Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/06—Electron- or ion-optical arrangements
  • H01J49/062—Ion guides
  • H01J49/063—Multipole ion guides, e.g. quadrupoles, hexapoles
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/10—Ion sources; Ion guns
  • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
  • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
  • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/26—Mass spectrometers or separator tubes
  • H01J49/34—Dynamic spectrometers
  • H01J49/40—Time-of-flight spectrometers

Definitions

• This invention relates to mass spectrometers and ion sources therefor. More particularly, this invention is concerned with pulsed ion sources and the provision of a transmission device which gives a pulse ion source many of the characteristics of a continuous source, such that it extends and improves the application of Time of Flight Mass Spectrometry (TOFMS) and that it additionally can be used with a wide variety of other spectrometers, in addition to an orthogonal injection time-of-flight mass spectrometer.
• TOFMS Time of Flight Mass Spectrometry
• Ion sources for mass spectrometry may be either continuous, such as ESI (electrospray ionization) sources or SIMS (secondary ion mass spectrometry) sources, or pulsed, such as MALDI (matrix-assisted laser desorption/ionization sources).
• Continuous sources have normally been used to inject ions into most types of mass spectrometer, such as sector instruments, quadrupoles, ion traps and ion cyclotron resonance spectrometers.
• TOF mass spectrometers have several advantages over conventional quadrupole or ion trap mass spectrometers.
• One advantage is that TOF mass spectrometers can analyze a wider mass-to-charge range than do quadrupole and ion trap mass spectrometers.
• Another advantage is that TOF mass spectrometers can record all ions simultaneously without scanning, with higher sensitivity than quadrupole and ion trap mass spectrometers.
• a TOF mass spectrometer therefore has a large inherent advantage in sensitivity.
• TOF mass spectrometers encounter problems with many widely used sources which produce ions with a range of energies and directions.
• the problems are particularly acute when ions produced by the popular MALDI (matrix-assisted laser desorption/ionization) technique are used.
• MALDI matrix-assisted laser desorption/ionization
• photon pulses from a laser strike a target and desorb ions whose masses are measured in the mass spectrometer.
• the target material is composed of a low concentration of analyte molecules, which usually exhibit only moderate photon absorption per molecule, embedded in a solid or liquid matrix consisting of small, highly-absorbing species.
• the sudden influx of energy is absorbed by the matrix molecules, causing them to vaporize and to produce a small supersonic jet of matrix molecules and ions in which the analyte molecules are entrained. During this ejection process, some of the energy absorbed by the matrix is transferred to the analyte molecules. The analyte molecules are thereby ionized, but without excessive fragmentation, at least in the ideal case.
• the ions also appear as pulses, facilitating their convenient measurement in a time-of-flight spectrometer.
• the ions acquire a considerable amount of energy in the supersonic jet, with velocities of the order of 700 m/s, and they also may lose energy through collisions with the matrix molecules during acceleration, particularly in high accelerating fields.
• a partial solution to the problem is provided by a reflecting spectrometer, which partially corrects for the velocity dispersion, but a more effective technique is the use of delayed extraction, either by itself or in combination with a reflector.
• delayed extraction the ions are allowed to drift for a short period before the accelerating voltage is applied.
• This technique partially decouples the ion production process from the measurement, making the measurement less sensitive to the detailed pattern of ion desorption and acceleration in any particular case. Even so, successful operation requires careful control of the laser fluence (i.e. the amount of power supplied per unit area) and usually some hunting on the target for a favorable spot.
• a pulse source such as a MALDI source
• spectrometer instruments in a manner which more completely decouples the spectrometer from the source and provides a more continuous ion beam with smaller angular and velocity spreads.
• TOF mass spectrometer with both continuous and pulsed sources, for example both ESI and MALDI sources, so either source can be selected.
• a mass spectrometer system comprising: a pulsed ion source, for providing pulses of analyte ions; a mass spectrometer; an ion path extending between the ion source and the mass spectrometer; and an ion transmission device located in said ion path and having a damping gas in at least a portion of the ion path, whereby there is effected at least one of: a reduction in the energy spread of ions emitted from said ion source; conversion of pulses of ions from the ion source into a quasi- continuous beam of ions; and at least partial suppression of unwanted fragmentation of analyte ions.
