Mass Spectrometry. Anders Malmendal. 1. Physical principles. Mass Spectrometry

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1 1. Physical principles Mass spectrometry is based on the laws determining the motions of charged particles. In a mass spectrometer, the motions of these particles are determined by the particle mass and by the interactions between the particle charge and the applied electric and magnetic fields. Since the experiments are performed under strong vacuum, any type of friction is negligible. In most cases gravity can also be ignored. By using the laws described below, a number of different ways to measure the mass-to-charge ratio can be deduced. The equation of motion We start looking at a particle of mass m. Anders Malmendal Figure 2.1. A) A particle with a certain mass m and a certain velocity v. B) A force F is applied parallel to the direction of motion. C) A force F is applied perpendicular to the direction of motion. If a force F (in Newton, N = kgm/s 2 ) acts on the particle, it accelerates with an acceleration a, where (2.1) When the force has been applied for a time t the particle has a velocity v And it has moved a distance d (2.2) (2.3) The energy of motion of an object, i.e. the kinetic energy (in Joule, J = N m = kg m 2 s 2 ) is defined as (2.4) If an accelerating force is applied to a particle that is not moving or applied to a moving particle in the direction of the motion (Figure 2.1B), the kinetic energy is increased by 2

2 (2.5) if the force is applied while the particle travels the distance d. If a force F is applied perpendicular to the direction of motion of a particle (Figure 2.1C), the acceleration is also perpendicular to the direction of motion and the motion follows a circular trajectory with the radius (r), (2.6) In this case the velocity does not change, and the kinetic energy is unaltered. Charge moving through an electric field Figure 2.2. Two charged plates with a potential difference V and a distance d. An electric field (E, measured in V/m (= N/C)) can be obtained by placing two plates with a potential difference V (a voltage) at a distance d (Figure 2.2): (2.7) If an object with a charge q (in Columb, C) is placed in such a field, the object experiences a force F: (2.8) Charge moving through a magnetic field The physical laws described above are all rather intuitive, but when it comes to charged particles in magnetic fields it is less so. There is no force acting on a charged particle in a magnetic field if the particle does not move. However, if the particle moves (Figure 2.3; the magnetic field points out of the paper), the object experiences a so called Lorenz force perpendicular to both the motion and the magnetic field. (2.9) where B is the field strength (in Tesla, T = kg/cs) and! is the angle between the magnetic field and the direction of motion. If the particle moves perpendicular to a magnetic field (Figure 2.3; the magnetic field points out of the paper), this simplifies to: Simple m/z measurements (2.10) As will be described in chapter 3, these properties can be used to determine the mass-to-charge ratio m/z of ionized particles. The ions are first accelerated by an electric field. In time of flight (TOF) instruments, the velocity is used to determine m/z. In sector instruments, measurements of the deflection of the charged particles in a magnetic field are used. Irrespective of the method a mass spectrum as in figure 1.2, with intensity as a function of m/z is obtained. Units of mass and charge Molecular charge is often expressed as z units of the charge of an electron e. where z is dimensionless and e = 1.602!10-19 Columb, C. (2.11) Molecular mass can be expressed in molecular units such as Daltons (Da), atomic mass units (u) and grams per mole (g/mol), where 1 Da = 1 u = 1 g/mol = 1.660!10-27 kg. To go from mass in Da and charge in elementary units to metric units (kg and C) you can use: (2.12) Figure 2.3. When a charged particle moves through a magnetic field (pointing out of the paper), the particle experiences a force perpendicular to both the motion and the magnetic field 3 4

3 Resolution The resolution R is the ability to resolve two neighbouring signals, defined as (2.13) where "(m/z) is the difference in m/z between two neighbouring signals of equal intensity, at (m/z) 1 and (m/z) 2, with 10% signal overlap (Figure 2.4). A resolution of 10,000 allows one to clearly distinguish a mass difference of 0.01%, which means a mass difference of 0.01 Da for ions with a mass of 100 Da (e.g and Da), or a mass difference of 1 Da for ions with a mass of 10 kda (e.g and kda). Figure 2.4. Resolution 2. Mass Analysis In the mass analyzer, the ions are separated according to their mass-to-charge ratio (m/z). Figure 3.1. Acceleration by two plates with a potential difference. Source: Ziuzdak. Before arriving at the actual mass analyzer, the ions are accelerated out of the ion source by a potential difference V acc. Depending on the ionization mode and on the sign of the accelerating potential, positive or negative ions (usually [M+H] + or [M+H] ) are selected. For proteins, the positive ion mode is usually used, while the negative ion mode is used for nucleic acids. The kinetic energy of an ion of charge q accelerated by a potential difference V acc is (3.1) according to equations 2.5 and 2.8. Since the kinetic energy depends on the mass and the velocity (equation 2.4), the ions of different m/z values have different velocities v, after acceleration out of the ion source. (3.2) Time-of-Flight The simplest way of measuring the mass-to-charge ratios of ions that have been accelerated as described above is thus to measure their velocities. Figure 3.2. A naïve picture of a TOF mass analyzer. Source: Markides & Gräslund. In a time-of-flight (TOF) analyzer (Figure 3.2), which was first successfully used as an ion analyzer in the 1950s, this is done by measuring the time it takes for each ion to travel a certain distance: high m/z ions take longer to reach the detector than low m/z ions. 5 6

