JP2005166639A - Mass spectrometer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass spectrometer including an ion source and a field-free or drift area, and an ion mirror including a reflectron. <P>SOLUTION: Metastable parent ions which spontaneously fragment by Post Source Decay whilst passing through the field free or drift region 5 are arranged to enter the ion mirror 7, and reflected by the reflectron towards an ion detector 8 when the reflectron is maintained at a certain voltage. The process is then repeated with the reflectron being maintained at a slightly lower voltage. Two related sets of time of flight or mass spectral data are obtained for two different voltage settings of the reflectron. From the two data sets, different times of flight for the same species of fragment ion are determined. The mass to charge ratio of the parent ion which fragmented to produce the particular species of fragment ion can then be determined from the times of flight of the fragment ions. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a mass spectrometer and a method of mass spectrometry.

  Matrix-assisted laser ionization (“MALDI”) is a method of generating ions of an analyte material. It is a particularly successful technique for generating ions of large organic and biochemical molecules, which has been largely unsuccessful in many other ionization techniques. This material to be analyzed is dissolved in a suitable solvent. One drop of that solution and one drop of another solution of the appropriate matrix material are then placed on the surface of the sample or target plate so that the two solutions can be mixed. The resulting solution is then evaporated and the remaining matrix material and the material to be analyzed form small crystals. The sample or target plate is then placed in a mass spectrometer and the sample or target plate is irradiated with a pulsed laser. The crystals are usually irradiated with ultraviolet (UV) light, while infrared (IR) light can be used for certain matrix materials.

  Since ions are generated using a pulsed laser beam, the generated ions are generated in short pulses. A particularly convenient type of mass spectrometer for analyzing ions generated from a pulsed ion source is a time-of-flight (“TOF”) mass spectrometer.

  Linear time-of-flight mass analyzers are known that the pulse of ions is accelerated at a high voltage, typically between 10 kV and 30 kV. The time that ions pass through the flight tube and reach the ion detector is recorded. Since all the ions are accelerated to approximately the same kinetic energy, then the resulting velocity of the ions is inversely proportional to the square root of their mass, and it is assumed that the ions are all charged separately. Thus, the time for ions to reach the ion detector is also proportional to the square root of their mass.

  In a MALDI ion source, ions having a range of velocities are desorbed from the surface of the sample or target plate. The average velocity of desorbed ions is determined almost independently of the mass to charge ratio of the ions, and is typically 300-600 m / s. The actual average speed of the desorbed ions depends on the laser power used and the size and nature of the sample and matrix crystals. It has been observed that desorbed ions tend to have a fairly wide range of velocities near the average velocity. As a result, ions accelerated to a time-of-flight mass spectrometer typically have a wide range of ion energies that can cause problems when using a time-of-flight mass spectrometer.

  In a linear time-of-flight mass spectrometer, the time for an ion to reach the ion detector depends on the energy of the ion. Thus, when the ions emitted from the ion source have a range of kinetic energy, then they will also have a range of arrival times. This results in a wide mass peak and insufficient mass resolution.

  Attempts to address this problem are known by using a reflectron that reflects ions at approximately 180 ° and that passes back through a portion of the flight tube to the ion detector. Thus, ions having a relatively higher initial kinetic energy before entering the reflectron penetrate further into the reflectron before being reflected. Therefore, ions having a relatively higher kinetic energy travel a greater distance apart. In this regard, the faster and more energetic ions initially can move a greater distance before impacting the ion detector. If the mean flight path in the reflectron is properly prepared, then they have ions up to the first approximation when they reach the acceleration region of the time-of-flight mass analyzer. It can be arranged to reach the ion detector substantially independently of the kinetic energy. Therefore, using a reflectron results in a narrower observation mass peak and improved mass resolution. Thus, a MALDI ion source incorporating a reflectron and coupled to a time-of-flight mass analyzer can achieve a higher mass resolution than a MALDI ion source coupled to a linear time-of-flight mass analyzer without a reflectron. Is possible.

  A MALDI time-of-flight mass analyzer incorporating a reflectron is also capable of separating and analyzing fragment ions that originate from the parent ions and decompose naturally during flight. Such parent ions are usually metastable ions, and the in-flight decomposition process is commonly referred to as post-source decomposition (“PSD”). The decomposition of the parent ions is also induced by collisions with gas molecules (eg in fragmentation or collision cells). Such a process is commonly referred to as collision-induced dissociation (“CID”).

  Fragment ions generated in the field-free flight region are believed to maintain essentially the same velocity as their corresponding parent ions until the first approximation (the result of the energy released during the decomposition reaction is actually The speed of the fragment ions may increase or decrease very slightly.) Thus, until the first approximation, the fragment ions arrive at the ion detector of the linear time-of-flight mass spectrometer without a reflectron substantially simultaneously with the corresponding unfragmented parent ion. Parent ions and corresponding fragment ions are therefore not substantially separated in time using a linear time-of-flight mass spectrometer without a reflectron. The situation is different when using a mass spectrometer incorporating a reflectron. A fragment ion has approximately the same velocity as its corresponding parent ion, but because of its low mass, then, of course, the fragment ion should have a kinetic energy lower than that of its corresponding parent ion. It is. For example, if a parent ion has a parent ion fragment that has a mass-to-charge ratio of 2000 and becomes a fragment ion that has a mass-to-charge ratio of 1000, then the fragment ion is originally possessed by the parent ion. Has only half of the kinetic energy. The kinetic energy ratio of fragments and parent ions will be the same ratio as their mass. The fragment ion has a lower kinetic energy than its corresponding parent ion, and the fragment ion penetrates to a shallower depth in the reflectron, so the overall path will naturally be shorter. As a result, in a mass spectrometer incorporating a reflectron, if fragment ions are formed by either CID or PSD, then such fragment ions are not correspondingly associated fragmented. Arrives at the ion detector before the parent ion. If the reflectron is optimized to reflect lower energy fragment ions, then the more energetic parent ions are not reflected by the reflectron, so such parent ions are Disappear for the system. Thus, it is possible to separate fragment ions from the corresponding unfragmented parent ion using a suitably prepared time-of-flight mass analyzer incorporating a reflectron, and recording the fragment ions separately It is possible to perform mass spectrometry.

  Analysis of fragment ions is particularly useful for structure determination and identification of the corresponding parent ion. For biopolymer ions, it is possible to infer their molecular sequences from fragment ion and parent ion data.

  A time-of-flight mass analyzer incorporating a reflectron can be used to analyze PSD fragment ions. In a linear field reflectron, the optimum energy concentrated on the ion detector is achieved when the time of flight in the reflectron is approximately equal to the total time of flight in the field free region upstream and downstream of the reflectron. Is done. The time of flight of fragment ions in the reflectron region depends on the depth of penetration of the fragment ions into the reflectron. For relatively low energy fragment ions, the penetration depth into the reflectron can be increased so that the penetration depth of the ions is close to the maximum. This can be achieved by reducing the reflectron voltage. The reflectron voltage may be advanced by multiple voltage settings, for example. From step to step, a 25% reduction in reflectron voltage may be used with a more concentrated concentration on fragment ions having a lower mass to charge ratio and thus lower kinetic energy. From step to step, extracted data (or segments of individual mass spectra) related to ions focused by the reflectron are then merged or bound together to fragment a particular parent ion. Form a single or mixed mass spectrum associated with all the various fragment ions generated from

  Known MALDI time-of-flight mass spectrometers used to analyze fragment ions include a timed electrostatic deflection system or ion gate located in the flight tube upstream of the time-of-flight mass analyzer. The ion gate is arranged to pass only ions having a specific velocity through it. The timing of the ion gate is such that only parent ions having a narrow mass-to-charge ratio are transmitted by the ion gate. Any of the fragment ions generated by fragmentation of the parent ion upstream of the ion gate move at essentially the same speed as the corresponding unfragmented parent ion. Thus, such fragment ions are also transmitted by the ion gate substantially simultaneously with the associated unfragmented parent ion. Thus, by using the ion gate, fragment ions generated from only one specific parent ion (or from a smaller number of parent ions) can be recorded.

  Known mass spectrometers have a number of problems associated with using timed ion gates to select specific ions. Timed ion gates have the disadvantage that they can disrupt the movement of important ions, ie those ions that are intended to be transmitted by the ion gate. The transmitted ions can also be accelerated or decelerated axially and / or radially from the ion gate by an electric field. The fast electron pulses needed to control the ions at the gate may also be too slow or go too far and oscillate. This adversely affects both the mass resolution of the parent ion and the fragment ions and the overall ion transfer of the mass spectrometer. Low energy fragment ions are particularly vulnerable to the effects of the electrostatic field from the ion gate.

  Known ion gates, such as those used in conventional mass spectrometers, include the Bradbury Nielson ion gate. The Bradbury Neilson ion gate includes parallel wires with alternating polarity voltage applied to a continuous wire to minimize the static field. Since some ions strike the wire and are neutralized, such an arrangement has the problem that the parallel wires can reduce ion transmission.

  Other effects resulting from the use of ion gates are also undesirable. For example, ions deliberately deflected by an ion gate strike other parts of the mass spectrometer, and scattered ions (or other secondary ions can be obtained by sputtering, secondary ion emission, surface induced decomposition or similar processes. Target piece). As a result, in complex mixtures, the observation of fragment ions from a slightly weaker parent ion from a slightly weaker parent ion is caused by scattering or more abundant ions intentionally when the ion gate is closed. The presence of secondary ions is obscured.

  Another problem with using a timed ion gate is that one fragment ion spectrum can only be recorded at one time for a particular parent ion. Thus, for example, to characterize a complex mixture of peptide ions by PSD, by reducing the voltage applied to the reflectron, the individual parent peptide ions in the mixture are transmitted one after the other, and for each parent ion It is necessary to set up the ion gate to record its corresponding fragment ion spectrum separately. Therefore, it takes a significant amount of time to obtain fragment ion spectra for all parent ions. Furthermore, the normal approach consumes a relatively small amount of sample before all parent peptide ions are analyzed. This problem is further compounded by the fact that not all parent peptide ions provide more useful fragment ions for PSD. However, it is not known which parent peptide ions give the most useful data until after all parent ions have been analyzed individually. As a result, much time and sample is consumed, and PSD fragment ion data is obtained from parent peptide ions that are less productive or less relevant. In some cases, all samples are consumed before obtaining useful or interesting data.

  On the other hand, if a timed ion gate is not incorporated into a normal mass analyzer, then all fragment ions resulting from the fragmentation of all multiple parent ions are transmitted and recorded simultaneously. Thus, if the sample to be analyzed contains a complex mixture of different parent peptide ions, then the resulting mass spectrum is very flooded with mass peaks, and very large numbers Since it is not known which of the observed fragment ions corresponds to which parent ion, analysis is impossible. As a result, it is not possible to associate an observed fragment ion with a particular parent ion, and therefore no useful information can be obtained when a conventional mass spectrometer is used without an ion gate.

  Accordingly, it has been desirable to provide an improved mass spectrometer and method of mass spectrometry.

According to one aspect of the invention, a method of mass spectrometry is provided, the method comprising:
Providing a time-of-flight mass spectrometer including an ion mirror;
Keeping the ion mirror in a first setting;
Acquiring a first time-of-flight or mass spectral data when the ion mirror is in a first setting;
Keeping the ion mirror at a different second setting;
Acquiring a second time-of-flight or mass spectral data when the ion mirror is in a second setting;
Determining a first time of flight of a first fragment ion having a certain mass or mass to charge ratio when the ion mirror is in a first setting;
Determining a different second time-of-flight of a first fragment ion having the same certain mass or mass-to-charge ratio when the ion mirror is in a second setting; and fragmenting the first fragment Determining either the mass or mass-to-charge ratio of the parent ion that produced the ion and / or the mass or mass-to-charge ratio of the first fragment ion from the first and second time-of-flight. including.

  The ion mirror preferably includes a reflectron, which may be either a linear electric field reflectron or a non-linear electric field reflectron.

  The method further includes providing an ion source and a drift or flight space upstream of the ion mirror, wherein when the ion mirror is in a first setting, the first potential difference is the ion It is preferred that the second potential difference be maintained between the ion source and the drift or flight space when the source and the drift or flight space are maintained and the ion mirror is in a second setting.

  In one embodiment, the first potential difference is substantially the same as the second potential difference.

  In another embodiment, the first potential difference is substantially different from the second potential difference. The difference between the first potential difference and the second potential difference is p% of the first potential difference or the second potential difference, where p is (i) <1; (ii) 1-2; (iii) 2-3 ; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; ( It is preferably within a range selected from the group consisting of xviii) 45-50; and (xix)> 50.

  The difference between the first potential difference and the second potential difference is (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150- 200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V ; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; ( xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3- 4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV ; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; ( (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28- 29 kV; (l) 29-30 kV; and (li)> 30 kV.

  The first potential difference and / or the second potential difference are: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii ) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii ) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22 -23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 is within a range selected from the group consisting of kV; (l) 29-30 kV; and (li)> 30 kV Preferred.