• the invention has particular applicability to time of flight mass spectrometers. As these require a pulsed beam, conventional teaching is that a pulsed source should be coupled maintaining the pulsed characteristics.
• a pulsed source should be coupled maintaining the pulsed characteristics.
• the present inventors have now realised that there are advantages to, in effect converting a pulsed beam into a continuous, or at least quasi- continuous, beam, and than back into a pulsed beam.
• the advantages are: improvement in beam quality through collisional damping; decoupling of the ion production from the mass measurement; ability to measure the beam current by single-ion counting because it is converted from a few large pulses to many small pulses, for example from about 1 Hz. to about 4 kHz., or a factor of 4,000; compatibility with a continuous source, such as ESI, offering the possibility of running both sources on one instrument.
• the invention also has applicability to mass spectrometers that work with or require a continuous beam.
• a pulsed source can indeed be used with such spectrometers.
• the ion source provides the analyte for ionization by radiation, and there is provided a source of electromagnetic radiation, more preferably a pulsed laser, directed at the ion source, for generating radiation pulses to cause desorption and ionization of analyte molecules.
• the ion source comprises a target material composed of a matrix and analyte molecules in the matrix, the matrix comprising a species adapted to absorb radiation from the radiation source, to promote desorption and ionization of the analyte molecules.
• the transmission device comprises a multipole rod set.
• Collisional damping can also be accomplished in a chamber where no RF field is present providing there is enough buffer gas pressure.
• ions with reduced velocities can be moved to the exit of the chamber by gas flow drag or a DC electrostatic field.
• Combinations of electrostatic fields, RF fields and gas flow can also be implemented in a collisional damping chamber.
• Another advantage of the invention is that the collisional cooling of the ions helps to reduce the amount of fragmentation of the molecular ions. It is usually desirable to produce a simple mass spectrum containing only ions representative of molecular species. In typical MALDI ion sources, therefore, the laser power must be carefully optimized so that it is close to the threshold of ionization in order to reduce fragmentation. The inventors have observed, however, that the presence of a gas around the sample surface greatly assists in reducing fragmentation, even at relatively high laser power. Presumably this is due to the effect of collisions with gas molecules which remove internal energy from the desorbed species before they can fragment. This means that the laser power can be increased in order to improve the ion signal strength, without causing excessive decomposition.
• the mass spectrometer system can include a continuous ion source, and means for selecting one of the pulsed ion source and the continuous ion source, and this then provides the characteristics of two separate instruments in one instrument.
• the two ion sources can comprise a MALDI source and an ESI source.
• Another aspect of the present invention provides a method of generating ions and delivering ions to a mass spectrometer, the method comprising the steps of:
• step (3) comprises providing an RF rod set within the transmission device.
• a DC field can be provided between the ion source and the spectrometer to promote movement of ions towards the spectrometer.
• the method can include providing two or more rod sets in the ion transmission device, and operating at least one rod set with a DC offset to enable selection of ions with a desired mass-to-charge ratio.
• a potential difference can be provided between two adjacent rod sets sufficient to accelerate ions into the downstream rod set, to cause collisionally induced dissociation in the downstream rod set.
• a pulsed laser for each laser pulse, a plurality of pulses of ions are delivered into the time-of-flight mass spectrometer.
• the ions can first pass through one or more differentially pumped regions that provide a transition from the pressure at the ion source to pressure in the spectrometer.
• the ion source may be at atmospheric pressure or at least at a pressure substantially higher than that in downstream quadrupole stages and in the mass spectrometer. At least one of these regions can be without any rod set and ion motion towards the mass spectrometer is then driven by gas flow and /or an electrostatic potential.