4 From equation 3.2 we can derive the expression; (3.3) for the velocity of an ion of mass m and charge q and the time t spent in the drift region of length L. With an accelerating voltage of 20 kv and a drift tube of 1 meter, a singlycharged ion of mass 500 Da will have a velocity of approximately 9!10 4 m/s and the time spent in the drift tube will be 1!10-5 s. For practical purposes where the exact values of many of the constants are unknown, one uses a formula like (3.4) where a and b are constants for a given set of instrument conditions, determined experimentally from flight times of ions with known mass. For a TOF instrument, the resolution is proportional to t/"t. Linear TOF instruments are capable of attaining a resolution of The ions analysed by TOF must be pulsed into the analyzer, usually at rates between 10 and 10,000 Hz. Major limitations in achieving high resolution are the spread in time, space and kinetic energy of the initial ion packet. As will be described in the next chapter, ions in e.g. a MALDI-TOF instrument are volatilized and ionized by a laser pulse. The time difference "t in formation of two ions caused e.g. by the length of the laser pulse, will remain the same during the flight to the detector. One way to increase the resolution is to increase the flight time by reducing the acceleration voltage or to increase the length of the drift tube, since "t would remain the same but t become greater (see equation 3.3). Another way of overcoming this is to use delayed extraction (DE), where the accelerating field is not turned on until the laser pulse has ended. In this way ions volatilized at the start and end of the pulse will start the acceleration process at the same time, which significantly reduces the spread in time. energies. Ions can also be generated with different initial kinetic energy. This spread in kinetic energy can be partially compensated by using a device called a reflectron as shown in figure 3.3, which has an electric field that becomes stronger and stronger as one moves deeper into it. Ions of higher energy will penetrate deeper into the reflectron before they are turned around. If the reflectron is appropriately tuned, ions with the same m/z ratio will arrive at the detector at the same time irrespective of the initial kinetic energy. Fragments of metastable ions generated through fragmentation of ions in the drift region (after acceleration) cannot be distinguished from the original ion in a linear TOF (without reflectron), because once they are accelerated their velocity remains the same. Fragments generated prior to reflection have the same velocity as their parent ion, but a reduced energy which causes a time difference compared to the intact ions. The reflectron also elongates the flight tube. Reflectron TOF instruments are capable of a resolution of over 10,000. TOF is ideal for pulsed ion sources, like MALDI (see below), and the MALDI- TOF mass spectrometers have become very popular in life sciences, but TOF can also be used with continuous ionization sources (EI, ESI, FAB, etc.), using pulsed deflection or pulsed extraction. The mass range of TOF analyzers is virtually unlimited and the practical upper limit is determined by the ionization process and the detector. The combinations of time-of-flight mass spectrometry with MALDI and ESI (see below) have produced effective tools in the laboratory of biochemists, due to their relatively low cost, high sensitivity, speed and ease of operation. Sector Instruments Another way of separating ions of different m/z is by measuring their deviation in a magnetic field. This is the classical way to separate molecules based on different size and charge, and was first described in 1912 by J.J. Thompson (Nobel Prize laureate in 1906 for investigations of the conduction of electricity by gases). Figure 3.3. Skematic of a TOF instrument with a reflectron. Source: Caprioli & Sutter. Ions that are not formed at the same location, due to the spread of the sample on the target or width of the ion beam, will be accelerated to different kinetic Figure 3.4. Magnetic sector instrument The first mass spectrometers used fixed magnetic and electric fields to separate ions of different mass and energy. Using mass spectrometers it was proven that the noble gas Ne in fact was composed of two different isotopes of masses 20 and 22 Da, providing the final proof of the atomic theory of matter. When entering a homogenous magnetic field on a trajectory perpendicular to the magnetic field strength B, the ion experiences the Lorentz force F that is 7 8

5 perpendicular both to B and v as described in chapter 2. By combining equation 2.10 and 3.2 we can calculate the force F acting perpendicular to the direction of motion of an ion accelerated by a potential difference V acc to be (3.5) When this equation is combined with equation 2.6, we can see that the ion follows a trajectory along a circle with a radius r. (3.6) On a magnetic sector instrument, r is a fixed value that is given by the geometry of the magnet. V acc is usually kept constant during the acquisition while the magnetic field strength B is adjusted (scanned) in order to successively transmit ions of different m/z values through a slit matching the magnet radius r (see figure 3.4). Quadrupole Mass Filter The resolution of a quadrupole mass filter depends on the number of cycles experienced by the ions while they are in the analyzer ("t), where the time, t, spent in the analyzer depends on its length and the velocities of the ions (t=l/v). The resolution will thus increase with increasing m/z, as ions of higher m/z have lower velocity. On the other hand, the transmission efficiency, and thus the sensitivity, will decrease, due to the longer time ions of higher masses spend in the quadrupole. Apart from the low cost and small size, one of the advantages of a quadrupole mass filter over a sector instrument is the low voltage applied to the ion source, i.e., kinetic energies of the ions on the order of 5-10 ev, compared with several kev for a sector instrument. This eliminates high voltage problems and makes interfacing to GC and LC easier. Other advantages are good transmission efficiency, high scan speed, and wide acceptance angle for incoming ions to give high sensitivity. Quadrupole Ion Trap Figure 3.7. Quadrupole mass filter. Source The basic principles of the quadrupole mass filter were published in the early 1950s (Steinwedel 1953). It has become one of the most widely used types of mass spectrometers because of its ease of use, small size and relatively low cost, e.g. making it the ideal bench top analyser. Mass separation in a quadrupole mass filter is based on achieving a stable trajectory for ions of specific m/z values in the electrostatic field of four parallel rods, which each can be as small as a pen. To one pair of diagonally opposite rods a potential is applied consisting of a constant part U and an oscillating part V with a frequency " in the radio frequency (rf) range. To the other pair of rods, a potential of opposite sign is applied. The potentials! o applied to the two rods (see figure 3.7) are given by. (3.7) At given values of U, V and ", only certain ions will have stable trajectories through the quadrupole. The range of ions of different m/z values, capable of passing through the mass filter, depends on the ratio of U to V. All other ions will have trajectories with large amplitudes, and will be lost. Figure 3.8. Quadrupole ion trap. Source: Caprioli & Sutter. The quadrupole ion trap is based on the same principle as the quadrupole mass filter, except that the quadrupole field is generated in three dimensions. This trap consists of a ring electrode and two end caps as shown in figure 3.8. The quadrupole field is most homogeneous in the centre of the trap. Therefore a moderator gas like helium is often introduced into the trap to dampen the oscillations of the ions and concentrate them in the centre. The ion trap can store ions over a long period of time, making it possible to study e.g. gas phase reactions. The ion trap has excellent MS/MS capabilities (see below). The ion trap has very high sensitivity since all ions formed in theory can be detected. Space charge effects (ion-ion coulombic interactions) reduce the accuracy of mass assignment for an ion trap. 9 10