  When the ion mirror is in the first setting, the first electric field strength or field gradient is at least 10%, 20%, 30%, 40%, 50%, 60%, 70% of the length of the ion mirror, When kept along 80%, 90%, 95% or 100% and the ion mirror is in the second setting, the second electric field strength or field gradient is at least 10% of the length of the ion mirror 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%.

  The first electric field strength or electric field gradient may be substantially the same as the second electric field strength or electric field gradient. Alternatively, the first electric field strength or electric field gradient may be substantially different from the second electric field strength or electric field gradient.

  The difference between the first electric field strength or electric field gradient and the second electric field strength or electric field gradient is q% of the first or second electric field strength or electric field gradient, and q is (i) < 1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8 ; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; Preferably, it is within a range selected from the group consisting of (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50.

  The difference between the first electric field strength or electric field gradient and the second electric field strength or electric field gradient is (i) <0.01 kV / cm; (ii) 0.01-0.1 kV / cm; (iii) 0.1-0.5 kV / cm; (iv) 0.5-1 kV / cm; (v) 1-2 kV / cm; (vi) 2-3 kV / cm; (vii) 3-4 kV / cm; (viii) 4-5 kV / cm; (ix) 5-6 kV / cm; (x) 6-7 kV / cm; (xi) 7-8 kV / cm; (xii) 8-9 kV / cm; (xiii) 9-10 kV / cm; (xiv) 10-11 kV / cm; (xv) 11-12 kV / cm; (xvi) 12-13 kV / cm; (xvii) 13-14 kV / cm; (xviii) 14-15 kV / cm; (xix) 15-16 kV / cm; (xx) 16-17 kV / cm; (xxi) 17-18 kV / cm; (xxii) 18-19 kV / cm; (xxiii) 19-20 kxx / cm; (xxiv) 20-21 kV / cm; (xxv) 21-22 kV / cm; (xxvi) 22-23 kV / cm; (xxvii) 23-24 kV / cm; (xxviii) 24-25 kxx / cm; (xxix) 25-26 kV / cm; (xxx) 26-27 kV / cm; (xxxi) 27-28 kV / cm; (xxxii) 28-29 kV / cm; (xxxiii) 29-30 kV / cm; and (xxxiv)> 30 kV / cm may be selected.

  The first electric field strength or electric field gradient and / or the second electric field strength or electric field gradient are: (i) <0.01 kV / cm; (ii) 0.01-0.1 kV / cm; (iii) 0.1-0.5 kV / cm; (iv) 0.5-1 kV / cm; (v) 1-2 kV / cm; (vi) 2-3 kV / cm; (vii) 3-4 kV / cm; (viii) 4-5 kV / cm; (ix) 5-6 kV / cm; (x) 6-7 kV / cm; (xi) 7-8 kV / cm; (xii) 8-9 kV / cm; (xiii) 9-10 kV / cm; (xiv) 10-11 kV / cm; (xv) 11-12 kV / cm; (xvi) 12-13 kV / cm; (xvii) 13-14 kV / cm; (xviii) 14-15 kV / cm; (xix) 15-16 kV / cm; (xx) 16-17 kV / cm; (xxi) 17-18 kV / cm; (xxii) 18-19 kV / cm; (xxiii) 19-20 kV / cm; (xxiv) 20-21 kV / cm; (xxv) 21-22 kV / cm; (xxvi) 22-23 kV / cm; (xxvii) 23-24 kV / cm; (xxviii) 24-25 kV / cm; (xxix) 25-26 kV / cm; (xxx) 26-27 kV / cm; (xxxi) 27-28 kV / cm; (xxxii) 28-29 kV / cm; (xxxiii) 29-30 kV / and preferably within the range selected from the group consisting of (xxxiv)> 30 kV / cm.

  In the preferred method, when the ion mirror is at a first setting, the ion mirror is held at a first voltage, and when the ion mirror is at a second setting, the ion mirror is at a second voltage. Kept. The first voltage may be substantially the same as the second voltage, or may be substantially different from the second voltage.

  In a preferred embodiment, the difference between the first voltage and the second voltage is r% of the first voltage or the second voltage, where r is: (i) <1; (ii) 1-2; iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x ) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50.

  The difference between the first voltage and the second voltage is as follows: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150- 200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; ( xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3- 4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV ; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; ( (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28- Preferably selected from the group consisting of 29 kV; (l) 29-30 kV; and (li)> 30 kV.

  The first voltage and / or the second voltage are: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii ) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii ) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22 -23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 preferably within a range selected from the group consisting of kV; (l) 29-30 kV; and (li)> 30 kV Arbitrariness.

  The preferred method is that when the ion mirror is in a first setting, the ion mirror is kept at a first potential relative to the potential of the ion source, and when the ion mirror is in a second setting, the ion mirror is It further includes providing an ion source that is maintained at a second potential relative to the potential of the ion source. The first potential may be substantially the same as the second potential, or may be substantially different from the second potential.

  In a preferred embodiment, the difference between the first potential and the second potential is s% of the first potential or the second potential, and s is: (i) <1; (ii) 1-2; iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x ) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50.

  The potential difference between the first potential and the potential of the ion source and / or the potential difference between the second potential and the potential of the ion source is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv ) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv ) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19 -20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) It is preferably within a range selected from the group consisting of 28-29 kV; (l) 29-30 kV; and (li)> 30 kV.

  The first potential and / or the second potential are: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii ) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii ) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22 -23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 preferably within a range selected from the group consisting of kV; (l) 29-30 kV; and (li)> 30 kV Arbitrariness.

  Preferred methods include: (i) an electrospray (“ESI”) ion source; (ii) an atmospheric pressure chemical ionization (“APCI”) ion source; (iii) an atmospheric pressure photoionization (“APPI”) ion source; (iv) Laser desorption ionization (“LDI”) ion source; (v) Inductively coupled plasma (“ICP”) ion source; (vi) Electron impact (“EI”) ion source; (vii) Chemical ionization (“CI”) ion (Viii) Field ionization (“FI”) ion source; (ix) Fast atom bombardment (“FAB”) ion source; (x) Liquid secondary ion mass spectrometry (“LSIMS”) ion source; (xi) Large Atmospheric pressure ionization (“API”) ion source; (xii) field desorption (“FD”) ion source; (xiii) matrix-assisted laser ionization (“MALDI”) Further includes providing a and (xiv) a silicon on desorption / ionization ( "DIOS") ion source selected from the group consisting of an ion source; emissions source.

  The ion source may be a continuous ion source or pulsed ions.

  The method further includes providing a drift or flight space upstream of the ion mirror, wherein the ion mirror is at a first potential relative to the drift or flight space potential when the ion mirror is in a first setting. Preferably, when the ion mirror is in a second setting, the ion mirror is maintained at a second potential relative to the drift or flight space potential. In this embodiment, the first potential may be substantially the same as the second potential, or may be substantially different from the second potential.

  In a preferred embodiment, the difference between the first potential and the second potential is t% of the first potential or the second potential, where t is: (i) <1; (ii) 1-2; iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x ) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50.

  The difference between the first potential and the second potential is as follows: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150- 200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V ; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; ( xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3- 4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV ; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; ( (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28- Preferably within a range selected from the group consisting of 29 kV; (l) 29-30 kV; and (li)> 30 kV .

  The first potential and / or the second potential are: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii ) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii ) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22 -23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 preferably within a range selected from the group consisting of kV; (l) 29-30 kV; and (li)> 30 kV Arbitrariness.

  In the preferred method, when the ion mirror is in the first setting, ions having a certain mass-to-charge ratio and / or a certain energy penetrate at least a first distance through the ion mirror, and the ion mirror When the setting is 2, ions having the certain mass-to-charge ratio and / or the certain energy pass through the ion mirror at least at different second intervals.

  The difference between the first interval and the second interval is u% of the first interval or the second interval, and u is (i) <1; (ii) 1-2; (iii) 2- 3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x) 9-10 ; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; It is preferably within a range selected from the group consisting of (xviii) 45-50; and (xix)> 50.

  In the preferred method, the step of determining the first time of flight of the first fragment ion and the step of determining the second time of flight of the first fragment ion include the first time of flight or Identify, determine, identify, or search for a first fragment ion in mass spectral data, as well as identify, determine, and identify a corresponding first fragment ion in the second time-of-flight data Or include searching.

  In this embodiment, the step of identifying, determining, identifying or searching for the first fragment ion in the first time-of-flight or mass spectral data may be done manually and / or automatically. Preferably, the step of identifying, determining, identifying or searching for the first fragment ion in the second time-of-flight or mass spectral data is preferably done manually and / or automatically.

  The step of identifying, determining, identifying, or retrieving a first fragment ion in the first and / or the second time-of-flight or mass spectral data is in the first time-of-flight or mass spectral data. Preferably comparing the pattern of the isotope peak with a pattern of isotope peaks in the second time-of-flight or mass spectral data.

  In a preferred embodiment, the step of comparing the pattern of isotope peaks comprises comparing the relative intensity of the isotope peaks and / or the distribution of the isotope peaks. The step of identifying, determining, identifying, or searching for a first fragment ion in the first and / or the second time-of-flight or mass spectral data also includes the first time-of-flight or mass spectrum. Comparing or instead of the intensity of the ions in the data with the intensity of the ions in the second time-of-flight or mass spectral data may be included.

  The step of identifying, determining, identifying or retrieving a first fragment ion in the first and / or the second time-of-flight or mass spectral data results from the first time-of-flight or mass spectral data. Comparing the width of one or more mass spectral peaks in the first mass spectrum with the width of one or more mass spectral peaks in the second mass spectrum resulting from the second time-of-flight or mass spectral data It is preferable to include.

  The preferred method further comprises obtaining a parent ion mass spectrum. Preferably, the method further comprises determining the mass or mass to charge ratio of one or more parent ions from the parent ion mass spectrum.

  In this embodiment, the method may further comprise determining a time of flight of the one or more fragment ions from the first time of flight or mass spectral data. The method includes the mass or mass to charge ratio that a potential first fragment ion would have, the mass or mass to charge ratio of the parent ion determined from the parent ion mass spectrum, and a first flight. Preferably, the method further comprises predicting based on the time of flight of the fragment ions determined from time or mass spectral data.

  In another embodiment, the method comprises determining the mass or mass to charge ratio that a potential first fragment ion would have of one or more parent ions determined from the parent ion mass spectrum. Predicting based on the mass or mass to charge ratio and the time of flight of the one or more fragment ions determined from the first time of flight or mass spectral data.

  The method preferably includes determining a time of flight of the one or more fragment ions from the second time of flight or mass spectral data. This is because the potential second fragment ion would have the mass or mass to charge ratio, the mass or mass to charge ratio of the parent ion determined from the parent ion mass spectrum, and the second flight. Preferably, the prediction includes based on the time of flight of the fragment ions determined from time or mass spectral data.

  In another embodiment, the method includes determining the mass or mass to charge ratio that a potential second fragment ion would have from one or more of the parent ions determined from the parent ion mass spectrum. Predicting based on a mass to charge ratio and a time of flight of the one or more fragment ions determined from the second time of flight or mass spectral data.

  The preferred method is characterized in that the expected mass or mass to charge ratio of one or more possible first fragment ions is the expected mass of one or more possible second fragment ions. Comparing or contrasting with a mass or mass to charge ratio.

  The method may also include the predicted mass or mass-to-charge ratio of one or more of the potential first fragment ions such that the expected of one or more of the potential second fragment ions. In the first time-of-flight or mass spectral data as related to the same species of fragment ions in the second time-of-flight or mass spectral data Identifying, determining or identifying the fragment ions of x is (i) <0.001; (ii) 0.001-0.01; (iii) 0.01-0.1; (iv) 0.1-0.5; (v) 0.5-1.0; (vi) 1.0-1.5; (vii) 1.5-2.0 preferably within a range selected from the group consisting of: (viii) 2-3; (ix) 3-4; (x) 4-5; and (xi)> 5.

  Determining from the first and second time-of-flight the mass or mass-to-charge ratio of a parent ion that has been fragmented to yield the first fragment ion comprises the mass or mass-charge of the first fragment ion Preferably, the method comprises determining the mass to charge ratio of the parent ion that has been fragmented to yield the first fragment ion, either independently of the ratio information or without the need for the information.

  In a preferred embodiment, the parent ion that fragmented to yield the first fragment ion, independently of or without the information of the mass or mass to charge ratio information of the first fragment ion. The step of determining the mass or mass-to-charge ratio includes determining the expected mass or mass charge of the parent ion that has been determined that one or more parent ion mass peaks have fragmented to yield the first fragment ion. Determining from the parent ion mass spectrum whether it is observed within y% of the ratio. y is (i) <0.001; (ii) 0.001-0.01; (iii) 0.01-0.1; (iv) 0.1-0.5; (v) 0.5-1.0; (vi) 1.0-1.5; (vii) 1.5-2.0 (viii) 2-3; (ix) 3-4; (x) 4-5; and (xi)> 5.