• Figure 1 shows a block diagram of a mass spectrometer system
• FIG. 2 is a schematic diagram showing a MALDI-TOF mass spectrometer with orthogonal injection of the MALDI ions into the spectrometer through a collisional damping interface (quadrupole ion guide) according to the present invention
• Figure 3 shows a mass spectrum of a mixture of several peptides and proteins leucine-enkephalin-Arg (Le-R), substance P (Sub P), melittin (ME), CD4 fragment 25-58 (CD4), and insulin (INS) ) produced in the spectrometer of Figure 2
• Figure 4 shows plots of transit times through the interface for different ions;
• Figure 5 shows a mass spectrum of substance P
• Figure 6 shows a mass spectrum of a tryptic digestion of citrate synthase
• Figure 7A shows a schematic of part of spectrometer of Figure 2, showing the collisional interface and indicating applied voltages
• Figures 7B, 7C and 7D show different operating regimes of the mass spectrometer of Figure 2;
• Figures 8A, 8B, 8C, and 8D are mass spectra obtained from substance P recorded in the different operation regimes, according to Figures 7B, 7C, and 7D;
• Figure 9 shows the behaviour of the ion current from a single target spot as a function of time; and Figure 10 shows schematically combined ESI and MALDI sources for a mass spectrometer.
• Figure 11 shows a MALDI-QqTOF mass spectrometer utilizing a collisional damping interface including extra ion manipulation stages which are added between the interface and the time-of-flight mass spectrometer;
• Figure 12 shows mass spectra obtained on a MALDI-QqTOF of
• Figure 13 shows an alternative collisional damping setup for the MALDI-QqTOF mass spectrometer of Figure 11, where ion velocities are partially damped in a region without RF fields;
• Figure 14 shows an experimental apparatus which was used to investigate the effect of pressure and electric field strength on the MALDI ion current;
• Figure 15 is a graph showing the total ion current produced by MALDI source shown in Figure 14 as a function of voltage difference applied at different pressures in the chamber.
• the first embodiment shown in Figure 1 is a block diagram of a general mass spectrometer system.
• 1 represents any sort of pulsed ion source (for instance MALDI)
• 2 is a collisional focusing chamber or region filled with a buffer gas and with a multipole 3 driven at some RF voltage.
• This is followed by an optional manipulation stage 4 and then a mass analyzer 5.
• the collisional ion guide 3, in accordance with the present invention spreads the pulsed ion beam in time, and improves its beam quality (i.e. space and velocity distributions) by damping the initial velocity and focusing the ions toward the central axis.
• the beam is then quasi-continuous and may enter an optional manipulation stage 4, where ions can be subjected to any sort of further manipulation.
• stage 4 the resultant ions are analyzed in the mass analyzer 5.
• a simple example of further manipulation in stage 4 is dissociation of the ions by collisions in a gas cell, so that the resulting daughter ions can be examined in the mass analyzer. This may be adequate to determine the molecular structure of a pure analyte. If the analyte is a complex mixture, stage 4 needs to be more complicated. In a triple quadrupole or a QqTOF instrument (as disclosed in A. Shevchenko et al, Rapid Commun. Mass Spectrom.
• stage 4 would include a quadrupole mass filter for selection of a parent ion of interest and a quadrupole collision cell for decomposition of that ion by collision-induced dissociation (CID). Both parent and daughter ions are then analyzed in section 5, which is a quadrupole mass filter in the triple quadrupole, or a TOF spectrometer with orthogonal injection in the QqTOF instrument. In both cases stages 1 and 2 would consist of a pulsed source and a collisional damping ion guide. - li ⁇
• the collisional focusing chamber 2 is shown with a multipole rod set 3, which could be any suitable rod set, e.g. a quadrupole, hexapole or octopole.
• the particular rod set selected will depend upon the function to be provided.
• a radio frequency ring guide could be used for the collisional focusing device, and ion creation could be performed within the volume defined by the radio frequency field in order to contain the ions.
• FIG. 2 shows a preferred embodiment of a MALDI-TOF mass spectrometer 10 according to the present invention.
• the spectrometer 10 includes a conventional MALDI target probe 11, a shaft seal chamber 12, pumped in known manner, and a target installed in the target-holding electrode 13.
• a mixture of the sample to be investigated and a suitable matrix is applied to the sample probe following the usual procedure for preparing MALDI targets.
• a pulsed laser 14 is focused on the target surface 15 by lens 16, and passes through a window 17.