6 Ion Cyclotron Resonance Figure 3.9. Ion cyclotron mass spectrometer. Source: Caprioli & Sutter. The ion cyclotron resonance (ICR) mass spectrometer is based on technology developed in the 1950s, Fourier transform techniques (FT-MS) and the development of external ion sources. Like the ion trap, FT-MS is capable of storing ions within a cell. It consists of three pairs of parallel plates arranged as a cube, used for trapping, excitation or detection, respectively, as shown in figure 3.9. The ions are trapped in a field of a superconducting magnet similar to an NMR spectrometer perpendicular to the trapping plates. An outstanding feature of FT-MS is the extremely high resolution; for example, using electrospray ionization, a resolution of over 2!10 6 has been achieved. Another important feature of FT-MS is its MS/MS capability. Sensitivity Some analysers, e.g. time-of-flight, integrates the ion beam, i.e., record all of the ions all of the time. Others, e.g. magnetic sector and quadrupole filters, scan through the m/z values and only record a fraction of the ions. Thus, at any given time, only ions within a certain m/z range are focused on the detector. Since just a subset of the ions hit the detector, a scanning analyser is less sensitive than an integrating analyser. On the other hand, scanned analysers can select a few specific ions for monitoring, rather than scanning over the entire m/z range. This can be used to increase the sensitivity and to select specific ions for e.g. fragmentation in an MS-MS experiment (see below). The m/z of the ions to be monitored must then of course be known in advance. To increase the sensitivity, spectra can be measured over a longer time so that more ions have time to be detected, or several spectra can be recorded and added together. If the measurement time or the number of experiments are increased by a factor N, the signal intensity will increase N times. Since the noise is random it will only increase with a factor. Thus the signal-to-noise S/N is increased by. TOF Fast detection (100 spectra/s) Magnetic sector Classical reference instrument Spectrum collected in one shot Unlimited m/z Good resolution Needs pulsed source Quadrupoles Small and inexpensive Suitable for MS-MS Scanning (jumped) Trap can be used for storage and reactions Limited resolution (unit resolution 0.3 m/z units) Peak height function of mass Molecular weight Best quantification High resolution (slit width) Large and expensive Scanning (jumped) Ion cyclotron resonance Highest resolution (10 6 ) Non destructive detection Advanced manipulation of ions Well suited for pulsed sources Very large and expensive Table 3.1. Comparison of mass analyzers The molecular mass of a compound can be expressed in several ways. The nominal molecular mass is calculated using the atomic mass numbers, i.e., C = 12 Da, H = 1 Da, etc (Table 3.2). The nominal masses are usually not useful in mass spectrometry other than to provide a 'ballpark' estimation of the molecular mass. The exact mass of hydrogen ( 1 H) is ~ Da. Even relatively small molecules such as a protein of mass 10 kda typically have hydrogen atoms, which results in a mass that is 4-5 Da higher than the nominal value. The monoisotopic molecular mass of a compound can be calculated from the exact atomic masses, i.e., C= Da, H= Da, etc (Table 3.2). But the monoisotopic mass calculation does not take into account the presence of other naturally occurring isotopes such as 13 C, 2 H, etc. (Table 3.2). 13 C for example is relatively abundant, constituting about 1.1% of the carbon atoms in biological samples. Consequently, real samples contain a distribution of isotope mixtures, with an average molecular mass that is higher than the monoisotopic mass. Element Mass number Isotopic mass (Da) H Abundance (%) Average mass (Da) C N O P S