  If one parent ion mass peak is observed within y% of the expected mass or mass to charge ratio of the parent ion determined to fragment to yield the first fragment ion, then the Preferably, the mass or mass to charge ratio of the parent ion mass peak is a more precise determination of the mass or mass to charge ratio of the parent ion that has been fragmented to yield the first fragment ion.

  In another embodiment, more than one parent ion mass peak is observed within y% of the expected mass or mass to charge ratio of the parent ion determined to fragment to yield the first fragment ion. If so, then a determination is made as to which observed parent ion mass peak corresponds to or is related to the parent ion most likely to fragment to yield the first fragment ion. In such a method, a determination as to which observed parent ion mass peak corresponds to or is related to the parent ion most likely to fragment to yield the first fragment ion is the ion Preferably, this is done by reference to the third time-of-flight or mass spectral data acquired when the mirror is held at a different third setting.

  The mass or mass-to-charge ratio of the observed parent ion mass peak corresponding to or related to the parent ion most likely to have fragmented to yield the first fragment ion is fragmented to produce the first fragment ion. It is preferable to have a more precise determination of the mass or mass to charge ratio of the parent ion that yields the fragment ions.

  In a preferred embodiment, a more precise determination of the mass or mass to charge ratio of the first fragment ion is made using the more accurate determination of the mass or mass to charge ratio of the parent ion.

From another aspect of the present invention, a mass spectrometer is provided, the mass spectrometer including a time-of-flight mass analyzer,
The time-of-flight mass analyzer includes an ion mirror, and in use, the ion mirror is initially maintained at a first setting to obtain a first time-of-flight or mass spectral data, and the ion mirror In a second time, kept at a different second setting to obtain a second time-of-flight or mass spectral data;
When the mass spectrometer is in use:
(A) a first time of flight of a first fragment ion having a certain mass or mass to charge ratio when the ion mirror is held at the first setting;
(B) a different second time of flight of first fragment ions having the same constant mass or mass to charge ratio when the ion mirror is held at the second setting;
(C) the mass or mass-to-charge ratio of the parent ion and / or the mass or mass-charge of the first fragment ion that fragmented from the first and second time of flight to produce the first fragment ion; Determine the ratio.

  According to a preferred embodiment, a MALDI time-of-flight mass spectrometer that includes PSD and / or CID fragment ion spectra from different parent ions, but does not require or require the use of a timed ion gate. Can be obtained simultaneously. Thus, according to the preferred embodiment, all the problems associated with normal placement requiring the use of timed ion gates can be prevented. A preferred method for interpreting the recorded data is also disclosed.

  According to a preferred embodiment, the voltage applied to the reflectron that forms part of the time-of-flight mass spectrometer is the simultaneous or otherwise post-source decomposition fragment resulting from fragmentation of the parent ion. Preferably, the ions are programmed to change in a particular sequence such that they can be acquired substantially simultaneously. The recorded data is then preferably processed to determine the fragment ion mass to charge ratio and to predict the corresponding parent ion mass to charge ratio for each of the observed fragment ions.

  With the preferred multiplex system, PSD data can be acquired much faster and with significantly less sample consumption than a normal system. The removal of the timed ion gate also results in a mass spectrometer that is inexpensive, has low manufacturing complexity, and is fairly easy to operate. Advantageously, the PSD data acquired according to a preferred embodiment is from all the parent ions in the sample, as in the case of a normal arrangement with a timed ion gate. Not only from ions. Thus, PSD data can be acquired with significantly less sample consumption and significantly improve the detection limit acquired with the preferred embodiment.

  In a preferred embodiment, the time of flight of PSD fragment ions is determined by subtracting the reflectron voltage from a first voltage level to a relatively close second voltage level. The second voltage level is preferably about 4-5% lower than the first voltage level. A relatively small change (eg, 4-5%) in the applied reflectron voltage is hereinafter referred to as the smaller decrease (or step). The greater change (eg, 25%) in the reflectron voltage used to optimally reflect different energy fragment ions is hereinafter referred to as the greater decrease (or step).

  Observed by acquisition of two similar mass spectra at two slightly different reflectron voltages (ie, the reflectron voltage changes only by a smaller decrease or step). It allows not only the mass-to-charge ratio of fragment ions, but also the corresponding parent ion (from which the fragment ions are derived and precisely determined).

  Once mass spectral data for ions having a particular range of energy is acquired, the reflectron voltage is then preferably reduced by a larger decrease or step. The process of precisely determining the parent and fragment ion mass to charge ratio is then preferably repeated. The reflectron voltage is then preferably reduced by another larger reduction amount or step, and the process is preferably repeated a number of times, so that ions are scattered across the important mass to charge ratio range. Are subjected to mass spectrometry.

  Although not as much as the previous embodiment, according to a preferred embodiment, there may be no step of reducing the reflectron voltage by a smaller reduction amount or step. Instead, for each of the observed fragment ions in the corresponding mass spectrum, using the selected data acquired after the reflectron voltage has been decreased by a continuous larger decrease or step. The parent and fragment ion mass to charge ratio may be calculated.

  Various embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings. The drawings are as follows.

A preferred embodiment will now be described with reference to FIG. FIG. 1 shows a preferred MALDI time-of-flight PSD mass spectrometer. The laser light 1 is preferably directed onto the sample or target plate 2 and is preferably kept at the voltage V _S. Ions are preferably generated in the sample or target plate 2 by a MALDI process. A two-stage delayed extraction (or time lag focus) device 3 may be provided between the sample or target plate 2 and a field-free or drift region 5, and if provided, one of the ion sources 4. It is thought to form a part. The delayed extraction device 3 preferably increases the energy of ions initially desorbed from the sample or target plate 2 with relatively low energy. Ions appearing from the ion source 4 are preferably accelerated into a field-free or drift region 5 arranged downstream of the ion source 4. The delayed extraction device 3 can cause slower ions to catch up with faster ions in the field-free or drift region 5 by increasing the energy of ions with less energy.

  The field-free or drift region 5 preferably includes a flight tube that may be grounded with respect to the ion source 4. However, according to a preferred embodiment, not as much as the other previous embodiments, the flight tube may be kept at a relatively high voltage and the ion source 4 may be grounded. According to other embodiments, the flight tube and / or ion source 4 may be maintained at other different potentials or voltages.

According to a preferred embodiment, the parent ions originating from the ion source 4 and passing through the field-free or drift region 5 preferably have a kinetic energy approximately equal to eV _s electron volts.

  Parent ions may be intentionally fragmented by CID (in an optional collision or fragmentation cell 6 that may be provided in the field free region 5). However, since the metastable parent ion passes through the field-free or drift region 5 without the aid of a collision or fragmentation cell 6, the metastable parent ion is additionally or alternatively naturally fragmented by PSD. More preferably.

  The fragment ions formed by CID and / or more preferably by PSD preferably emerge from said field-free or drift region 5 and then pass through ion mirror 7 or otherwise to ion mirror 7. It is preferable to enter. The ion mirror 7 preferably includes a reflectron. The ion mirror 7 can reflect at least some of the fragment ions from the inside of the ion mirror 7 toward the ion detector 8 (preferably arranged downstream of the ion mirror 7). Are preferably arranged. The ion detector 8 may include, for example, a microchannel plate ion detector.

  The ion mirror 7 initially causes fragment ions (which would have less kinetic energy than the corresponding unfragmented parent ion) to be substantially reflected by a delayed electric field within the ion mirror 7. While it may be held at voltage, potential, electric field strength or electric field gradient, unfragmented parent ions (which will have a relatively high kinetic energy) are not reflected by the ion mirror 7. Let's go. Thus, initially, relatively few or substantially no unfragmented parent ions are reflected by the ion mirror 7 and, therefore, if not all, most of the fragmented No parent ions can persist through the ion mirror 7 without being reflected, and thus may be arranged not to be of the system.

  Once the most energetic fragment ions are optimally reflected by the ion mirror 7 and then mass analyzed, the maximum ion mirror or reflectron voltage, potential, field strength or field gradient is then In a series of smaller and larger reduction amounts or steps, it is preferable to gradually reduce as described in more detail below. By reducing the reflectron voltage, potential, electric field strength or electric field gradient in this manner, less energy fragment ions can be optimally reflected by the ion mirror 7. With increasingly lower reflectron voltages, potentials, field strengths or field gradient settings, very few, if not, parent fragments, if any, will be reflected by the ion mirror 7. Thus, the resulting mass spectrum will be almost exclusively related to fragment ions.

  Said embodiment comprises changing the voltage, potential, electric field strength or electric field gradient of the ion mirror 7 or reflectron, while the voltage of the ion source 4 and / or the field free or drift region 5 Alternatively, the potential remains substantially constant, but according to other embodiments, the potential of the ion mirror 7 or reflectron is either the ion source 4 and / or the field free or drift region 5. More generally, may be varied. That is, the potential of the ion source 4 and / or the field-free or drift region 5 may vary, while, for example, the voltage, potential, electric field strength, or electric field gradient of the ion mirror 7 or reflectron is , May remain substantially constant. According to an embodiment, the potential of the ion source 4 and / or the field free or drift region 5 and / or the ion mirror 7 may vary.

  FIG. 2 shows how the ion mirror or reflectron voltage, potential, electric field strength or electric field gradient gradually decreases over time in a series of smaller and larger reductions according to a preferred embodiment. Explain. Initially, first time-of-flight or mass spectral data is acquired, while the ion mirror or reflectron 7 is relative to the potential of the field-free or drift region 5 (preferably maintained at ground). Therefore, it is preferable that the first voltage, the potential, the electric field strength, or the electric field gradient VR1 be maintained at a relatively high level. Since VR1 is relatively high, then the first time-of-flight or mass spectral data preferably includes a relatively large portion of energetic fragment ions. This is because the ion mirror 7 or reflectron is preferably set at a voltage, potential, electric field strength, or electric field gradient so that relatively energetic fragment ions are optimally reflected. Lower energy fragment ions are also reflected. Certain low energy parent ions may also be reflected by the ion mirror 7 and thus may be observed in the first time-of-flight or mass spectral data, and may, but not necessarily, be particularly Not intended.

  When the first time-of-flight or mass spectral data is used to generate a mass spectrum, then only a limited portion of the mass spectrum will yield potentially useful information. This is because the ion mirror 7 or reflectron was maintained at a voltage, potential, field strength or field gradient optimized to reflect fragment ions having a relatively narrow range of mass to charge ratios. Thus, the resulting time-of-flight or mass spectral data segment provides useful information, and this usable portion of the mass spectrum is preferably related to the relatively energetic fragment ions, and the less energetic parent. It may contain some of the ions.

  According to a preferred embodiment, once the first set of time-of-flight or mass spectral data is acquired, then the maximum reflectron voltage, potential, electric field strength or electric field gradient is then slightly lower. It is preferable to decrease the second voltage setting VR1 ′ by a smaller decrease amount (for example, only 4 to 5%). Since the reflectron voltage, potential, electric field strength or electric field gradient is not greatly reduced, then essentially the same fragment ions are still optimally reflected by the ion mirror 7 or reflectron. Will be done. Second time-of-flight or mass spectral data is then preferably acquired, while the ion mirror 7 or reflectron is held at this slightly lower second voltage, potential, electric field strength or electric field gradient VR1 ′. Be drunk. However, although essentially the same fragment ions are optimally reflected, the voltage, potential, electric field strength or electric field gradient applied to the ion mirror 7 or reflectron has been reduced so that a specific mass-to-charge ratio is achieved. An increase in the observed time of flight of ions having can be recognized. As a result, a difference in time of flight of ions having a specific mass to charge ratio at the two slightly different reflectron voltages, potentials, field strengths or field gradient settings VR1 and VR1 'will be observed. That difference in time of flight can be used to provide an accurate prediction or estimate of the mass-to-charge ratio of the parent ions that fragment to yield the observed fragment ions. This prediction or estimate of the mass to charge ratio of the parent ion can be obtained solely from the time-of-flight data associated with fragment ions and does not require a parent ion scan to be performed. In a manner similar to the first time-of-flight or mass spectral data, the second time-of-flight or mass spectral data segment will provide usable information. The useful portion of the second time-of-flight or mass spectral data generally preferably corresponds to essentially the same usable portion of the first time-of-flight or mass spectral data.

  First and second time-of-flight or mass at two slightly different reflectron voltages or slightly different potentials for the ion source 4 and / or field-free or drift region 5 (or field strength or field gradient) Spectral data acquisition makes it possible to calculate the mass to charge ratio of the fragment ions (which are optimally reflected by the ion mirror 7 or reflectron). Similarly, the mass-to-charge ratio of the parent ions that fragment to yield the fragment ions can also be determined precisely or additionally.

  In order to observe and identify fragment ions over a wide range of mass-to-charge ratios and determine the mass-to-charge ratio of the parent ions corresponding to such fragment ions, the maximum reflectron voltage is reduced to each of the smaller reduction amounts. After that, it is preferable to gradually reduce the larger reduction amount. Each of the larger reduction amounts may be, for example, a decrease in the reflectron voltage, potential, electric field strength or electric field gradient for the ion source 4 and / or field free or drift region 5 of about 25%, or the ion mirror 7. May include a decrease in the maximum potential.