• the laser beam is indicated at 20, and the laser is run at a repetition rate of anywhere from below a few Hz to tens of kHz, more specifically in this embodiment tested at a rate of 13 Hz.
• An inlet 18 is provided for nitrogen or other neutral gas.
• Each laser shot produces a plume of neutral and charged molecules. Ions of the sample analyte are produced and entrained in the plume which expands into vacuum chamber 30, which contains two quadrupole rod sets 31 and 32.
• Chamber 30 is pumped by a pump (not shown) connected to port 34 to about 70 mTorr but the pressure can be varied over a substantial range by adjusting the flow of gas through a controlled leak valve 18. Pressures of up to 1 atmosphere could also be used in the ion generation region, by putting the ionization region in a chamber which is upstream of chamber 30, and providing a small aperture through which ions are pulled into chamber 30. Lower pressures could be used, and an important characteristic is the product of pressure and rod length.
• a total length x pressure value of at least 10.0 mTorr-cm could be used, although a value of 22.5 mTorr-cm, as in U.S. patent 4,963,736, is preferred.
• the gas in chamber 30 (typically nitrogen or argon or other suitable gas, preferably an inert gas) will be referred to as a damping gas or cooling gas or buffer gas.
• the quadrupole rod sets 31 and 32 were made of rods 4.45 cm in length and 11 mm in diameter, and were separated by 3 mm, i.e. the spacing between rods on adjacent corners of the rod set.
• the quadrupoles 31 and 32 are driven by a power supply which provides operating sine wave frequencies from 50 kHz to 2 MHz, and output voltages from 0 to 1000 volts peak-to-peak. Typical frequencies are 200 kHz to 1 MHz, and typical voltage amplitudes are 100 to 1000 V peak-to-peak. Both quadrupoles are driven by the same power supply through a transformer with two secondary coils. Different amplitudes may be applied to the quadrupoles by using a different number of turns in the two secondary coils. D.C. Bias or offset potentials are applied to the rods of quadrupoles 31 and 32 and to the various other components by a multiple-output power supply.
• the RF quadrupoles 31 and 32 with the damping gas between their rods can be run in an RF-only mode, in which case they serve to reduce the axial energy, the radial energy, and the energy spreads, of the ions which pass through it, as will be described. This process substantially spreads the plume of ions out along the ion path, changing the initial beam, pulsed at about 13 Hz, into a quasi-continuous beam as described in more detail below.
• the first quadrupole 31, can also be run in a mass-filtering mode by the application of a suitable DC voltage.
• the second quadrupole 32 can then be used as a collision cell (and an RF-guide) in collision-induced dissociation experiments (see below).
• the ions pass along an ion path 27 and through a focusing electrode 19 and then pass through orifice 38, into a vacuum chamber 40 pumped by a pump (not shown) connected to a port 42. There, the ions are focused by grids 44 through a slot 46 into an ion storage region 48 of a TOF spectrometer generally indicated at 50.
• ions are extracted from the storage region 48 and are accelerated through a conventional accelerating column 51 which accelerates the ions to an energy of approximately 4000 electron volts per charge (4 keV).
• the ions travel in a direction generally orthogonal to the ion path 27 between the ion storage region, through a pair of deflection plates 52.
• the deflection plates 52 can serve to adjust the ion trajectories, so that the ions are then directed toward a conventional electrostatic ion mirror 54, which reflects the ions to a detector 56 at which the ions are detected.
• the ions are detected using single-ion counting and recorded with a time-to-digital converter (TDC).
• TDC time-to-digital converter
• the accelerating column 51, plates 52, mirror 54 and detector 56 are contained in a main TOF chamber 58 pumped to about 2 x 10" 7 Torr by a pump (not shown) connected to a port 60.
• a pump not shown
• the size of the aperture which admits ions from the storage region into the spectrometer must clearly be much smaller than this to maintain a uniform extraction field, particularly if a slit is placed between the target and the TOF axis. This further reduces the sensitivity.
• the well-defined target-plane perpendicular to the TOF axis allows a combination of time-lag focusing (delayed extraction with optimized values of delay and applied voltage) and electrostatic focusing (optimized value of the reflector voltage) in an ion mirror to produce resolution well above 10,000 in some cases.