7 Cl K Table 3.2. Masses of the elements As an example, consider the measurement of the molecular mass of bovine proinsulin, a small protein with the elemental formula C 381 H 586 N 107 O 114 S 6. The molecular mass distribution of the resolved isotope pattern is shown in figure Figure MALDI-TOF spectrum of bovine proinsulin (R = 9500). Source: Caprioli & Sutter. Some interesting points can be made. First, the monoisotopic mass for the [M+H] + ion of this protein is Da, and the corresponding signal has the lowest intensity of the isotope peaks shown in the figure. If the mass would have been higher, e.g., 20 kda, the corresponding monoisotopic [M+H] + ion would have had insufficient intensity to be recorded. Second, as the molecular mass of a protein, or other macromolecule, increases, the mass width of the isotope distribution increases markedly; for bovine proinsulin, eleven different mass peaks extending over 10 Da can be measured. At higher masses, say 100 kda, this isotope spread would extend over 40 Da. Furthermore, each peak is itself composed of many different isotopic species. Thus, the highest peak at m/z 8681 is composed of 10 different isotopic species. Overall, the pattern shown in figure 3.10 contains 62 different isotopic species. In very special cases where it is necessary to measure one or more of these individual isotopic peaks, instruments having very high resolving capabilities must be used, and even then it is often not possible to resolve them all. Figure MALDI-TOF spectrum of bovine proinsulin (R = 720). Source: Caprioli & Sutter. It is not necessary to resolve the different isotope peaks in order to measure a useful value for the molecular mass of a molecule. The spectrum given in figure 3.11 also shows the [M+H] + region for bovine proinsulin, but recorded at much lower resolution (720 vs. 9500). This spectrum shows one single broad peak including all the individual isotopic species. A calculation of the average mass from this peak gives a m/z value of for the [M+H] + species of this protein, which is precisely the value one obtains for the weighted average of the resolved isotopic peaks. The cost in ion intensity to resolve the isotope pattern is high. Often when the resolution is increased in a scanned analyzer, a high mass peak observable at low resolution disappears before the pattern is resolved. This is because as the resolution is increased, the number of ions that comes through to the detector is drastically decreased, and if too few ions are transmitted no signal is recorded. Mass spectrometric measurements of the molecular masses of proteins are usually performed under low resolution conditions, i.e., the resolution is in the range. Today, these are most commonly performed with ESI (see below) on quadrupole instruments and MALDI (see below) on time-of-flight instruments. For ESI, mass measurement accuracy is generally about 0.005% in cases where good signal strengths are produced and for MALDI, about 0.05%. To calculate molecular masses from high-molecular weight molecules, isotope-averaged atomic masses of elements are used, giving average chemical masses for the molecules of interest. For low molecular mass measurements, for example for molecules below 1000 Da, the isotope peaks can most often be resolved and the monoisotopic mass is the most abundant. In this case, monoisotopic masses should be used to calculate molecular mass. Amino Acid Monoisotopic mass (Da) Average mass (Da) Gly G Ala A Ser S Pro P Val V Thr T Cys C

8 Ile I Leu L Asn N Asp D Gln Q Lys K Glu E Met M His H Phe F Arg R Tyr Y Trp W H 2O Table 3.3. Residue masses for amino acids For the calculation of molecular masses of peptides, either monoisotopic masses or average masses could be used, depending on the resolution of the instrument and the size of the peptide, as discussed earlier. Table 3.3 lists the common amino acid residue masses, i.e., as they appear in a peptide. Molecular masses are calculated by summing these masses and adding the masses of the terminal groups, usually ~1 Da (H) for the amino end and ~17 Da (OH) for the carboxyl end. In positive ionization mode, the molecular species recorded is often [M+H] +, and so one additional Da must be added for H +, or 23 Da for [M+Na] +, etc. 3. Ionization The utilization of mass spectrometry to investigate biological processes dates back to the late 1930s and early 1940s with the use of stable isotopes and isotope ratio mass spectrometers. These tools were essential in discovering the dynamic state of living organisms. Over the intervening decades, mass spectrometry continued to be of great utility in the elucidation of the structures of biological molecules through analysis of fragmentation patterns and accurate mass measurements. Complex mixtures of compounds that either were volatile or could be derivatized to enhance volatility could be analyzed by combined gas chromatography and mass spectrometry (GC/MS). Samples may be introduced in gas, liquid or solid states. In the latter two cases volatilization must be accomplished either prior to, or accompanying ionization. Simple and rather harsh ionization techniques to produce ions out of small molecules in the gas phase, like electron impact ionization (EI) and chemical ionization (CI) have been around for quite long. But only recently, processes such as FAB, MALDI and ESI made it possible to make the biomolecules leave the aqueous phase and go into gas phase. Electron Impact Figure 4.1. Schematic picture of an EI source. Source: Sheehan. The simplest and harshest ionization technique is electron impact ionization, EI, where volatile substances are ionized by letting the gaseous sample collide with an electron beam (Figure 4.1). The electrons are emitted by heating a filament in the ion source and are accelerated by a potential difference between the filament and the electron trap (Figure 4.1). This potential difference is usually set to 70 V, resulting in an electron energy of 70 ev (~1.12!10-17 J). Upon impact with a 70 ev electron, the gaseous molecule may lose one of its electrons to become a positively charged radical ion, M + e - # M + + 2e - where M + is termed the molecular ion. It carries an unpaired electron and can occupy various excited states. If these excited states contain enough energy, bonds will break and fragment ions and neutral fragment particles will be 15 16

