997).For example, abundant X or A cross-ring fragment ions may provide considerable detail of a glycan structure.
The MS/MS technique can be conducted using a reflection MALDI/TOF/TOF instrument as illustrated in (Figure2.13?). After the sample ions are extracted from the source 1, a precursor ion is selected by a timed-ion selector in the TOF 1 region. The fragmentation of a selected precursor ion then occurs in the collision cell filled with a gas. The precursor ion and fragment ions are analyzed by the TOF 2 region. The MS/MS technique of MALDI-TOF/TOF has been used in the investigation of linear oligosaccharides and branched high-mannose-type N-glycans derived from ribonuclease B (Mechref et al., 2003) and the characterization of neutral carbohydrates (Spina et al., 2004; Morelle et al., 2004). In addition, by using a combination of permethylation and tandem MS with CID, glycan structural information has been obtained (Mechref et al., 2003; Solouki et al., 1988; Harvey et al., 2004)
2.9 (c) ESI Q-TOF Mass Spectrometry
In 1946, William E. Stephens of the University of Pennsylvania proposed the concept of TOF MS. Since then a number of TOF instruments have been constructed with increasing sophistication and capabilities. Some of the important figures responsible for such advances were William C. Wiley and I. H. McLaren of Bendix Corp., Detroit, Michigan U.S.A and Boris A. Mamyrin of the Physical-Technical Institute, Leningrad, Soviet Union. According to Biemann, the first TOF instruments were poor because"their performance in resolution was so poor that they never lived up to even single-focusing magnetic instruments," but adds that, "this analyzer has been greatly improved recently...to almost match the most sophisticated, and very expensive, double-focusingmassspectrometers." http://masspec.scripps.edu/mshistory/perspectives/sborman.php#link1).
In a TOF analyzer, ions are separated by differences in their velocities as they move in a straight path toward a collector in order of increasing mass-to-charge ratio. TOF MS is fast, it is applicable to chromatographic detection, and it is now used for the determination of large biomolecules, among other applications (Wolff and Estephens, 1953).
The ESI-QTOF hybrid mass spectrometer combines the benefits of ESI and the quadrupole-TOF (QTOF) mass analyzer, thus complementing the MALDI-TOF/TOF tandem mass spectrometry. The ESI-QTOF technique has been most useful in sequencing of very low abundance glycans.
Samples dissolved in solvent emerging from a liquid chromatography (LC) column or syringe are introduced to the ESI source through a capillary at high voltage and at atmospheric pressure. The sample solution eventually emerges as tiny droplets at the needle tip and they have a strong positive or negative charge due to the strong electric field. At first, when it emerges from the tip, the charged liquid forms a cone shape known as a Taylor cone before the droplets burst away (Cole, 2010). The droplets then pass through a curtain of heated inert gas, usually nitrogen, to remove the remaining solvent molecules. When the electric field on their surface is large enough, ion desorption from the droplet surface will occur before the ions enter the QTOF mass analyzer (De Hoffinann & Stroobant, 2007). One of the advantages of this method is that it is soft ionization, that is, the molecules remain intact (Gross, 2004).
More importantly, ESI has the capability to produce multiply charged ions that is extremely helpful in characterizing biomolecules with high masses.
Usually, ESI is connected to a LC system which enables direct analysis of LC separated biomolecules in mass spectrometers. As an example, the nano-Iiquid chromatography interface (nano-LC) on ESI is excellent for glycosylation site specific analysis as well as the detection of other protein modifications such as phosphorylation and alkylation (Thomsson et al., 2000; Dell & Morris, 2001). However, manual injection into the ESI needle via a syringe is also excellent for ESI especially when involving very low amounts of samples or when LC separation is not required.
Ionisation of glycans and glycoconjugates in conventional ESI were not as good compared with peptides and proteins until the introduction of nano ESI that produces smaller droplets with better ion signals (Wilm & Mann, 1996). This is because as Karas et al., (2000) point out, smaller droplets reduce the hydrophilicity of oligosaccharides and thus increases surface activity rather than volatility and resembles the effects of glycan derivatisation.
The QTOF mass analyzer was first proposed by H.R. Morris (Morris et al., 1996) and at present is the most commercially successful hybrid system (Gross, 2004). The QTOF mass analyzer has four cylindrically shaped rod electrodes with the pairs of opposite rods being each held at the same potential and commonly assembled in three sets to form an instrument with ion activation capability. A direct current voltage and an oscillating radio-frequency are applied to each pair of opposite rods creating an electric field that acts as a mass filter. The quadrupole allows high-speed scanning at relatively high pressure that is ideal for the continuous beam of ions from the ESI source (Dell et aI., 2008). The additional reflectron TOF mass analysis immediately after the quadrupole analysis improves the ion detection, transmission, resolution and mass accuracy of a quadrupole instrument (Morris et aI., 1997). However, initially it was impossible to directly combine a TOF analyzer and a pulsed instrument, with continuous ionisation from an electro spray source. It was solved by the orthogonal arrangement of the quadrupole and TOF analyzers (Morris et aI., 1996). The design was originally devised by Dawson and Guilhaus (1989) and was effective because of the incorporation of an ion modulator/pusher at the interface.
The first quadrupole analyzer, as illustrated in Figure 1.16, ? is a radio frequency (RF)-only quadrupole (Q0) where ions are collimated and transferred into the adjacent high vacuum region of the second quadrupole, which is known as the mass filter quadrupole (Ql). In the MS mode, Q 1 transmits ions over a wide mass range, whilst in the MS/MS mode, ions are resolved according to their m/z. This is followed by the third quadrupole (Q2), which is an RF-only quadrupole filter inside a collision cell. In the MS mode, Q2 only focuses on the ion beam, which is similar to Q0. However, in the MS/MS mode, Q2 transmits ions through the collision cell that contains the collision gas (nitrogen) for fragmentation to occur. Precursor and/or fragment ions then enter the ion modulator in the TOF analyzer. A push-out pulse applied orthogonally to the ion beam direction then extracts the ions to pass through the TOF tube. Ions are separated according to their m/z value before being detected and recorded (Cole, 2010). The TOF analyzer contains a reflectron which is similar to that of MALDI-TOF/TOF-MS as discussed earlier.
Last but not least it is instructive to note that developments in hybrid mass spectrometry have had a tremendous impact on the sciences, such that Fenn, who incidentally, produced the first spectra of proteins above 20 kDa in 1988 (Fenn et al., 1989) together with Tanaka shared the Noble Prize for Chemistry in 2002 for their outstanding work in analyzing biomolecules using mass spectrometry. In addition, in 2004, Tanaka supplemented his contribution to the field of mass spectrometry by inventing the MALDI-quadrupole ion trap (QIT)-TOF MS (Ojima et al., 2005).