• FIG. 3 shows a spectrum of an equimolar mixture of several peptides and proteins from mass 726 to 5734 Da in an ⁇ -cyano-4-hydroxy cinnamic acid matrix. The spectrum was acquired in a single run and shows uniform mass resolution (M/ ⁇ M F HM ) of about 5000 throughout the mass range. Using a simple external calibration with substance P and melittin, the mass determination for each of the molecular ions is accurate within about 30 ppm.
• the peaks for the various substances are identified as: peak 60 for Leucine-enkephalin; peak 61 for substance P; peak 62 for Melittin; peak 63 for CD4 fragment 25-28; and peak 64 for insulin. All peaks are identified both on the overall spectrum and as an enlarged partial spectrum.
• the resolution demonstrated in Figure 3 is rather ⁇ close to the resolution obtainable with the same instrument using an ESI source.
• the entrance orifice was made slightly larger than normally used in ESI, approximately 1 mm diameter as compared to a normal diameter of around 1/3 mm, to make adjustments easier in the preliminary experiments. This does not appear to have been necessary so it is reasonable to expect improved resolution if a smaller orifice is used. Resolution up to 10,000 has been obtained with ESI ions in the same instrument and in the MALDI-QqTOF instrument described below.
• the decreasing relative intensity of the molecular ions with mass is to some extent a reflection of the decreasing detection efficiency with increasing mass. Detection efficiency depends strongly on velocity, which decreases with mass for singly-charged ions at a given energy. In this embodiment the energy of singly-charged ions is only about 5 keV (compared to 30 keV in typical MALDI experiments), so the detection efficiency limits the practical range of application to less than about 6000 Da.
• the relative intensities of the molecular ion peaks in Fig. 3 is consistent with that observed from the same sample when analyzed in a conventional MALDI experiment using 5 kV acceleration.
• the detection efficiency in the present embodiment can be increased by increasing the voltage which accelerates the ions into the spectrometer, or by increasing the voltage on the detector.
• the collisional cooling spreads the ions out along the ion beam axis changing the initial beam pulsed at 13 Hz into a quasi-continuous beam.
• Figure 4 shows the count rate as a function of time after the laser pulse; i.e. the distribution of transit times through the ion guide.
• the width of the time distribution is on the order of 20 ms which represents an increase in the time spread by a factor of at least 10 7 as each laser pulse is about 2 ns long. It will be appreciated that it is not necessary to produce a time distribution of the order of 20 ms; for example the quasi-continuous pulse could be as short as 0.1 ms.
• Dispersion along the axis is a disadvantage in orthogonal-injection MALDI without cooling, but with the present invention, since optimum extraction conditions do not depend on the time delay after the laser shot, multiple injection pulses into the TOF storage region 48 can be used for each laser shot. In the present embodiment, 256 injection pulses into the TOF storage region 48 were used for every laser shot. The losses are then determined by the duty cycle of the instrument which in this case is about 20%.
• the duty cycle is the percentage of the time that ions can be injected from the storage region into the TOF spectrometer; here, it effectively means the fraction of the time, the TOF storage region 48 is available to accept ions.
• a quasi-continuous beam is in fact an advantage in this mode of operation.
• ions are ejected from the target probe with every laser shot at a repetition rate of 13 Hz, but as a result of spreading along the beam axis or ion path 27 (and some losses) approximately 2 to 5 ions are injected into the instrument with every injection pulse less than one ion on average of a particular species.
• TDC Time to Digital Converter
• single-ion counting eliminates problems with detector shadowing from intense matrix peaks, and problems with peak saturation which require attention in conventional MALDI because of the strong dependence of the signal on laser fluence and the shot-to-shot variation.
• single-ion counting places much more modest demands on the detector and amplifier time resolution because the electronic reduction and digitization of the pulse is quite insensitive to the detector pulse shape.
• the losses can be compensated to a large extent by the higher repetition rate and higher fluence of the laser.