9 formed. With an electron energy of 70 ev, enough energy is transferred to most molecules to cause extensive fragmentation. The fragmentation can be reduced by choosing an electron energy close to the ionization potential of the neutral molecule (typically ev for simple organic molecules). All ions are accelerated out of the ion source by an electric field produced by the potential difference applied to the ion source and an electrode placed before the analyzer. Depending on the lifetime of the excited state, fragmentation will either take place in the ion source giving rise to stable fragment ions, or to metastable ions that fragment on the way to the detector. If no fragmentation occurs before the ion strikes the detector, a signal for the molecular ion is generated. Fast atom bombardment (FAB) In 1981 fast atom bombardment ionization, FAB, was introduced. In FAB, polar and thermally labile compounds (< 16 kda; e.g. small biomolecules) are volatilized and ionized by bombardment with accelerated atoms or ions of e.g. argon, cesium or xenon. The molecules are mixed with a non-volatile environment that protects the molecular ions during the ionization process. Figure 4.2. EI fragmentation of lactic acid. Source: Sheehan. The mass spectrum obtained from recording all of these ions contains signals of varying m/z and intensities, depending on the numbers of ions that reach the detector. The fragmentation pathways of the molecular ion depend on the structure of the molecule, and similar structures give similar mass spectra. As an example, the ionization and fragmentation of lactic acid by EI is shown in figure 4.2. Mass spectral libraries that contain hundreds of thousands of spectra are available, and can be used to help identify unknown substances and search for common substructures. Though EI is excellent for characterizing small molecules, it is not useful for large biomolecules such as peptides and proteins, since the obtained fragments are too small to be traced back to the original molecule. Chemical ionization (CI) In the 1960s chemical ionization (CI) for the first time made it possible to ionize thermo-labile biomolecules. In CI, abundant reagent gas ions like and are first formed by electric discharge in a reagent gas like ammonia and methane. The reagent ions can then in turn ionize the volatilized molecules of interest. M + # MH + + CH 4 Figure 4.3. FAB ionization process. Source: Caprioli & Sutter. The general process leading to the formation of the molecular ion is depicted in figure 4.3 and involves several different mechanisms. The phase transition, i.e., the conversion of liquid sample to gaseous ions on bombardment, is believed to result from a redistribution of momentum from the bombarding high energy particle through a cascade of collisions within the matrix. The formation of protonated molecular ions (M+H) + or other cationized species such as (M+Na) + in the positive ion mode, and (M-H) - in the negative ion mode, can be attributed to both gas and liquid phase reactions. Doubly-charged ion species and dimeric cluster ions of the analyte are occasionally observed. In a FAB analysis, a sample is typically dissolved in an appropriate matrix, a viscous solvent, in order to keep the sample in the liquid state. Some of the more common liquid matrices are glycerol, 1-thioglycerol, a mixture of dithiothreitol and dithioerythritol, 3-nitrobenzyl alcohol, and triethanolamine. One major role of the matrix is to keep the sample in a liquid state as it enters the high vacuum ion source. The matrix also reduces damage to the analyte caused by the high kinetic energy (8-10 kev) of the bombarding particles. The major advantage of FAB is that it is easy and fast to operate, and the spectra are simple to interpret. However, one of the major disadvantages of the FAB technique is that it requires a high concentration of the organic liquid matrix (typically 80 to 95%), giving only moderate sensitivity. Matrix cluster ions can, in some cases, dominate the mass spectrum, and damage to the matrix caused by particle bombardment gives intense chemical background. It has been shown that discrimination in ionization efficiency of one analyte over another, due to differences in hydrophobicity and surface activity causes problems when analyzing mixtures quantitatively. In some cases, the matrix reacts directly with the analyte, forming radical anions or causing reduction of the analyte. The desorption process produces many neutral molecules in addition to the ions. The detection limit of standard FAB for molecular mass determination of most peptides is approximately 20 pmol. However, this amount varies significantly 17 18

10 depending on the chemical and physical properties of the peptide. The upper mass limit that is considered routine is about 5 kda. In continuous-flow (CF) FAB, the probe continuously delivers sample solution to the target. As a consequence, less organic matrix is necessary to keep the sample in the liquid state, which results in an increase in the signal-to-noise ratio due to lower chemical background. A more uniform ionization is also achieved. A major advantage of CF-FAB is its usefulness for flow-injection analysis and on-line reaction monitoring, as well as coupling to HPLC and capillary electrophoresis (see below). Matrix assisted laser desorption ionisation (MALDI) During the 1980s several groups tried to solve the volatilization/ionization problem of mass spectrometry using laser light as an energy source. By focusing a light beam onto a small spot of a liquid or solid sample, one hoped to be able to vaporize a small part of the sample without causing chemical degradation. In 1985, it was shown that an absorbing matrix could be used to volatilize small analyte molecules. A breakthrough for the laser desorption method in its application to large biomolecules was reported in 1987, when Koichi Tanaka (Nobel Prize laureate in 2002 for developments in the mass spectrometry technique) at the Shimadzu Corp. in Kyoto presented results of a mass spectrometric analysis of an intact protein. In , Tanaka presented ionization of proteins of up to 100 kda (e.g. Tanaka et al. 1988). Tanaka showed that gaseous macromolecular ions could be formed using a low-energy (nitrogen) laser, a fact that was not expected at that time. A nitrogen laser beam has a wavelength of 330 nm, which is not absorbed by the aromatic groups in proteins and peptides. This is important for avoiding fragmentation. The principle is illustrated in figure 4.4. The currently dominating laser desorption method is matrix assisted laser desorption ionisation, MALDI (Karas & Hillenkamp 1988). The actual desorption and ionization mechanism of MALDI is still being investigated. The laser power absorbed by the matrix, (typically on the order of 10 6 W/cm 2 ) leads to intense heating and generation of a plume of ejected material that rapidly expands and undergoes cooling. The phase transition (evaporation and sublimation) is probably the rate determining step in ion formation. Generation of ions is believed to arise through ion/molecule reactions in the gas phase. Depending on the matrix used, enough energy can be transferred to the molecule to break weak bonds. Figure 4.5. A MALDI spectrum of horse myoglobin in sinapinic acid. The major peaks (from left to right) correspond to the [MH 2] 2+, [M+H] + and [M 2H] + ions. The insert shows peaks corresponding to the [M+H] + ion with matrix molecules bound. Source: Caprioli & Sutter. Generally, the [M+H] + ion, or [M+Na] +, [M+K] +, etc., are preferentially formed in the positive ion mode, and [M-H] - ion in the negative ion mode. These peaks are often accompanied by weaker peaks corresponding to the [MH 2] 2+ and [M 2H] + ions, etc (Figure 4.5). Peaks corresponding to matrix molecules bound to these ions may also be detected. Figure 4.6. MALDI sample plate. Various types of physical/chemical matrices have been, and are continuously being developed. The crystallization is done outside the mass spectrometer on a sample plate (Figure 4.6) that is introduced into the spectrometer. Common matrices are listed in Table 4.1. Figure 4.4. The MALDI process. Source: Markides & Gräslund