  FIG. 2 after the ion mirror 7 or reflectron is held at the second voltage, potential, electric field strength or electric field gradient VR1 ′ and second time-of-flight or mass spectral data is acquired at this setting. In the particular example shown, the reflectron voltage, potential, electric field strength or electric field gradient is then reduced by a larger reduction amount, for example by a reduction of 25% with respect to the new third voltage VR2. It is preferable to do so. The third time-of-flight or mass spectral data is then preferably acquired with this third reflectron voltage, potential, electric field strength or electric field gradient VR2. Similar to the first smaller decrease (when the reflectron voltage, potential, electric field strength or electric field gradient is reduced from VR1 to VR1 ′), the reflectron voltage, electric potential, electric field strength or electric field gradient is At that time, it is preferable to decrease the same amount by the same smaller decrease amount (for example, only 4 to 5%) with respect to the fourth voltage, potential, electric field strength, or electric field gradient VR2 ′. The fourth time-of-flight mass spectral data is then preferably acquired with this fourth reflectron voltage, potential, electric field strength or electric field gradient VR2 '.

  The larger reduction amount (for example, 25% reduction amount) that sandwiches the decrease in the reflectron voltage, potential, electric field strength, or electric field gradient by the smaller reduction amount (for example, 4-5% reduction amount). The reflectron voltage, potential, electric field strength or electric field gradient reduction process is performed several times until sufficient time-of-flight or mass spectral data is acquired or obtained throughout the desired mass-to-charge ratio range. , Preferably continued. According to a preferred embodiment, the usable portion or segment of time-of-flight or mass spectral data acquired at each of the reflectron voltages or associated ion mirror potentials is obtained from each of the time-of-flight or mass spectral data sets. It may be selected. Multiple usable portions or segments of data can then be used to allow one or more composite mass spectra to be formed.

  Decreasing the relative potential of the ion mirror 7 or reducing the reflectron voltage (for example, only 25% for each of the larger reduction amounts) is the voltage ratio VR2 / VR1 = 0.75. Is shown in this example and is described in connection with FIG. Similarly, the voltage ratio VR3 / VR2 = 0.75, and more generally the voltage ratio VRN / VRN-1 = 0.75. Similarly, reducing the reflectron voltage by 4% for each of the smaller reductions means the voltage ratio VR1 '/ VR1 = 0.96. Similarly, the voltage ratio VR2 '/ VR2 = 0.96 and more generally the voltage ratio VRN' / VRN = 0.96.

  According to other embodiments, the larger reduction amount or step and / or the smaller reduction amount or step in the ion mirror or reflectron voltage or relative potential may be smaller or larger than the above. . For example, the smaller decrease or step in the ion mirror reflectron voltage, relative potential, potential, electric field strength or electric field gradient is <1%, 1-2%, 2-3%, 3-4%, 4- It may be 5%, 5-6%, 6-7%, 7-8%, 8-9%, 9-10% or> 10%. The greater reduction or step in the ion mirror or reflectron voltage, relative potential, potential, field strength or field gradient is <10%, 10-15%, 15-20%, 20-25%, 25-30. %, 30-35%, 35-40%, 40-45%, 45-50% or> 50%.

  According to an embodiment, in order to acquire a mass spectrum over the desired mass to charge ratio range, the ion mirror or reflectron voltage or relative potential is reduced by a 10-20 larger reduction or step, Each larger reduction amount or step may include a 10-20 smaller reduction amount or step, where appropriate. As a result, to obtain a complete PSD spectrum with sufficient data to determine all the mass to charge ratios of the fragment ions and their corresponding parent ions over the important mass to charge ratio range, The ion mirror reflectron voltage or relative potential may therefore be reduced, for example 20 to 40 times overall.

According to a preferred embodiment, the ion mirror or reflectron voltage or relative potential varies, preferably decreases, so that two (or more) independent sets of time-of-flight or mass spectral data are Acquired with slightly different ion mirror or reflectron voltages or relative potential settings. By solving two simultaneous equations by measuring two different times of flight T _f , T _f ′ for the same species of fragment ions, with two slightly different ion mirrors or reflectron voltages or relative potential settings It is possible to infer both the observed mass-to-charge ratio of the fragment ions and also the mass-to-charge ratio of the parent ion that fragmented to yield the fragment ions.

In a mass spectrometer incorporating a reflectron according to a preferred embodiment, the time of flight T _f of the fragment ions is given by:

Where M _p is the mass of the parent ion alone charged, M _d is the mass of the separately charged daughter ion or fragment ion observed, and the coefficients a and b are the mechanical coefficients. And depends on the specific voltage applied to the ion optics of the mass spectrometer and the dimensions of the mass spectrometer.

  The first part of the expression

Represents the time of flight of the fragment ions from the ion source 4 through the field-free or drift region 5 to the entrance to the ion mirror 7 or reflectron. Second part of the formula

Represents the further time of flight of the fragment ions that once enter the ion mirror 7 or reflectron, reverse direction and reflect back out of the ion mirror 7 or reflectron. The coefficient b is inversely proportional to the ion mirror or reflectron voltage or relative potential. Thus, as the ion mirror or reflectron voltage decreases, the fragment ions will spend longer in the ion mirror 7 reflectron and thus the factor b will increase.

  The coefficients a and b can be calculated if all machine parameters are known. However, it is more preferred that the coefficients a and b are measured or determined empirically using appropriate calibration compounds. For example, the time of flight of a number of known PSD fragment ions from the calibration compound at each of different ion mirror or reflectron voltages, relative potentials, potentials, field strengths or field gradient settings may be measured. The coefficients a and b are then preferably measured empirically for each of the different ion mirror or reflectron settings using the above formula. Until the first approximation, the coefficient a is considered constant with respect to the ion mirror or reflectron voltage or relative potential, and therefore the coefficient a is not necessarily recalculated for each ion mirror or reflectron voltage setting. There is no need.

  When the ion mirror or reflectron voltage or relative potential is decreased by a smaller decrease or step (eg, by a decrease of 4-5%), with a corresponding increased coefficient b ′, The longer flight time Tf ′ at which fragment ions occur can then be measured. Therefore, the three coefficients a, b and b 'can be measured based on experiments. Once these mechanical coefficients are determined for more than one, two or two ion mirror or reflectron voltages, relative potentials, potentials, field strengths or field gradient settings, then PSD spectra from unknown substances (ie , Time of flight or mass spectral data) can then be acquired. The PSD spectrum for an unknown material may be acquired with substantially the same ion mirror or reflectron voltage or relative potential settings used in calibration. However, according to other embodiments, the PSD data of the unknown sample is an ion mirror or reflectron voltage that is slightly or substantially different from the voltage or relative potential setting at which the mechanical coefficient is determined, or It may be obtained with a relative potential setting. Accordingly, the machine coefficients a, b and b 'may be determined with reference to calibration curve interpolation or calibration curves. Once the mechanical coefficient is determined, the PSD spectrum (ie, time of flight or mass spectral data) is then analyzed to determine the mass to charge ratio of the observed fragment ions, and / or Alternatively, the mass to charge ratio of the parent ion (from which the fragment ion was derived) can be determined.

When the ion mirror or reflectron voltage or relative potential changes (eg, decreases), then the change that occurred in the time of flight for a particular species of fragment ion? It will be appreciated that T _f (eg, increase) is proportional to the change in coefficient b, which depends on the ion mirror or reflectron voltage or relative potential.

In the above formula,? b = b′−b. T _f ? Since T _f , a, b, and b ′ (hence,? B) are known, at that time, by solving the two simultaneous equations, the mass-to-charge ratio M _{d of the} fragment ions and the corresponding parent Both ion mass-to-charge ratios M _p can be determined. The parent ion mass to charge ratio Mp and the fragment ion mass to charge ratio M _d are as follows.

By predicting or estimating the mass to charge ratio of the parent ions that have been fragmented to yield the observed fragment ions, a normal parent ion mass spectrum may then be acquired, acquired, or referenced. The parent ion mass to charge ratio predicted based on the PSD acquisition of the fragment ions may then be matched or compared with the parent ion observed in the parent ion mass spectrum. The mass to charge ratio of the parent ion is predicted, and then the predicted parent ion is matched with the actual parent ion in the parent ion mass spectrum, where the mass to charge ratio M _{p of the} parent ion in the above formula is By using the determined value based on the experiment, it is possible to improve the determination of the mass-to-charge ratio M _d of the fragment ions. As a result, both the mass to charge ratio of the parent ion and the mass to charge ratio of its corresponding fragment ion can be determined very precisely.

  To illustrate the effectiveness of the preferred embodiment, a 10 pmol tryptic protein digest of alcohol dehydrogenase (ADH1 (yeast)) obtained from Waters Inc. (Milford, USA) was analyzed.

  FIG. 3 shows a calibrated parent ion mass spectrum of the various peptide ions resulting from digestion of ADH. The parent ion mass spectrum was acquired and calibrated in the usual manner.

  Prior to analyzing a sample of ADH according to a preferred embodiment, the mass spectrometer was first calibrated. To calibrate the mass spectrometer for multiplex PSD, 10 pmol of a single specific peptide ACTH (adrenocorticotropic hormone, clip 18-39) was loaded. ACTH was used because the PSD fragmentation spectrum for ACTH was known from studies based on previous experiments. At that time, a first PSD fragmentation mass spectrum of ACTH was acquired and a second PSD fragmentation mass spectrum was acquired by reducing the reflectron voltage by a smaller decrease of approximately 4%.

  FIG. 4A shows an uncalibrated mass spectrum segment acquired when a (maximum) voltage of 13000 V is applied to the reflectron 7 of a mass spectrometer, according to a preferred embodiment. The reflectron voltage, potential, field strength or field gradient was such that only a few PSD fragment ions were optimally reflected by the reflectron 7. FIG. 4B shows that when the voltage, potential, electric field strength or electric field gradient subsequently applied to the reflectron decreases by a smaller decrease of approximately 4% with respect to the (maximum) voltage of 12500 V, A corresponding uncalibrated mass spectrum segment acquired is shown. The acceleration voltage for the data shown in FIGS. 4A and 4B was 14059V. The portion or segment of the time-of-flight or mass spectral data shown in FIGS. 4A and 4B corresponds to energetic fragment ions as optimally concentrated by the reflectron 7.

The x-axis scales shown in FIGS. 4A and 4B are arbitrary units that are uncalibrated and proportional to the square root of the flight time of the fragment ions. For a given known fragment peak or fragment ion, the calibration factor when the reflectron voltage is set to 13000 V using the time of flight T _f , T _f ′ at two different reflectron voltages (13000 V and 12,500 V). a and b were calculated, and the calibration factors a and b ′ were calculated when the reflectron voltage was set to 12500V. Therefore, the mechanical coefficients a, b, b ′ and? b was determined for both reflectron voltage settings.

  Once the mass spectrometer is calibrated with the ACTH sample at two different reflectron voltages, potentials, field strengths or field gradient settings, the ADH sample is then analyzed and the method of the preferred embodiment is Can be identified as being ADH. A sample of the ADH digestion product is loaded onto the sample or target plate 2 of the mass spectrometer according to a preferred embodiment, and time-of-flight or mass spectral data is calibrated using the ACTH sample. Acquired under the same experimental conditions used for For the analysis of the ADH sample at 13000V and 12,500V reflectron voltages, two resulting uncalibrated mass spectra are shown in FIGS. 5A and 5B, respectively.

The x-axis scales in FIGS. 5A and 5B are uncalibrated and simply indicate arbitrary units proportional to the square root of the flight time of the fragment ions. Times of flight T _f , T _f ′ for fragment peaks or fragment ions of the same species, so? The value of T _f was determined after first determining and identifying or contrasting matching fragment peaks or corresponding fragment ions in the two mass spectra. Some of the peaks that have been determined to correspond or correspond to the same type of fragment ion are shown in FIGS. 5A and 5B connected by arrows. The mass to charge ratio of the fragment ions and the corresponding parent ion mass to charge ratio were then calculated for each of the observed fragment ions.

  The process of identifying peaks or fragment ions in the two different mass spectra (obtained at slightly different ion mirror reflectron voltages or relative potentials) corresponding to or related to fragment ions of the same species May be performed by visual inspection or more preferably by automatic determination.

  If the ion mirror or reflectron voltage, relative potential, potential, electric field strength or electric field gradient decreases by a smaller decrease or step (eg 4-5% decrease or step) then a certain constant It is known that fragment ions having a mass to charge ratio will now spend a longer time in the ion mirror 7 or reflectron. Thus, the observed mass peaks corresponding to the fragment ions all appear to be shifted in the same direction, ie longer flight time. Based on similarities in the height and / or width of the observed mass peaks in the two mass spectra, the peaks are similar in relation to the fragment ions of the same species in the two different mass spectra. In addition, it can additionally be identified or adapted. According to a particularly preferred embodiment, fragment ions of the same species can be distinguished in the two mass spectra by comparing or contrasting the pattern of isotope peaks in the two mass spectra.