• the repetition rate was 13 Hz, but can easily be increased to 20 Hz with the current laser, or in principle up to at least 100 Hz before the counting system becomes saturated.
• the usual MALDI experiment is run at about 1 or 2 Hz.
• the laser fluence in a conventional MALDI experiment must be kept close to threshold to achieve the best performance, the threshold being the minimum energy necessary to cause vaporization of the sample to produce a useful signal using a conventional transient recording with analog to digital conversion.
• the laser fluence can be increased to the fluence at which the ion production process saturates.
• FIG. 6 shows the spectrum 85 obtained from a tryptic digest of citrate synthase again showing the uniform mass resolution over the mass range; the inset 86 shows the spectrum obtained from 20 fmoles applied to the target.
• the laser is simply set to maximum fluence (several times the usual threshold) and left while the target is moved to a fresh position occasionally. This means that alternative targets can easily be tried (including insulating targets), and alternative lasers with different wavelengths or pulse widths can be used.
• the decoupling of the ion production from the mass measurement also provides an opportunity to perform various manipulations of the ions after ejection but before mass measurement.
• One example is parent ion selection and subsequent fragmentation (MS/MS). " This is most suitably done with an additional quadrupole mass filter as described below, but even in the present embodiment of Figure 2, some selectivity and fragmentation is possible.
• Figures 7A, 7B and 7C show three different modes of operation of the instrument shown in Figure 2.
• the reference numerals of Figure 2 are provided along the z axis to indicate correspondence between potential level and the different elements of the apparatus. Voltages for the quadrupole sections 31, 32 are indicated respectively at JJ-, (t) and U 2 (t).
• Figure 7A shows the simple collisional ion guide mode that was used in obtaining the results shown in Figures 4-6.
• the same amplitudes of RF voltage and no DC offset voltages are applied to different sections of the quadrupole. Potential differences in the longitudinal direction are kept small to minimize fragmentation due to CID.
• Figure 7B shows a mass filtering mode, which is analogous to the same filtering mode implemented in conventional quadrupole mass filters.
• a DC offset voltage V is added to the first section of the quadrupole to select an ion of interest, while the second section again acts as an ordinary ion guide since there is no CID because of the small potential difference between the sections.
• the amplitude of the voltage applied in the second quadrupole section 32 is only one third of the voltage applied in the first section 31.
• Figure 7C is an MS-MS mode which differs from the mode of Figure 7B by a higher potential difference between the quadrupole sections 31, 32, so ions are accelerated in that region and enter the second section with high kinetic energy, the additional energy being indicated as ⁇ collision energy.
• the second section acts as an collision cell and parent ions are decomposed there by collisions with the buffer gas (CID).
• the amplitude of the RF voltage in the second section is only one third of the amplitude of the RF voltage in the first section, which allows daughter ions much lighter than the parent ions to have stable trajectories and to be transmitted through the second quadrupole.
• Figure 8 shows examples of the spectra obtained in the different modes illustrated in Figure 7, and in particular gives an example of possible beam manipulation. All the spectra were acquired using the same initial sample.
• Figure 8A is a mass spectrum where ions were cooled in a collisional focusing ion guide (the mode of Figure 7A).
• Figure 8B is an example where ions of interest were selected in the first quadrupole 31 and cooled in the second quadrupole 32 section (the mode of Figure 7B). Once ions of interest have been selected, they can be used for fragmentation in CID to obtain detailed information on composition and structure.
• Figure 8C presents an MS/MS spectrum of substance P obtained in this way.
• Molecular ions of substance P are selected in the first quadrupole section and fragmented by collisions in the second quadrupole section (according to the mode of Figure 7C).
• the potential difference, ⁇ collision energy, between the first and second quadrupoles was 100V.
• the intensities of the fragment ions were small in comparison with intensity of the primary ion so the region inside dotted lines is expanded by a factor of 56.
• Figure 8D shows the spectrum obtained in the same mode but where the potential difference between the quadrupoles 31, 32 was 150 V. In this case, more fragment ions are observed and the parent ion peak is substantially reduced.
• Figure 9 shows how long a signal from the same spot on a MALDI target can last.
• a given spot was irradiated by a series of shots from the laser, running at 13 Hz.