11 Matrix Analytes Laser wavelength (nm) Molecular weight (Da) $-cyano-4-hydroxycinnamic acid sinapinic acid (3,5-dimethoxy-4- hydroxycinnamic acid) 2,5-dihydroxybenzoic acid 3-hydroxypicolinic acid proteins, oligosaccharides 337 * proteins, industrial polymers 337, proteins, oligosaccharides, oligonucleotides, sulfonic acids oligonucleotides, glycoproteins Table 4.1. MALDI matrices 337, 355, 2940 * , 308, MALDI is very fast and sensitive, and can be implemented on small and relatively inexpensive instruments that do not require extensive expertise in mass spectrometry. Such instruments allow easy access to singly charged ionization of intact biomolecules in complex matrices, and are ideally suited for biological scientists that need molecular mass information more quickly and more accurately than can be obtained by gel electrophoresis. MALDI is able to detect fmol of peptide, or less. Since most MALDI instruments use TOF analyzers (Figure 4.8), mass range is practically unlimited. The MALDI technique is now able to detect biomolecules over 300 kda in size, and has found very many applications, including the analysis of proteins and oligonucleotides. MALDI is perhaps the most forgiving of all the ionization techniques in terms of salts and other contaminants interference with the production of ions. Mass spectra have been produced from samples containing isotonic salt solutions and 2M urea. Since conversion of the TOF of an ion to an m/z value is dependent on calibration standards, internal standards added to the sample is the best method of calibration. Mass measurement accuracies of ± 0.01%, in the best case, can be achieved with MALDI for the analysis of peptides although not at the conditions used to obtain optimum sensitivity. Ion suppression effects make it inadvisable to attempt a complete analysis of unknown peptide mixtures by direct analysis of the bulk sample. Figure 4.7. Sinapinic acid mass spectra at low (lower trace) and high (upper trace) laser power. Source: Caprioli & Sutter. MALDI produces a relatively intense matrix background, generally below m/z 1000, which can be minimized electronically. This chemical background depends on the matrix and laser wavelength and power chosen (Figure 4.7). The best resolution is obtained when a laser power close to the threshold level for producing ions is used, i.e. at low signal strength. Figure 4.8. A MALDI TOF mass spectrometer. Source: Caprioli & Sutter. Figure 4.9. MALDI spectra for cytochrome c prepared using a sinapinic acid matrix. The sample was air dried in a dry environment (A), and using a hot air drier (B). Source: Caprioli & Sutter. Care must be taken in preparing samples as illustrated in figure 4.9, showing MALDI spectra for cytochrome c prepared using the same sinapinic acid matrix, but different amounts of patience. Electrospray ionisation (ESI) Initial experiments by the physicist John Zeleny in 1917 preceded the first description by Malcolm Dole in 1968 of the electrospray principle. However, the breakthrough of ESI came in 1988, when John Fenn (Nobel Prize laureate in 2002 for developments in the mass spectrometry technique) showed ESI spectra of polypeptides and proteins of up to 40 kda (Fenn et al 1988). Fenn showed that a molecular-weight accuracy of 0.01% could be obtained by applying a signal-averaging method to the multiple ions formed in the ESI process. The findings were based on earlier developments when electrospray and mass spectrometry for the first time were successfully combined in Fenn s laboratory

12 protein-protein complexes. It is so mild that viral material can remain viable after an electrospray ionization (ESI) process (Bothner et al 1998). Figure Electrospray ionization ESI. Modified from: Sheehan. The ESI source design is simple, with spray formation occurring in a strong electric field as shown in figure The ion are then accelerated by the so called cone voltage, the potential difference over which acceleration occurs. Figure ESI spectrum of horse myoglobin. Source: Caprioli & Sutter. In Figure 4.12, a protein of molecular weight Da is analyzed with ESI. The process results in over 15 differently charged peaks. The resulting mass/charge ratio equals and can thus be easily analyzed in any mass analyzer. The signals are distributed according to (4.1) Figure The electrospray ionization process. Source: Sheehan. In a proposed mechanism (Figure 4.11), desolvation of the droplets aided by the heated bath gas (usually nitrogen), leads to an increasing charge density on the droplet surface that will eventually cause a Coulombic explosion that leads to individual ions. In a way the electrospray chamber works like an electrolysis cell, where one half-cell reaction takes place at the stainless steel needle / liquid interface with transport of electrons via the power supply to the counter electrode (in the positive mode), and transport of positively charged droplets and ions through the gas phase to the counter electrode. Independent of the exact mechanism, ions are formed at atmospheric pressure and enter a cone shaped orifice, which acts as a first vacuum stage where they undergo so-called free jet expansion. A skimmer then samples the ions and guides them to the mass spectrometer. Spray formation is the crucial part of the ESI technique. It is usually advisable to filter all the solvents and high concentrations of electrolytes should be avoided because they can lead to electrical breakdown and unstable operating conditions. where M represents the molecular mass of the uncharged molecule and m+ the mass of the charged adduct (e.g. H+, Na+, and NH4+). This distribution of ions permits the calculation of the molecular mass of the original analyte from any two neighbouring ions at m/z values (m/z)i and (m/z)i+1, with zi and zi+1 charges, respectively. Since zi+1 = zi+1, the equations can be solved to give both zi and M. (4.2) (4.3) (4.4) When the charges are known the molecular mass can be calculated with high accuracy: (4.5) ESI is the least invasive of all ionization methods. It allows studies of molecular complexes that only have weak non-covalent interactions, such as 23 24