  The accuracy of the mass-to-charge ratio of the expected parent ion, determined solely from the PSD (ie, time of flight or mass spectrum) fragment ion data for the ADH sample, is described in more detail below with respect to the results shown in FIG. As explained, it was determined to be +/- 1% if not better. Such an error window is comparable to the parent ion resolution obtained using a conventional mass spectrometer with an ion gate. However, the same level of accuracy was advantageously obtained using a mass spectrometer without an ion gate.

  According to a preferred embodiment, for each fragment peak or fragment ion, its corresponding parent ion mass to charge ratio was predicted. The most intensely observed experimentally in the corresponding normally acquired parent (or precursor) ion mass spectrum (eg, located within 1% or 2% error window for the expected parent ion mass to charge ratio) It is preferred that a strong peak or parent ion is assumed to correspond to the expected parent ion. The mass to charge ratio of the parent ion determined to correspond to the expected parent ion and determined experimentally from the parent ion mass spectrum is then assumed to be the most accurate value of the mass to charge ratio of the parent ion. Also good. The parent ion mass-to-charge ratio precisely determined empirically may then be considered particularly precise, in which case the mass of the observed fragment ions can be determined using or reverting to the above simultaneous equations. The charge ratio can be determined more precisely. The accuracy of the mass measurement of the fragment ions according to this approach is at least as accurate if not less accurate than is possible with a conventional mass spectrometer. Typical errors in determining the fragment ion mass are less than 1 Dalton and preferably less than 0.5 Dalton.

  According to a more preferred embodiment, the data from the parent ion mass spectrum corresponds to fragment ions of the same species in the two mass spectra acquired at slightly different ion mirror or reflectron voltage or relative potential settings, Or it may be used to identify associated mass peaks. The parent ion mass spectrum may be analyzed, for example, to provide a list of known parent ion mass to charge ratios. Fragments observed in the first mass spectrum acquired at the first ion mirror or reflectron voltage or relative potential, respectively, using the parent ion mass to charge ratio determined empirically, then in the above simultaneous equations, respectively. Some or all of the theoretically possible mass to charge ratios that each of the ions would have may be calculated based on the determined time of flight of a particular fragment ion. Similarly, each of the parent ion mass-to-charge ratios determined empirically can be used to determine whether each of the fragment ions observed in the second mass spectrum acquired at the second ion mirror or reflectron voltage or relative potential. , Some or all of the theoretically possible mass-to-charge ratios that you might have may be calculated based on the determined time of flight of a particular fragment ion. Thus, for each observed fragment ion, a whole series of theoretically possible candidate fragment ion mass to charge ratios may be calculated. The number of theoretically possible candidate fragment ion mass to charge ratios preferably corresponds to the number of parent ions observed. Comparing each other within a specific mass-to-charge ratio window compatible with the expected accuracy of mass-to-charge ratio measurements by comparing a list of theoretically possible candidate fragment ion mass-to-charge ratios for both mass spectra It is then possible to look for a theoretically possible fragment ion mass to charge ratio in each of the mass spectra. In this way, the identification of fragment ions of the same species in two mass spectra acquired with slightly different ion mirror or reflectron voltages or relative potentials can be more easily automated.

  To illustrate a preferred process for identifying that fragment ion mass peaks in two mass spectra acquired with slightly different ion mirror or reflectron voltages or relative potentials coincide with fragment ions of the same species, FIG. Each of the fragment ions observed in the mass spectrum resulting from the PSD of the peptide ion derived from ADH as shown in FIGS. 5B and 5B is the parent peptide of the tryptic digest product of the ADH protein as shown in FIG. It may be assumed that it originates from one of the four most intense parent peptide ions observed in the ion mass spectrum. By applying the simultaneous equations, four different tentative fragment ion mass-to-charge ratios may be suggested for each of the observed fragment ions in the mass spectra shown in FIGS. 5A and 5B. However, only one of the four tentative fragment ion mass-to-charge ratios will be accurate in practice.

  According to a preferred embodiment, matching the expected fragment ion mass to charge ratio within a certain tolerance (eg, within +/− 1 dalton) may be required for the same candidate parent ion. The fragment ion mass charges that are most closely matched for the same parent ion indicate an exact match.

  In certain instances, for example, if there are a large number of different parent ions, it is possible that two unrelated fragment ions will obviously appear (incorrectly) in relation to the same parent ion. However, such potential inaccurate identification is preferably prevented, for example, by also comparing the peak intensity and / or peak sharpness or profile from the two fragmentation mass spectra. Inaccurate identification may additionally or alternatively correspond to a second or further smaller decrease or step of the ion mirror or reflectron voltage, relative potential, potential, electric field strength or electric field gradient, It may also be prevented by acquiring (or further) PSD mass spectra. That is, each of the larger reductions in the ion mirror or reflectron voltage or relative potential, in the same way as in the preferred embodiment, sometimes results in two or more smaller reductions rather than just one. May be put in. The third (or further) time-of-flight data or data from the mass spectrum is then processed and used in the same way, or else confirming the results from the two first PSD mass spectra. Also good. A third (or further) time-of-flight data or mass spectral data set may also be used to resolve the two fragment peaks if they overlap in one of the mass spectra.

  FIG. 6 illustrates three parent peptide ions and corresponding fragment ions observed from analyzing an ADH peptide mixture, according to a preferred embodiment. The calculated mass of each fragment ion based on the experiment was compared against the theoretical (or textbook) mass of the fragment ion. The theoretical (or textbook) mass of the fragment ions was calculated from their known sequence. The parent and fragment ions were also matched to the theoretically derived peptide fragment mass using MASCOT (RTM) database search software from Matrix Science Ltd, UK. ADH1_yeast was clearly identified from experimental PSD fragmentation data. Probability based on a Mowse score of 81 indicated that the fragmentation data presented almost certainly from ADH since a score> 32 indicates the first positive identification of the protein. The confident identification of the protein is due to the identification of the fragmentation data. Identification of the protein (ie, presenting only the three parent ion masses) by peptide mass fingerprinting alone was not possible using MASCOT (RTM).

  FIG. 7 shows a multiplex PSD spectrum of uncalibrated ADH with comments. The spectrum shows the different fragment ions formed due to the PSD of the three parent peptide ions listed in FIG. 6 and adapted using MASCOT (RTM). The x-axis scale is uncalibrated and simply indicates arbitrary units proportional to the square root of the flight time of the fragment ions. In this example, the data was acquired by reducing the reflectron voltage by a smaller decrease of 4%. A number of different fragment ions were observed and identified. The reflectron voltage is gradually reduced by a larger decrease of 25%, so that fragment ions having a lower mass-to-charge ratio (ie, lower energy fragment ions) are gradually introduced into the ion mirror 7 or It was collected optimally by the reflectron.

  A mixture of two peptides angiotensin (MH + 1296.7) and substance P (MH + 1347.7) with a fairly similar mass to charge ratio was also analyzed according to the preferred embodiment. Both peptides were similarly uniquely identified in an obvious manner by putting the PSD fragmentation data into MASCOT (RTM).

  Another experiment was performed with a tryptic digest of what was originally thought to be the protein ADH1. When the parent ion mass spectrum of the sample was acquired, the resulting mass spectrum showed a strong peptide peak at (MH + 2477.1). However, the expected parent ion spectrum for ADH1 is well known (see FIG. 3) and when the sample is related to the digestion product of ADH1, no parent ion with a mass to charge ratio of 2477.1 is observed. This is clear from FIG. Therefore, the sample cannot be thanks to trypsin digestion of ADH1. After further analysis using a mass spectrometer according to a preferred embodiment, the resulting PSD fragmentation data was used to unambiguously identify the trypsin digestion product in relation to the protein ADH2. ADH2 is similar to ADH1 except that the amino acid is slightly different in a part of the protein sequence. A normal MALDI MS / MS experiment was then performed using a mass filter, when a particular parent ion was selected that fragmented to yield MS / MS mass spectral data. These experiments confirmed that the sample was ADH2, not ADH1 originally thought.

  Data based on further experiments will report here which emphasizes the power of the preferred embodiment and uniquely identifies the sample with minimal sample consumption. Six segment multiplex PSD fragmentation data was obtained from tryptic digestion of 5 pmol ADH. The PSD fragmentation data was then entered into a peak fit and parent ion identification algorithm. A list of parent ions obtained from the parent ion scan was also obtained. A fragmentation ion peak list was generated and then searched against the database using MASCOT (RTM) ion search (Matrix Science). MASCOT (RTM) correctly identified ADH with the prospect of being based on a Mowse score of 190 (which shows very high (ie, obvious) certainty).

  To obtain this fit, MASCOT (RTM) correctly identified the five parent peptides from ADH, all of which are top-ranking, i.e. they are all independently against the data in the database. It was the best fit. These five parent peptides are shown in FIG. Note that three of these five parent peptide ions are shown and described above with respect to FIG.

  Using a preferred multiplex technique, fragmentation data was acquired for a parent peptide ion having an integer mass of 2312 Da and the sequence ATDGGAHGVINVSVSEAIAESTR to further demonstrate the quality of the data that can be obtained. The resulting fragmentation data adapted with MASCOT (RTM) is shown in FIG.

  An advantageous feature of the preferred multiplex technique is that a substantial amount of noise is preferably filtered out of the fragmentation mass spectrum. The noise is reduced because certain fragment ions must be observed at the exact location in the two related fragmentation mass spectra, so noise peaks that occur simultaneously in this way are only with low statistical probability. Due to the fact that it is clear that it is unlikely to happen. Consequently, as can be seen from the fragmentation data shown in FIG. 9, the ratio of correctly identified peaks to the total number of observed peaks presented is very high.

  In this particular experiment, only six segments of PSD fragmentation data are recorded, i.e., the reflectron voltage is at six major reductions, with six smaller reductions at its core. , Went down. PSD data was acquired every time the reflectron voltage dropped. According to another embodiment, 12 or more segments of PSD fragmentation data (i.e., the reflectron voltage is twelve larger reductions, where twelve smaller reductions are in place). May be acquired to acquire fragmentation mass spectral data over an important typical mass range (which may have dropped). Nevertheless, the six segments were sufficient to obtain an effective range of approximately 70% of the important mass range, and were easy enough to identify samples related to ADH by category. To further illustrate this, FIG. 10 shows all fragments that theoretically arise from the fragmentation of the parent peptide derived from ADH with an integer mass of 2312 daltons. FIG. 10 also highlights these theoretical fragments that fit exactly to the fragment ions observed based on experiments. As can be seen, 16 of the 23 possible y-series fragment ions were precisely matched. A significant number of b-series fragment ions were also compatible. The ability to fit such a large number of the fragment ions to the theoretical data demonstrates that proteins can be identified with a very high level of confidence, according to preferred embodiments.

  The peak fitting and parent identification algorithm used by the preferred embodiment is repeated through each of the peaks in the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is decreased by a smaller decrease, and then , Acquired when the ion mirror or reflectron voltage or relative potential is a slightly higher voltage or relative potential, i.e., the ion mirror or reflectron voltage or relative potential before decreasing by the smaller decrease amount. It is preferred to attempt to match these peaks with the peaks in the fragment ion spectrum. Instead, a preferred algorithm is repeated through each of the peaks in the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is reduced by a larger reduction, after which the ion mirror or Fragment ion spectrum acquired when the reflectron voltage or relative potential is decreased by a smaller decrease, i.e., at the ion mirror or reflectron voltage or relative potential before decreasing by a larger decrease. An attempt may be made to match these peaks with the peaks at. The algorithm then identifies the parent ion for each pair of fitted peaks, eg, as described below.

  For at least some of the parent ions acquired from a parent ion scan, corresponding to the peak from the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is decreased by the smaller decrease Considering a single fragment ion, an estimate of the time of flight of the corresponding fragment ion in the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is slightly higher may be made. Thus, when there are 10 parent ions, then estimate 10 to the corresponding fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is a slightly higher voltage or relative potential. May be performed for the time of flight of the corresponding fragment ions at. These 10 estimates are then compared with, for example, the actual time of flight of the fragment ions measured when the ion mirror or reflectron voltage or relative potential was a slightly higher voltage or relative potential. Also good. Any of these fragment ions, found within a pre-determined tolerance of 10 estimates (eg, tolerance on the order of +/− 150 ppm), is then considered to be a potential accurate match. preferable. Several potential accurate matches are found, so additional criteria may be used to determine if the potential fit is accurate. According to one embodiment, the peak from the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is reduced by a smaller reduction amount is the ion mirror or reflectron voltage or relative While the potential may be matched to the strongest potential-matching peak from the fragment ion spectrum acquired when it is a slightly higher voltage or relative potential, other methods of determining an exact fit can be used. It may be used.