• the laser intensity was two or three times the "threshold" intensity.
• the sample lasted for about one minute.
• the shape of the curve suggests that the laser shots dig ' deeper and deeper into the sample until it is exhausted. At that point the laser irradiates the metallic substate, so no signal is observed.
• continuous sources such as electrospray ionization (ESI)
• ESI electrospray ionization
• MALDI pulsed sources
• the present invention enables two such sources to be available in one instrument.
• the MALDI probe 11 in Figure 2 can be replaced by an ESI source to enable measurement of ESI spectra in the instrument.
• the instrument would then be essentially the same as the one illustrated in the paper Krutchinsky A.N., Loboda A.V., Spicer V.L., Dworschak R., Ens W., Standing K.G., Rapid Commun. Mass Spectrom. 1998, 12, 508-518. This change could of course be carried out by actually taking off one source and replacing it by the other, but a number of more convenient arrangements can be provided.
• FIG 10 shows a further embodiment where the electrospray ion source 94 is attached to the input of a collisional damping interface 92, including a quadrupole, or other multipole, rod set 93.
• a MALDI ion source 94 is introduced on a probe 95 that enters from the side, and can be displaced in and out; for this purpose, a shaft end 96 is slidingly and sealingly ' fitted into the housing of the collisional interface 92.
• the MALDI ion source 94 is similar to the one shown in Figure 2 except in this case the sample is deposited onto a flat surface machined on the side of the probe shaft 95, instead of onto the end of a cylindrical probe.
• the sample is irradiated by a laser with corresponding optics, generally indicated at 97, and ions are transmitted to a spectrometer indicated at 98.
• a laser with corresponding optics, generally indicated at 97
• ions are transmitted to a spectrometer indicated at 98.
• shaft 96 is pulled out far enough to clear the path of the ESI ions.
• the MALDI ion source 94 is operating the shaft 96 is inserted back so the MALDI target 94 is in the central position.
• MALDI and ESI techniques are often considered to be complementary methods for biochemical analysis, so many biochemical or pharmaceutical laboratories have two instruments in use.
• the cost of a combined instrument is expected to be little more than half the cost of two separate instruments.
• similar procedures for ion manipulation, detection and mass calibration could be used, since the ion production is largely decoupled from the ion measurement. This would simplify the analysis and processing of the separate spectra and their comparison.
• the apparatus could include a single multipole rod set as shown in Figure 1, or two rod sets as shown in Figure 2. While quadrupole rod sets are preferred, other rod sets, such as hexapole and octopole are possible, and the rod set can be selected based on the known characteristics of the different rod sets. Additionally, it is possible that three or more rod sets could be provided. Further, while Figure 2 shows the two rod sets, 31 and 32 provided in a common chamber, the rod sets could, in known manner, be provided in separate chambers operating at different pressures, to enable different operations to be preformed.
• the MALDI target is provided at 100 and generates an ion beam indicated at 102.
• the MALDI target 100 is located in a differentially pumped chamber 104 connected to a pump as indicated at 106 in known manner.
• a first rod set Q0 is located in the chamber 104.
• An aperture and an interquad aperture plate 108 provides communication through to a main chamber 110. Again, in known manner a pump connection is provided at 112.
• a short rod set 111 within the main chamber 110, there is a short rod set 111, sometimes referred to as "stubbies", provided for the purpose collimating the beam.
• a first quadrupole rod set in the chamber 110 is indicated at Ql and a second rod set at Q2.
• the rod set Q2 is located in a collision cell 114 provided with a connection, indicated at 116, for a collision gas.
• ions pass through a grid and then an aperture into the storage region 48 of the TOF instrument, again indicated at 50.
• a TOF instrument 50 is provided with a liner 118 around the flight region.
• the differentially pumped chamber 104 is maintained at pressure of around 10" 2 torr.
• the main chamber 110 is maintained at a pressure of around 10 -5 torr, while the collision cell 114 is maintained at a pressure of around 10 -2 torr.
• the pressure in the collision cell 114 can be controlled by controlling the supply of nitrogen to it through connection 116.