13 Thus deconvolution of the charge pattern allows determination of the mass of the uncharged protein to far higher accuracy than data for a single charge species. The charge state distribution of large molecules is not always reproducible, because it can readily be changed by relatively small changes in analysis conditions, changing ph, adding solvents or salts, partial denaturation of the protein, breaking disulfide bonds, etc. injected into a continuous-flow of this mixture, or be contained in the effluent of an HPLC column or CE capillary. The sensitivity of ESI is quite good, with 100 fmol detection levels (10-13 mol) for many peptides. For ESI the mass range is quite high, and the molecular masses of proteins over 50,000 Da can be measured. As with most ionization techniques, ion suppression effects exist and render direct analysis of complex mixtures inadvisable. The ESI process may also be susceptible even to low amounts of salts, which can significantly decrease sensitivity. The attainable mass accuracy for measuring molecular masses of biomolecules with ESI on a magnetic sector instrument is typically about 0.001% and somewhat better when using internal calibration. One advantage of ESI over MALDI is that as a consequence of the multi-charging phenomenon, the instrument can be calibrated in the low m/z range, using singly-charged calibrants with very well defined masses. Nanospray ionisation Figure ESI spectrum of the +17 charge state of horse myoglobin. Source: Caprioli & Sutter. If high resolution capabilities are available, one may resolve the individual carbon isotope peaks of a given charge state. Figure 4.13 shows the high resolution ESI ion distribution of a single charge state of horse myoglobin. Since the mass of the different isotopic species differ by one Da, the isotope peaks should be 1/z units apart. The isotope peaks are approximately 0.06 m/z units apart in figure 4.13, indicating that they carry (approximately) 17 charges (0.06 % 1/17). A disadvantage of the wide distributions of multiply charged states is that mixtures of high mass samples give overlapping charge state distributions that may be difficult to assign to the individual components. Ionized peptides usually have only a few charges, typically 1-4 depending on size, frequency and relative positions of residues with basic groups, solution conditions, etc. If only one charged species for a peptide of unknown molecular mass is present it is relatively easy to determine the number of charges by simply measuring the m/z position of the first isotope peak. For example, a singly-charged ion of a molecule of molecular mass Da would be recorded in the spectrum at m/z ([M+H] + ) and its first isotope peak at m/z ; a doubly-charged ion [M+2H] 2+ of a molecule of molecular mass Da would also appear at m/z , but its first isotope peak would appear at m/z ; etc. ESI has been used in conjunction with all common mass analyzers, and has had a tremendous impact on the use of mass spectrometry in biological research. The sample is usually dissolved in a mixture of water and organic solvent, commonly methanol, isopropanol or acetonitrile. It can be directly infused, or Figure Nanospray ionization. Source: Miranker. The sensitivity of the ESI process is not increased by high mass flow. From microliter/minute flow rates, the sensitivity has been enhanced by lowering the flow rate to tenths of nanoliter/minute (Valakovic & McLafferty 1995, Wilm et al 1996). Sensitivities of attomole levels (10-18 mole % 1 million molecules) are commonly achieved in these low-flow rate applications. The sample Some limitations, such as avoiding certain reagents like SDS or other surfactants, phosphate buffers, and excessive amounts of any salts, apply to the samples for all ionization techniques. For ESI even moderate buffer and salt concentrations may adversely affect spray formation, while MALDI is more tolerant. Generally less than 1 mm salt should be used, and minimum buffer to achieve controlled buffer conditions (e.g. uniform oxidation state) should be used. Table 4.1 lists a few examples of what should and should not be in a MS sample. No interference Tolerable (< 50 mm) To be avoided TFA HEPES glycerole formic acid MOPS sodium azide &-mercaptoethanol Tris DMSO 25 26