  Some peaks from the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is decreased by a smaller decrease are the voltages that the ion mirror or reflectron voltage or the relative potential is slightly higher. Or it is possible to fit all to the same single peak from the fragment ion spectrum acquired when at relative potential. This is sometimes true, since the two peaks in the fragment ion spectrum can overlap (ie, they can probably not be resolved from each other in one of the spectra), but the law Rather, it is probably an exception. In order to prevent such complex fitting (inaccurate certainty), the process of fitting the peaks is slightly higher in the ion mirror or reflectron voltage or relative potential using the same fitting method as described above. Fit the fragment ion spectrum acquired when it is at voltage or relative potential to the peak from the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is decreased by the smaller decrease You may request further. In this embodiment, the pair of peaks from the two fragment ion spectra is the peak from the fragment ion acquired when the ion mirror or reflectron voltage or the relative potential is a slightly higher voltage or relative potential. Only when the ion mirror or reflectron voltage or relative potential is reduced by the smaller decrease and reversed to match the peak from the fragment ion obtained when it is reversed, it is determined to be exactly matched .

  The matched pair of fragment ions may then be used to estimate the parent ions from which they were generated. Any experimentally observed parent ion within a pre-determined tolerance (eg, +/− 1.5% of the expected parent mass) may be considered potential matched. In the same manner as above, the matched pair of fragment ions may be matched with the strongest potential-matching parent ion. Once this is complete, the mass-to-charge ratio of the parent ion matched to the pair of fragment ion peaks is used to calibrate the mass-to-charge ratio of the two matched fragment ion peaks, and the mass-to-charge ratio of the same fragment ions Two measurements that are preferably slightly different may occur. The average of the two mass to charge ratios of the two peaks and their respective intensities may then be determined.

  The monoisotopic mass is preferably measured for the parent ion observed based on experimentation. However, according to a preferred embodiment, only the average mass to charge ratio may be measured for PSD fragment ions, although not as much as in the previous embodiment where the resolution of the PSD fragmentation data is relatively low. The majority of database search engines, including MASCOT (RTM), require either an average mass for both parent and fragment mass, or a monoisotopic mass for both parent and fragment mass, i.e. they are The monoisotopic mass is not allowed to be used for the parent ion while the average mass is used for the fragment ion. Therefore, if necessary, it is preferable to apply the function to the average mass and convert it to the monoisotopic mass. This function can be obtained empirically by plotting the monoisotopic mass as a function of average mass for a number of common peptides. Different types of compounds (eg, polymers, sugars, etc.) may require different functions to be applied due to their specific isotope composition.

  Various further optimizations may be performed to further improve the speed of the preferred method, but the optimization does not directly affect the fitting process. For example, during the adaptation process, the peak from the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is reduced by the smaller decrease is represented by the ion mirror or reflectron voltage or relative potential. To fit the peak from the fragment ion spectrum acquired when it is at a slightly higher voltage or relative potential (with a smaller estimated mass or time of flight (since this is an inherent property of the multiplex technique)) It is preferable to simply try. This is preferred because fragment ions of the same species have a shorter flight time when the ion mirror or reflectron voltage, relative potential, potential, electric field strength or electric field gradient is increased. Thus, fragment ions of the same species are compared with the ion mirror or reflectron voltage compared to the fragment ion spectrum acquired when the ion mirror or reflectron voltage or relative potential is decreased by a smaller decrease. Or it will be detected in a shorter flight time in the fragment ion spectrum acquired when the relative potential is a slightly higher voltage or relative potential. Similarly, only peaks from fragment ion spectra corresponding to fragment ions having a mass to charge ratio within an optimally concentrated region of the ion mirror 7 or reflectron may be considered in the fitting process.

According to various embodiments, a method for determining whether a potential fit is an exact fit once some of the potential matches between the peak from the fragment ion spectrum and the parent ion are obtained. ,
(I) fitting a peak from a fragment ion spectrum to the strongest peak from another fragment ion spectrum, and then fitting one of these matched peaks to the strongest parent ion peak;
(Ii) fitting a peak from a fragment ion spectrum to the strongest parent ion peak and then fitting one of these peaks to the strongest fragment ion peak from another fragment ion spectrum. ;
(Iii) fitting a peak from a fragment ion spectrum to the one closest to the estimate of that peak [each peak estimate is derived from the corresponding peak for another fragment ion spectrum and a different parent ion peak Acquired];
(Iv) fitting a peak from a fragment ion spectrum to the strongest peak of another fragment ion spectrum, and then fitting one of these peaks to the closest estimate of the parent ion peak; And (v) fitting the peak from the fragment ion spectrum to the most intense parent ion peak and then fitting to the closest estimate of the fragment ion peak from another fragment ion spectrum.

  Embodiments may also contemplate using different instrument coupling structures. For example, a non-linear electric field reflectron may be used according to a preferred embodiment, although not as much as the previous embodiment.

  According to a preferred embodiment, the ion mirror or reflectron voltage or relative potential gradually decreases in use. However, this is not a problem and the ion mirror or reflectron voltage, relative potential, potential, electric field strength or electric field gradient is initially set relatively low, after which energetic fragment ions are increasingly optimally concentrated. Other embodiments that increase progressively to be reflected by the ion mirror 7 or reflectron are contemplated.

  Furthermore, although not as much as the previous embodiments, preferred embodiments are contemplated. In that embodiment, the ion mirror or reflectron voltage, relative potential, potential, electric field strength or electric field gradient is reduced in another manner (which may be linear or non-linear) or in virtually any manner. And / or increase. Thus, fragmentation data over some or all of the important mass or mass-to-charge ratio ranges can be used to maintain the maximum voltage or the maximum relative potential (at that voltage, the ion mirror 7 or the reflectron in multiple stages. As a result, it is clear that the fragment ions with different energies are preferably obtained by changing all of them optimally concentrated one after the other. The available data can then be used to form one or more hybrid mass spectra. However, the exact order of obtaining the segments of usable data may be different.

  Although the invention has been described with reference to preferred embodiments and other arrangements, various changes in form and detail may be made without departing from the scope of the invention as set forth in the claims. Will be understood by those skilled in the art.

FIG. 1 shows a MALDI time-of-flight mass spectrometer according to a preferred embodiment. FIG. 2 shows the electrical potential at which the ion source, field free region and reflectron are maintained according to a preferred embodiment. FIG. 3 shows a parent mass ion spectrum obtained using a conventional mass spectrometer of the parent peptide ion formed by digesting ADH by trypsin action. FIG. 4A shows a first uncalibrated PSD mass spectrum of the PSD fragment of the trypsin digestion product of ACTH acquired at a first reflectron voltage, and FIG. 4B shows that the reflectron is a second reflectron. FIG. 5 shows the corresponding second uncalibrated PSD mass spectrum of the PSD fragment of the tryptic digest product of ACTH, acquired when held at voltage (4% lower than the first reflectron voltage). FIG. 5A shows an uncalibrated PSD spectrum of the PSD fragment of the trypsin digestion product of ADH, acquired at a first reflectron voltage, and FIG. 5B shows that the reflectron has a second reflectron voltage (the first reflectron voltage). FIG. 4 shows the corresponding second uncalibrated PSD mass spectrum of the PSD fragment of the trypsin digestion product of ADH, obtained when held at 4% below 1 reflectron voltage. FIG. 6 shows the masses of three observed parent peptide ions obtained from digests of ADH and the corresponding observed fragment ion masses (which make the protein uniquely identifiable). Is sufficient). FIG. 7 shows commented uncalibrated mass spectra showing various PSD fragment ions due to the fragmentation of the three peptide ions derived from ADH listed in FIG. FIG. 8 shows five of the parent peptide ions obtained from tryptic digestion of ADH that were then accurately identified according to the preferred embodiment. FIG. 9 shows experimental MS / MS or fragmentation mass spectral data obtained by the preferred embodiment in connection with the fragmentation of the parent peptide ion of ADH having an integer mass of 2312 Da. FIG. 10 shows a, b and y series of fragment ions corresponding to the fragmentation of the parent peptide ion with an integer mass of 2312 Da, derived from trypsin digestion of ADH.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Laser beam 2 Sample or target plate 3 Delay extraction device 4 Ion source 5 Field free or drift area 6 Collision or fragmentation cell 7 Ion mirror 8 Ion detector

Claims (101)