• collisional damping of ions generating from the MALDI target 100 is accomplished by the relatively high pressure in the differentially pumped chamber 104. Ions then pass through into the quadrupole rod set Ql, which can be operated to mass select a desired ion.
• the mass selected ion is then passed to the collision cell 114, and the rod set Q2; potentials are such that ions enter the rod set Q2 with sufficient energy to effect collision induced dissociation.
• the fragment ions generated by this CID are then passed into the TOF instrument 50 for analysis.
• Typical spectra obtained in a MALDI-QqTOF instrument are presented in Figure 12.
• the spectrum shown in Fig. 12a was obtained when Ql was operated in a wide band mode, so all ions produced in the MALDI ion source were delivered to the TOF mass analyzer.
• Three peaks (121, 122, 123) in Fig 12a correspond to ions of leucine-enkephalin, substance P and mellitin respectively.
• Ql was operated in selection mode, the spectrum shown in Fig 12b is observed.
• Ql was set to select only ions of substance P (peak 122) located at m/z around 1347.7. Note that no other peaks or background were observed in the mass spectrum, as conditions in Ql prevented transmission of other ions.
• Figure 12c shows the result of selection at substance P (peak 122) and collisional induced dissociation of the substance F ions.
• Ql was set to selection mode as in Fig 12b but the potential difference between QO and Q2 was increased to promote CID.
• the peaks observed in the lower mass region are fragments of the substance P ions.
• the MALDI source is indicated at 130 and the ion beam at 132.
• a sampling cone 134 was placed between the MALDI source 130 and the rod set QO. This effectively separates the differentially pumped region into a first differentially pumped region 136 and a second differentially pumped region 138.
• These differentially pumped regions 136, 138 are provided with respective connections 137 and 139 to pumps.
• a short set of rods or stubbies 140 together with a rod set Ql are provided in a chamber here indicated at 142.
• the alternative collisional damping setup of Figure 13 has been implemented in MALDI-QqTOF instrument but can be used with any configuration of collisional RF ion guides such as the simpler geometry described earlier and shown in Figure 2.
• some collisional damping is accomplished in the first region or chamber 136 where almost no RF field is present.
• Nitrogen is supplied to this chamber 136, as in Figure 2, and is also supplied to the chamber 104 in Figure 11; this is comparable to Figure 2, although the nitrogen connection is not shown in these later figures.
• the pressure in this first differentially pumped region or chamber 136 is typically higher than in the second differentially pump chamber 138 and ions are dragged towards the entrance of Q0 by the combination of a DC field and the gas flow.
• FIG 14 shows an apparatus used to study the effect of pressure and electric field on the intensity of the signal produced by MALDI.
• MALDI ions are generated at a target 150 by a pulsed UV-laser beam 152.
• the laser beam 152 passed through a lens 154 and a window 156, as in the spectrometer configurations described above.
• the window 156 is provided in a chamber 158, whose internal pressure can be varied in known manner (connections for pumps, etc. are not shown).
• a potential difference U between the target 150 and a collector electrode 162 is provided by a power supply 160.
• ions generated at the target 150 travel, as indicated at 164, to the collector electrode 162.
• An approximately homogeneous electric field is established in the region between the target 150 and the collector electrode 162.
• the field strength is proportional to the applied potential difference U.
• the distance between the target and collector was about 3 mm.
• the laser was operated at 20 Hz and the total ion current was measured using an amplifier 166.
• Figure 15 shows the dependence of the total ion current produced by MALDI at different pressures inside the chamber 158 as a function of the voltage applied between the target 150 and the collector electrode 162 shown in Fig. 14. It is apparent that ion yield decreases with increasing pressure, and there is a significant drop in yield between 14 and 47 Torr. However, the drop in yield can be recovered by raising the electric field strength.
• the MALDI technique can be used at any desirable pressure, even out of the range in which RF collisional multipoles can be implemented. Collisional damping of the ions can be accomplished at least partially in the region with no RF field adjacent to the sample target. The inventors believe that similar dependence of pressure and electric field can be observed in some other pulsed ion sources and these ionization techniques can be also used with collisional damping at higher pressures.

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