14 DTT NH 4Ac SDS volatile organic solvents octyl glucoside phosphate HCl NaCl NH 4OH urea acetic acid guanidine 4.Combined Techniques MS-MS or Tandem MS Table 4.1. What should and should not be in a MS sample. Although sensitivities are high, small volumes of solutions are used to load samples, often necessitating concentrations in the 'M range. sample treatment EI FAB MALDI ESI nano spray harsh quite gentle gentle very gentle very gentle MW limit hundreds of Da ten kda hundreds of kda hundreds of kda hundreds of kda pulsed / continuous both both pulsed both both LC/MS gas phase yes possible yes too slow Charges mostly 1 mostly 1 mostly 1 ~1 per kda ~1 per kda Figure 7.1. Schematic drawing of an MS-MS spectrum. MS1 shows the intact ions, and MS2 the fragments of these ions. Source: Ziuzdak. The ability of scanned analysers, e.g. quadrupole filter and sector instruments, to select ions of a certain m/z, allows for consecutive MS experiments in socalled tandem MS or MS-MS instruments. In such an instrument, ions of a certain m/z value can first be selected to be treated, e.g. fragmented by collision with an inert gas like argon or helium, so called collisionally induced decomposition (CID), and then the fragments can be analysed. If the first MS step is scanned a two-dimensional spectrum like that in figure 7.1 is obtained, where MS1 shows m/z of the intact sample molecules, and MS2 shows m/z of the fragments of the molecules at each m/z in MS1. Low MW background no yes yes no no Impurities very sensitive very sensitive Detection limit mol mol mol mol Preferred analyzer TOF quadrupole quadrupole Table 4.2. Comparison of the different ionization methods Figure 7.2. Triple quadrupole mass spectrometer. The spectrometers can be tandem either in space or in time. Tandem in space means having two mass spectrometers in series. Many combinations have been tried. One popular version is the triple quadrupole (QqQ) (Figure 7.2) where Q represents a quadrupole mass filter, and q a collision chamber. Here, an ion of interest generated in the ion source is preselected with the first mass filter Q1, CID takes place in the collision chamber q, and the fragmentation products are analyzed with the second quadrupole Q2. In this way it is possible, for example, to obtain sequence information from peptides in a mixture by selecting the ion corresponding to a certain peptide and analyzing the backbone fragments with Q2 (see below). This is termed a 'product ion' scan. Common 27 28

15 sub-structures can also be identified. The search for all of the precursors of a given ion is termed a 'parent ion' scan. This is achieved by keeping Q2 at the mass of the ion in question, and scanning Q1. The origin of neutral fragments, lost in the fragmentation process, can be identified by scanning Q1 and Q2 simultaneously, offset by the mass of the fragment. LC/MS If the different molecules in a mixture are separated by a liquid chromatography technique, e.g. HPLC, before analysis by MS, the sample gets more pure and a chromatographic dimension is added, i.e. each peak in the chromatogram has a mass spectrum associated with it (Figure 7.4). Figure 7.3. Q-TOF MS The Q-TOF (Figure 7.3) is a hybrid instrument that combines the ion selection capabilities of a quadrupole filter with the higher sensitivity of TOF. Tandem in time can be achieved on trapping devices such as quadrupole ion traps and ICR mass spectrometers. These instruments are in fact not limited to simple MS/MS experiments, but can achieve multiple MS/MS/MS.../MS measurements under optimal conditions. Figure 7.4. LC/MS of a mixture of metabolites. The main figure shows a normal UV trace of an LC experiment, and the insert the mass spectrum at a certain retention time. In an LC/MS experiment, mass spectra are continuously collected as the sample is eluted. LC/MS first appeared in the early 1970s. An interface is required because the LC operates at flow rates that are too high for the MS to accomodate. Since ESI ionizes the sample in a continuous liquid spray, it is the ideal ionization source for LC/MS. The high flow rates of the HPLC effluent, can be accommodated by using aids such as heated nebulizing gas to assist spray formation. Often an organic solvent, sometimes referred to as sheath liquid, is added to the HPLC effluent to establish a more stable spray. Interfaces to FAB and MALDI have also been developed. The combination of liquid chromatography and mass spectrometry is a very powerful analytical tool. It can provide a wealth of information on individual compounds even when present in complex mixtures, including identification by molecular mass, determination of structure through fragmentation, and quantification. Better chromatographic information is obtained with an analyzer that is specific for a given m/z value, and better mass spectrometry when the compounds in a mixture enter the spectrometer over time, increasing the efficiency of ionization. With LC/MS, desalting and preconcentration techniques are easily employed, increasing the efficiency and sensitivity of the mass spectrometer. Most importantly, the integrated system minimizes sample handling loss and maximizes efficiency of analyses. The mass spectrometer allows stable isotopically labelled compounds to act as quantitative references in LC separations, providing the high accuracy. However, LC/MS instruments 29 30

16 are more complex to operate and maintain, have a limited number of column types because they cannot be operated with all buffers (e.g., phosphate buffers are particularly troublesome), and are relatively expensive. 5. Protein and peptide applications After the introduction of the two complementary soft ionization processes ESI and MALDI in the mid-1980s, the techniques have matured into viable tools for the analysis of biomolecules and many other classes of molecules. Sequence Analysis Mass spectrometry can determine the sequence of a few picomoles of a peptide in the molecular mass range up to about 2500 Da. Different approaches are used, utilizing either specific enzymatic or chemical reactions to form truncated peptides and subsequent use of mass spectrometry to analyze the reaction products, or MS/MS methodology to perform ionization, peptide ion backbone cleavage, and identification of products. Depending on the ionization technique, peptide ions tend to fragment more or less at the peptide backbone, and produce major series of fragment ions. Although this happens to some extent as the result of internal energy from the ionization process, fragmentation is made more efficient by colliding the ionized molecular species (e.g., [M+H] + ) with a neutral gas molecule. The overall process is termed collision-induced decomposition (CID; see figure 7.1). Figure 8.1. Roepstorff nomenclature for peptide fragmentation. Source: Caprioli & Sutter. The types of fragmentation that occur as a result of this process are shown in figure 8.1, using the so called Roepstorff nomenclature. The N-terminal series (A, B, C) and C-terminal series (X, Y, Z) provide the major sequences of ions found in the spectrum of a peptide. In addition, another fragment ion series 31 32