A method of mass spectrometry,
Providing a time-of-flight mass analyzer including an ion mirror;
Keeping the ion mirror at a first setting;
Acquiring a first time-of-flight or mass spectral data when the ion mirror is in the first setting;
Keeping the ion mirror at a different second setting;
Acquiring a second time-of-flight or mass spectral data when the ion mirror is in the second setting;
Determining a first time of flight of a first fragment ion having a certain mass or mass to charge ratio when the ion mirror is in the first setting;
Determining a different second time of flight of first fragment ions having the same constant mass or mass to charge ratio when the ion mirror is in the second setting; and fragmenting the first Determine either the mass or mass to charge ratio of the parent ion that produced the fragment ion and / or the mass or mass to charge ratio of the first fragment ion from the first and second times of flight. A method comprising:   The method of claim 1, wherein the ion mirror comprises a reflectron.   3. The method of claim 2, wherein the reflectron comprises a linear electric field reflectron or a non-linear electric field reflectron.   A method according to any preceding claim, further comprising providing an ion source and a drift or flight space upstream of the ion mirror, wherein the ion mirror is at the first setting, When a first potential difference is maintained between the ion source and the drift or flight space and the ion mirror is at the second setting, a second potential difference is between the ion source and the drift or flight space. How to be kept in.   5. The method according to claim 4, wherein the first potential difference is substantially the same as the second potential difference.   The method according to claim 4, wherein the first potential difference is substantially different from the second potential difference.   7. The method according to claim 6, wherein the difference between the first potential difference and the second potential difference is p% of the first potential difference or the second potential difference, and p is (i) <1; ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix ) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a method within a range selected from the group consisting of> 50.   The method according to claim 6 or 7, wherein the difference between the first potential difference and the second potential difference is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x ) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700 -750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix ) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14 -15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 30 kV A method selected from the group.   The method according to any one of claims 6, 7 and 8, wherein the first potential difference and / or the second potential difference are: (i) <10 V; (ii) 10-50 V; (iii) 50 -100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1 -2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi ) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26 -27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; Beauty (li)> method is within a range selected from the group consisting of 30 kV.   A method according to any of the preceding claims, wherein when the ion mirror is at the first setting, the first electric field strength or electric field gradient is at least 10%, 20% of the length of the ion mirror. 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% and when the ion mirror is in the second setting, the second The electric field strength or gradient is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the ion mirror How to be kept.   11. The method of claim 10, wherein the first electric field strength or electric field gradient is substantially the same as the second electric field strength or electric field gradient.   11. The method of claim 10, wherein the first electric field strength or electric field gradient is substantially different from the second electric field strength or electric field gradient.   13. The method of claim 12, wherein a difference between the first electric field strength or electric field gradient and the second electric field strength or electric field gradient is a q of the first or second electric field strength or electric field gradient. % And q is (i) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii ) 6-7; (viii) 7-8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) A method within a range selected from the group consisting of 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50 .   The method according to claim 12 or 13, wherein the difference between the first electric field strength or electric field gradient and the second electric field strength or electric field gradient is: (i) <0.01 kV / cm; (ii) 0.01-0.1 kV / cm; (iii) 0.1-0.5 kV / cm; (iv) 0.5-1 kV / cm; (v) 1-2 kV / cm; (vi) 2-3 kV / cm; (vii) 3-4 kV / cm; (viii) 4-5 kV / cm; (ix) 5-6 kV / cm; (x) 6-7 kV / cm; (xi) 7-8 kV / cm; (xii) 8-9 kV / cm; (xiii) 9-10 kV / cm; (xiv) 10-11 kV / cm; (xv) 11-12 kV / cm; (xvi) 12-13 kV / cm; (xvii) 13-14 kV / cm; (xviii) 14-15 kV / cm; (xix) 15-16 kV / cm; (xx) 16-17 kV / cm; (xxi) 17-18 kV / cm; (xxii) 18-19 kV / cm; (xxiii) 19-20 kV / cm; (xxiv) 20-21 kV / cm; (xxv) 21-22 kV / cm; (xxvi) 22-23 kV / cm; (xxvii) 23-24 kV / cm; (xxviii) 24-25 kV / cm; (xxix) 25-26 kV / cm; (xxx) 26-27 kV / cm; (xxxi) 27-28 kV / cm; (xxxii) 28-29 kV / cm; (xxxiii) 29-30 kV / cm; and (xxxiv)> 30 kV / cm.   15. The method according to claim 12, 13 or 14, wherein the first electric field strength or electric field gradient and / or the second electric field strength or electric field gradient is: (i) <0.01 kV / cm; (ii) 0.01-0.1 kV / cm; (iii) 0.1-0.5 kV / cm; (iv) 0.5-1 kV / cm; (v) 1-2 kV / cm; (vi) 2-3 kV / cm; (vii) 3-4 kV / cm; (viii) 4-5 kV / cm; (ix) 5-6 kV / cm; (x) 6-7 kV / cm; (xi) 7-8 kV / cm; (xii) 8-9 kV / cm; (xiii) 9-10 kV / cm; (xiv) 10-11 kV / cm; (xv) 11-12 kV / cm; (xvi) 12-13 kV / cm; (xvii) 13-14 kV / cm; (xviii) 14-15 kV / cm; (xix) 15-16 kV / cm; (xx) 16-17 kV / cm; (xxi) 17-18 kV / cm; (xxii) 18-19 kV / cm; (xxiii) 19-20 kV / cm; (xxiv) 20-21 kV / cm; (xxv) 21-22 kV / cm; (xxvi) 22-23 kV / cm; (xxvii) 23-24 kV / cm; (xxviii) 24-25 kV / cm; (xxix) 25-26 kV / cm; (xxx) 26-27 kV / cm; (xxxi) 27-28 kV / cm; (xxxii) 28-29 kV / cm; (xxxiii) 29-30 kV / cm; and (xxxiv)> within a range selected from the group consisting of> 30 kV / cm.   The method according to any of the preceding claims, wherein when the ion mirror is at the first setting, the ion mirror is maintained at a first voltage and the ion mirror is at the second setting. When the ion mirror is maintained at a second voltage.   The method of claim 16, wherein the first voltage is substantially the same as the second voltage.   The method of claim 16, wherein the first voltage is substantially different from the second voltage.   19. The method of claim 18, wherein the difference between the first voltage and the second voltage is r% of the first voltage or the second voltage, and r is: (i) <1; ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix ) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a method within a range selected from the group consisting of> 50.   20. The method according to claim 18 or 19, wherein the difference between the first voltage and the second voltage is (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x ) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700 -750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix ) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14 -15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 30 kV Method selected from the group.   21. The method according to claim 18, 19 or 20, wherein the first voltage and / or the second voltage are (i) <10 V; (ii) 10-50 V; (iii) 50-100 V (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; ( x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV ; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; ( xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20- 21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV ; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 3 A method within a range selected from the group consisting of 0 kV.   A method according to any preceding claim, further comprising providing an ion source, wherein the ion mirror is at a first potential relative to the potential of the ion source when the ion mirror is in the first setting. A method wherein the ion mirror is maintained at a second potential relative to the potential of the ion source when the ion mirror is in the second setting.   23. The method of claim 22, wherein the first potential is substantially the same as the second potential.   23. The method of claim 22, wherein the first potential is substantially different from the second potential.   25. The method according to claim 24, wherein a difference between the first potential and the second potential is s% of the first potential or the second potential, and s is (i) <1; (ii ) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35 -40; (xvii) 40-45; (xviii) 45-50; and (xix) a method within a range selected from the group consisting of> 50.   26. The method according to claim 24 or 25, wherein a potential difference between the first potential and the potential of the ion source and / or a potential difference between the second potential and the potential of the ion source is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550- 600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11- 12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV ; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; ( xxxxv) 24-25 kV; (xxxxv i) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 30 kV A method that is within the selected range.   27. The method of claim 24, 25 or 26, wherein the first potential and / or the second potential is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; ( x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV ; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; ( xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20- 21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV ; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 3 A method within a range selected from the group consisting of 0 kV.   A method according to any of the preceding claims, further comprising providing an ion source, the ion source comprising: (i) an electrospray (“ESI”) ion source; (ii) atmospheric pressure chemical ionization ( ("APCI") ion source; (iii) atmospheric pressure photoionization ("APPI") ion source; (iv) laser desorption ionization ("LDI") ion source; (v) inductively coupled plasma ("ICP") ion source (Vi) electron impact (“EI”) ion source; (vii) chemical ionization (“CI”) ion source; (viii) field ionization (“FI”) ion source; (ix) fast atom bombardment (“FAB”); ) Ion source; (x) liquid secondary ion mass spectrometry (“LSIMS”) ion source; (xi) atmospheric pressure ionization (“API”) ion source; (xii) field desorption (“FD”) ) Ion source; (xiii) matrix-assisted laser ionization ( "MALDI") ion source; and (xiv) a silicon on desorption / ionization ( "DIOS") method selected from the group consisting of an ion source.   A method according to any of the preceding claims, further comprising providing a continuous ion source.   29. A method according to any of claims 1-28, further comprising providing a pulsed ion source.   A method as claimed in any preceding claim, further comprising providing a drift or flight space upstream of the ion mirror, wherein the ion mirror is in the first setting, A method wherein the ion mirror is maintained at a second potential relative to the drift or flight space potential when the drift mirror or flight space potential is maintained at a first potential and the ion mirror is at the second setting.   32. The method of claim 31, wherein the first potential is substantially the same as the second potential.   32. The method of claim 31, wherein the first potential is substantially different from the second potential.   34. The method according to claim 33, wherein a difference between the first potential and the second potential is t% of the first potential or the second potential, and t is (i) <1; (ii ) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35 -40; (xvii) 40-45; (xviii) 45-50; and (xix) a method within a range selected from the group consisting of> 50.   35. The method of claim 33 or 34, wherein the difference between the first potential and the second potential is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x ) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700 -750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix ) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14 -15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 30 kV Method is within a range selected from the group.   36. The method according to claim 33, 34 or 35, wherein the first potential and / or the second potential is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; ( x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV ; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; ( xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20- 21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV ; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li)> 3 A method within a range selected from the group consisting of 0 kV.   A method according to any preceding claim, wherein when the ion mirror is in the first setting, ions having a certain mass to charge ratio and / or certain energy are present at least in the ion mirror. A method of penetrating ions having a certain mass-to-charge ratio and / or the certain energy into the ion mirror at least different second intervals when penetrating the first interval and the ion mirror is in the second setting. .   38. The method of claim 37, wherein the difference between the first interval and the second interval is u% of the first interval or the second interval, and u is (i) <1; ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix ) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a method within a range selected from the group consisting of> 50.   A method according to any of the preceding claims, wherein the first time of flight of the first fragment ions is determined, and the second time of flight of the first fragment ions is determined. A step of identifying, determining, identifying or searching for a first fragment ion in the first time-of-flight or mass spectral data, and a corresponding first in the second time-of-flight data. A method comprising identifying, determining, identifying or searching for fragment ions.   40. The method of claim 39, wherein the step of identifying, determining, identifying or searching for a first fragment ion in the first time-of-flight or mass spectral data is performed manually and / or automatically. Wherein the step of identifying, determining, identifying or searching for the first fragment ion in the second time-of-flight or mass spectral data is done manually and / or automatically.   41. A method according to claim 39 or 40, wherein the step of identifying, determining, identifying or retrieving a first fragment ion in the first and / or the second time-of-flight or mass spectral data. Comparing the pattern of isotope peaks in the first time-of-flight or mass spectral data with the pattern of isotope peaks in the second time-of-flight or mass spectral data.   42. The method of claim 41, wherein comparing the pattern of isotope peaks comprises comparing the relative intensity of the isotope peaks and / or the distribution of isotope peaks.   43. A method according to any of claims 39 to 42, wherein the first and / or second time-of-flight or mass spectral data are identified, determined, identified, or The step of searching includes comparing the intensity of ions in the first time-of-flight or mass spectral data with the intensity of ions in the second time-of-flight or mass spectral data.   44. The method according to any of claims 39-43, wherein the first and / or second time-of-flight or mass spectral data are identified, determined, identified, or A step of searching for a width of one or more mass spectral peaks in the first mass spectrum resulting from the first time-of-flight or mass spectral data; Comparing the width of one or more mass spectral peaks in the two mass spectra.   A method according to any preceding claim, further comprising obtaining a parent ion mass spectrum.   46. The method of claim 45, further comprising determining the mass or mass to charge ratio of one or more parent ions from the parent ion mass spectrum.   48. The method of claim 46, further comprising determining a time of flight of one or more fragment ions from the first time of flight or mass spectral data. 48. The method of claim 47, comprising:
Based on the mass or mass-to-charge ratio of the parent ion determined from the parent ion mass spectrum and the time of flight of the fragment ion determined from the first time-of-flight or mass spectral data,
The method further comprising predicting the mass or mass to charge ratio that a potential first fragment ion would have. 48. The method of claim 47, comprising:
The mass or mass to charge ratio of one or more parent ions determined from the parent ion mass spectrum; and
Based on the time of flight of the one or more fragment ions determined from the first time of flight or mass spectral data,
The method further comprising predicting the mass or mass to charge ratio that a potential first fragment ion would have.   50. The method of any of claims 46-49, further comprising determining a time of flight of one or more fragment ions from the second time of flight or mass spectral data. 51. The method of claim 50, comprising:
The mass or mass-to-charge ratio of the parent ion determined from the parent ion mass spectrum;
Based on the second flight time or the fragment ion flight time determined from the mass spectral data,
A method further comprising predicting the mass or mass to charge ratio that a potential second fragment ion would have. 51. The method of claim 50, comprising:
The mass to charge ratio of one or more parent ions determined from the parent ion mass spectrum;
Based on the second flight time or the flight time of one or more of the fragment ions determined from the mass spectral data,
A method further comprising predicting the mass or mass to charge ratio that a potential second fragment ion would have.   53. The method of claim 51 or 52, wherein the predicted mass or mass to charge ratio of one or more possible first fragment ions is one or more possible second. Comparing or contrasting with the expected mass or mass-to-charge ratio of the fragment ions. 54. The method of claim 53, wherein the expected mass or mass to charge ratio of one or more of the potential first fragment ions is one or more of the potential second. Corresponding to within x% of the expected mass or mass to charge ratio of the fragment ions of
Further identifying, determining, or identifying a fragment ion in the first time-of-flight or mass spectral data as related to the same species of fragment ion in the second time-of-flight or mass spectral data Including methods.   55. The method of claim 54, wherein x is (i) <0.001; (ii) 0.001-0.01; (iii) 0.01-0.1; (iv) 0.1-0.5; (v) 0.5-1.0; (vi ) 1.0-1.5; (vii) 1.5-2.0; (viii) 2-3; (ix) 3-4; (x) 4-5; and (xi)> 5 Method. A method according to any preceding claim, wherein from the first and second time of flight, the mass or mass to charge ratio of a parent ion fragmented to yield the first fragment ion is determined. The step
The mass-to-charge ratio of the parent ion that is fragmented to yield the first fragment ion, independent of or without the information of the mass or mass-to-charge ratio information of the first fragment ion. A method comprising determining. 57. The method of claim 56, wherein the first fragment ions are fragmented independently of or without the information of the mass or mass to charge ratio information of the first fragment ions. The step of determining the mass to charge ratio of the resulting parent ion comprises:
Whether one or more parent ion mass peaks are observed within y% of the expected mass or mass to charge ratio of the parent ion determined to have fragmented to yield the first fragment ion Determining from a parent ion mass spectrum.   58. The method of claim 57, wherein y is (i) <0.001; (ii) 0.001-0.01; (iii) 0.01-0.1; (iv) 0.1-0.5; (v) 0.5-1.0; (vi ) 1.0-1.5; (vii) 1.5-2.0; (viii) 2-3; (ix) 3-4; (x) 4-5; and (xi)> 5 Method.   59. The method of claim 57 or 58, wherein one parent ion mass peak of the expected mass or mass to charge ratio of the parent ion determined to fragment to yield the first fragment ion. If observed within y%, then the mass or mass to charge ratio of the parent ion mass peak is equal to the mass or mass to charge ratio of the parent ion that fragmented to yield the first fragment ion. A more precise decision method.   59. The method of claim 57 or 58, wherein the expected mass or mass to charge ratio of the parent ion determined to have more than one parent ion mass peak fragmented to yield the first fragment ion. Then observed parent ion mass peak corresponds to or is associated with the parent ion most likely to fragment to give the first fragment ion How decisions are made.   61. The method of claim 60, wherein which observed parent ion mass peak corresponds to or is associated with the parent ion that is most likely fragmented to yield the first fragment ion. A method wherein the determination is made by referring to a third time-of-flight or mass spectral data acquired when the ion mirror is held at a different third setting.   62. The method of claim 60 or 61, wherein the observed parent ion mass peak corresponds to or is associated with the parent ion most likely to have fragmented to yield the first fragment ion. The mass or mass to charge ratio is a more precise determination of the mass or mass to charge ratio of the parent ion that has been fragmented to yield the first fragment ion.   63. The method of claim 59 or 62, wherein a more precise determination of the mass or mass to charge ratio of the first fragment ion is a more precise determination of the mass or mass to charge ratio of the parent ion. The method made using. A mass spectrometer including a time-of-flight mass analyzer,
The time-of-flight mass analyzer includes an ion mirror;
In use, for the first time, the ion mirror is kept at a first setting, acquiring a first time-of-flight or mass spectral data, and in the second time, the ion mirror is kept at a different second setting, Acquire two time-of-flight or mass spectral data,
When the mass spectrometer is in use,
(A) a first time of flight of a first fragment ion having a certain mass or mass to charge ratio when the ion mirror is held at the first setting;
(B) a different second flight time of the first fragment ions having the certain constant mass or mass to charge ratio when the ion mirror is held at the second setting;
(C) the mass or mass-to-charge ratio of the parent ion and / or the mass or mass-charge of the first fragment ion that fragmented from the first and second time of flight to produce the first fragment ion; Mass spectrometer to determine the ratio.   65. A mass spectrometer according to claim 64, wherein the ion mirror includes a reflectron.   66. A mass spectrometer as claimed in claim 65, wherein the reflectron comprises a linear electric field reflectron or a non-linear electric field reflectron. 67. A mass spectrometer as claimed in claim 64, 65 or 66, further comprising an ion source and a drift or flight space upstream of the ion mirror,
In use, when the ion mirror is at the first setting, a first potential difference is maintained between the ion source and the drift or flight space, and when the ion mirror is at the second setting, A mass spectrometer in which a two-potential difference is maintained between the ion source and the drift or flight space.   68. A mass spectrometer as recited in claim 67, wherein in use, the first potential difference is substantially the same as the second potential difference.   68. A mass spectrometer as recited in claim 67, wherein in use, the first potential difference is substantially different from the second potential difference.   70. A mass spectrometer according to claim 69, wherein, in use, the difference between the first potential difference and the second potential difference is p% of the first potential difference or the second potential difference, and p is (i ) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7 -8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30- 35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a mass spectrometer within a range selected from the group consisting of> 50.   The mass spectrometer according to claim 69 or 70, wherein, in use, the difference between the first potential difference and the second potential difference is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350- 400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V ; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7- 8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV ; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; ( (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; And (li)> mass spectrometer is selected from the group consisting of 30 kV.   72. A mass spectrometer according to claim 69, 70 or 71, wherein, in use, the first potential difference and / or the second potential difference is: (i) <10 V; (ii) 10-50 V; ) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350 -400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii ) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7 -8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii ) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l ) 29-30 kV; and (li) a mass spectrometer within a range selected from the group consisting of> 30 kV.   73. A mass spectrometer according to any of claims 64 to 72, wherein in use, when the ion mirror is in the first setting, the first electric field strength is at least 10% of the length of the ion mirror, Second, when kept along 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% and the ion mirror is in the second setting The electric field strength is kept along at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the ion mirror. Mass spectrometer.   74. A mass spectrometer as recited in claim 73, wherein in use, the first electric field strength is substantially the same as the second electric field strength.   74. A mass spectrometer as recited in claim 73, wherein, in use, the first electric field strength is substantially different from the second electric field strength.   76. The mass spectrometer of claim 75, wherein in use, the difference between the first electric field strength and the second electric field strength is q% of the first electric field strength or the second electric field strength, q (I) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7-8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; ( A mass spectrometer within a range selected from the group consisting of xv) 30-35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50.   77. The mass spectrometer according to claim 75 or 76, wherein, in use, the difference between the first electric field strength and the second electric field strength is: (i) <0.01 kV / cm; (ii) 0.01-0.1 kV (iii) 0.1-0.5 kV / cm; (iv) 0.5-1 kV / cm; (v) 1-2 kV / cm; (vi) 2-3 kV / cm; (vii) 3-4 kV / cm; (viii) 4-5 kV / cm; (ix) 5-6 kV / cm; (x) 6-7 kV / cm; (xi) 7-8 kV / cm; (xii) 8-9 kV (xiii) 9-10 kV / cm; (xiv) 10-11 kV / cm; (xv) 11-12 kV / cm; (xvi) 12-13 kV / cm; (xvii) 13-14 kV / cm; (xviii) 14-15 kV / cm; (xix) 15-16 kV / cm; (xx) 16-17 kV / cm; (xxi) 17-18 kV / cm; (xxii) 18-19 kV (xxiii) 19-20 kV / cm; (xxiv) 20-21 kV / cm; (xxv) 21-22 kV / cm; (xxvi) 22-23 kV / cm; (xxvii) 23-24 kV / cm; (xxviii) 24-25 kV / cm; (xxix) 25-26 kV / cm; (xxx) 26-27 kV / cm; (xxxi) 27-28 kV / cm; (xxxii) 28-29 kV (xxxiii) 29-30 kV / cm; and (xxxiv)> 30 kV / cm.   78. A mass spectrometer according to claim 75, 76 or 77, wherein, in use, the first electric field strength and / or the second electric field strength is (i) <0.01 kV / cm; (ii) 0.01-0.1 kV / cm; (iii) 0.1-0.5 kV / cm; (iv) 0.5-1 kV / cm; (v) 1-2 kV / cm; (vi) 2-3 kV / cm; (vii) 3-4 kV / cm; (viii) 4-5 kV / cm; (ix) 5-6 kV / cm; (x) 6-7 kV / cm; (xi) 7-8 kV / cm; (xii) 8-9 kV / cm; (xiii) 9-10 kV / cm; (xiv) 10-11 kV / cm; (xv) 11-12 kV / cm; (xvi) 12-13 kV / cm; (xvii) 13-14 kV / cm; (xviii) 14-15 kV / cm; (xix) 15-16 kV / cm; (xx) 16-17 kV / cm; (xxi) 17-18 kV / cm; (xxii) 18-19 kxx / cm; (xxiii) 19-20 kV / cm; (xxiv) 20-21 kV / cm; (xxv) 21-22 kV / cm; (xxvi) 22-23 kV / cm; (xxvii) 23-24 kxx / cm; (xxviii) 24-25 kV / cm; (xxix) 25-26 kV / cm; (xxx) 26-27 kV / cm; (xxxi) 27-28 kV / cm; (xxxii) 28-29 A mass spectrometer within a range selected from the group consisting of kV / cm; (xxxiii) 29-30 kV / cm; and (xxxiv)> 30 kV / cm.   79. A mass spectrometer according to any of claims 64 to 78, wherein, when in use, when the ion mirror is in the first setting, the ion mirror is maintained at a first voltage, and the ion mirror is A mass spectrometer wherein the ion mirror is held at a second voltage when set to 2.   80. A mass spectrometer as recited in claim 79, wherein in use, the first voltage is substantially the same as the second voltage.   80. A mass spectrometer as recited in claim 79, wherein in use, the first voltage is substantially different from the second voltage.   82. The mass spectrometer of claim 81, wherein in use, the difference between the first voltage and the second voltage is r% of the first voltage or the second voltage, and r is (i) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7- 8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35 (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a mass spectrometer within a range selected from the group consisting of> 50.   83. A mass spectrometer according to claim 81 or 82, wherein, in use, the difference between the first voltage and the second voltage is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350- 400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V ; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7- 8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV ; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; ( (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li) A mass spectrometer selected from the group consisting of> 30 kV.   84. A mass spectrometer according to claim 81, 82 or 83, wherein, in use, the first voltage and / or the second voltage are: (i) <10 V; (ii) 10-50 V; (iii ) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350 -400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii ) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7 -8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii ) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-3 A mass spectrometer within a range selected from the group consisting of 0 kV; and (li)> 30 kV.   85. A mass spectrometer as claimed in any of claims 64 to 84, further comprising an ion source, wherein, when in use, when the ion mirror is in the first setting, the ion mirror is a second to the potential of the ion source. A mass spectrometer wherein the ion mirror is maintained at a second potential relative to the potential of the ion source when the potential is maintained at one potential and the ion mirror is at the second setting.   86. A mass spectrometer as recited in claim 85, wherein in use, the first potential is substantially the same as the second potential.   86. A mass spectrometer as recited in claim 85, wherein in use, the first potential is substantially different from the second potential.   88. A mass spectrometer according to claim 87, wherein in use, the difference between the first potential and the second potential is s% of the first potential or the second potential, and s is (i) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7- 8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30-35 (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a mass spectrometer within a range selected from the group consisting of> 50.   89. A mass spectrometer according to claim 87 or 88, wherein in use, a potential difference between the first potential and the potential of the ion source and / or a difference between the second potential and the potential of the ion source. (I) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350-400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix ) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7-8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii ) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23 -24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and ( li) A mass spectrometer within a range selected from the group consisting of> 30 kV.   90. A mass spectrometer according to claim 87, 88 or 89, wherein in use, the first potential and / or the second potential is: (i) <10 V; (ii) 10-50 V; ) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350 -400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii ) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7 -8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii ) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29- A mass spectrometer within a range selected from the group consisting of 30 kV; and (li)> 30 kV.   93. A mass spectrometer as claimed in any of claims 64-90, comprising: (i) an electrospray ("ESI") ion source; (ii) an atmospheric pressure chemical ionization ("APCI") ion source; (iii) large Barometric pressure photoionization (“APPI”) ion source; (iv) Laser desorption ionization (“LDI”) ion source; (v) Inductively coupled plasma (“ICP”) ion source; (vi) Electron impact (“EI”) (Vii) chemical ionization (“CI”) ion source; (viii) field ionization (“FI”) ion source; (ix) fast atom bombardment (“FAB”) ion source; (x) liquid secondary ion Mass spectrometry (“LSIMS”) ion source; (xi) Atmospheric pressure ionization (“API”) ion source; (xii) Field desorption (“FD”) ion source; (xiii) Matrix support Laser ionization ( "MALDI") ion source; and (xiv) a silicon on desorption / ionization ( "DIOS") further comprises a mass spectrometer ion source selected from the group consisting of an ion source.   92. A mass spectrometer as claimed in any of claims 64-91, further comprising a continuous ion source.   92. A mass spectrometer as claimed in any of claims 64-91, further comprising a pulsed ion source. A mass spectrometer according to any one of claims 64-93,
Further comprising a drift or flight space upstream of the ion mirror, and when in use, when the ion mirror is in the first setting, the ion mirror is maintained at a first potential relative to the potential of the drift or flight space; and A mass spectrometer wherein the ion mirror is maintained at a second potential relative to the drift or flight space potential when the ion mirror is at the second setting.   95. A mass spectrometer as recited in claim 94, wherein in use, the first potential is substantially the same as the second potential.   95. A mass spectrometer as recited in claim 94, wherein in use, the first potential is substantially different from the second potential.   99. The mass spectrometer of claim 96, wherein in use, the difference between the first potential and the second potential is t% of the first potential or the second potential, and t is (i ) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7 -8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30- 35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix) a mass spectrometer within a range selected from the group consisting of> 50.   98. The mass spectrometer of claim 96 or 97, wherein in use, the difference between the first potential and the second potential is: (i) <10 V; (ii) 10-50 V; (iii) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350- 400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V ; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7- 8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV ; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; ( (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (Li)> 30 mass spectrometer is within a range selected from the group consisting of kV.   99. A mass spectrometer according to claim 96, 97 or 98, wherein, in use, the first potential and / or the second potential is: (i) <10 V; (ii) 10-50 V; (iii ) 50-100 V; (iv) 100-150 V; (v) 150-200 V; (vi) 200-250 V; (vii) 250-300 V; (viii) 300-350 V; (ix) 350 -400 V; (x) 400-450 V; (xi) 450-500 V; (xii) 500-550 V; (xiii) 550-600 V; (xiv) 600-650 V; (xv) 650-700 V; (xvi) 700-750 V; (xvii) 750-800 V; (xviii) 800-850 V; (xix) 850-900V; (xx) 900-950; (xxi) 950-1000 V; (xxii ) 1-2 kV; (xxiii) 2-3 kV; (xxiv) 3-4 kV; (xxv) 4-5 kV; (xxvi) 5-6 kV; (xxvii) 6-7 kV; (xxviii) 7 -8 kV; (xxix) 8-9 kV; (xxx) 9-10 kV; (xxxi) 10-11 kV; (xxxii) 11-12 kV; (xxxiii) 12-13 kV; (xxxiv) 13-14 kV; (xxxv) 14-15 kV; (xxxvi) 15-16 kV; (xxxvii) 16-17 kV; (xxxviii) 17-18 kV; (xxxix) 18-19 kV; (xxxx) 19-20 kV; (xxxxi) 20-21 kV; (xxxxii) 21-22 kV; (xxxxiii) 22-23 kV; (xxxxiv) 23-24 kV; (xxxxv) 24-25 kV; (xxxxvi) 25-26 kV; (xxxxvii ) 26-27 kV; (xxxxviii) 27-28 kV; (xxxxix) 28-29 kV; (l) 29-30 kV; and (li) a mass spectrometer within a range selected from the group consisting of> 30 kV.   100. A mass spectrometer as claimed in any of claims 64 to 99, wherein in use, when the ion mirror is in the first setting, ions having a specific mass to charge ratio and / or specific energy are When the ion mirror penetrates at least a first interval and the ion mirror is in the second setting, ions having the specific mass to charge ratio and / or the specific energy are at least different second interval penetrations into the ion mirror. Mass spectrometer.   101. The mass spectrometer of claim 100, wherein, in use, the difference between the first interval and the second interval is u% of the first interval or the second interval, and u is (i ) <1; (ii) 1-2; (iii) 2-3; (iv) 3-4; (v) 4-5; (vi) 5-6; (vii) 6-7; (viii) 7 -8; (ix) 8-9; (x) 9-10; (xi) 10-15; (xii) 15-20; (xiii) 20-25; (xiv) 25-30; (xv) 30- 35; (xvi) 35-40; (xvii) 40-45; (xviii) 45-50; and (xix)> 50 mass spectrometer within a range selected from the group consisting of> 50.

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