HIGH MASS ION DETECTOR

Abstract

A high-mass ion detector for a mass spectrometer, a mass spectrometer, and a method for detecting high-mass ions are provided. The high-mass ion detector includes a secondary electron multiplier (SEM) (47) for converting ions into electrons and for multiplying a certain number of electrons within the SEM, and an acceleration unit (41) for accelerating the high-mass ions, separated from the front face of the SEM by a distance of between 100 micrometers and 10 millimeters. The SEM includes a front face (45) and a rear face (46) where, during operation, the ions enter through the front face. The acceleration unit includes at least two electrodes (42, 44), and each pair of electrodes is separated by an insulating layer (43) for electrical insulation. Figure 4

Description

Title of the invention: HIGH MASS ION DETECTOR

[0001] The present disclosure relates to a high mass ion detector for a mass spectrometer, a mass spectrometer, and a method for detecting high mass ions which are provided. Background

[0002] Top-down proteomics is a method of protein identification that may use an ion trap mass spectrometer to store an isolated protein ion for mass measurement and analysis by tandem mass spectrometry (MS / MS), or may use other protein purification methods such as two-dimensional gel electrophoresis in conjunction with MS / MS. Top-down proteomics focuses on the characterization of intact biomolecules, which reduces sample complexity and facilitates the detection of changes at the protein level.The combination of a matrix-assisted laser desorption / ionization (MALDI) technique and a time-of-flight (TOF) mass analyzer allows the generation of gaseous ions in low charge states from high-mass biomolecules and their subsequent separation according to their mass-to-charge ratio (m / z), as high-mass ions drift more slowly in the flight tube than lighter ions. Matrix-assisted laser desorption / ionization time-of-flight (MALDLTOF) instruments are widely used for mass spectrometric analysis of macromolecules. For the determination of m / z values ​​of analytes using MALDLTOF instruments, the sample is mixed with a matrix that absorbs the energy of a pulsed laser beam and transfers it to the analyte molecules. This usually results in ions that are first accelerated and then passed through a flight tube of known length.Signal amplification is performed with a secondary electron multiplier, SEM, such as a discrete dynode or a microchannel plate, MCP, from which analyte ions release electrons upon impact. These electrons are multiplied by means of other dynodes or in individual channels of MCP units. With an anode behind the SEM, an electron shower can be detected.

[0003] For example, MCP detectors can be integrated into MALDLTOF instruments since MCP detectors are characterized by good time resolution and high sensitivity due to the amplification effect, which can be further improved by using several MCPs in series.

[0004] For the separation of individual analyte ions that differ in mass and charge in the flight tube of MALDLTOF devices, all ions can initially receive the same kinetic energy Ekin in an ion source by a voltage acceleration U applied between the MALDI sample plate and the electrode opposite, before entering a TOF mass analyzer without an electric field. On the basis of the equation, [Math 1] Ekin = qU=4 m-v2 = - high mass i°ns have a speed v in lower than that of low-mass ions and need more time t to travel the distance d of the TOF flight tube, so that the ions can be separated according to their mass m and charge q, where q is equal to an integer number z of electronic charges e. The respective m / z value of the analyte ions can be calculated from the time-of-flight determination.

[0005] Since the ion velocity is inversely proportional to the square root of the ion mass, high mass molecules exhibit comparatively low velocities. It is known that the probability of secondary electron generation by ion impact decreases as the ion velocity decreases, leading to a significant reduction in SEM detection efficiency while increasing the ion mass. This means that the conversion of ions to electrons is reduced and more secondary ions are generated by the impact of the analyte ion on the PCM. For this reason, the signal intensity decreases considerably for higher mass ions, leading to major limitations to MALDL TOF applications. This effect is further enhanced in complex samples, as small ions that reach the detector first can also lead to oversaturation of individual PCM channels.

[0006] Some solutions to address the mentioned inefficiencies of PCM detectors may include cryogenic detectors operating at temperatures < 100 mK. However, these designs have failed to gain traction in routine applications due to the complex and expensive cooling system and the small active surface area of ​​the detector. Some approaches to extend the accessible mass range are based on modifying a conventional PCM detector: either by modifying the detection mechanism for the electronic signal emerging from the backside of the PCM, or by converting the slow-moving high-mass ions into electrons or smaller secondary ions before they hit the frontside of the PCM.In general, the signal intensity from an MCP detector can be enhanced by increasing the velocity of the incoming charged particles, which can be achieved by accelerating them through a large electric potential. Ions can be accelerated in a commercial mass spectrometer ion source by voltages up to 30 kV. A combination of several electrode configurations can be found to enable . additional spatial focusing of the ion beam. Higher accelerating voltages in the ion source can create a risk of electrical discharges and lead to shorter flight times, which in turn requires very fast and expensive measurement equipment to achieve sufficient mass resolution. Abstract

[0007] The present disclosure provides examples of systems that at least partially overcome some of the aforementioned drawbacks.

[0008] In a first aspect, the invention relates to a high mass ion detector for a mass spectrometer, the high mass ion detector comprising: • a secondary electron multiplier, SEM, for converting ions into electrons and for multiplying a number of electrons within the SEM, which SEM comprises a front face and a back face where, in use, ions enter through the front face; and • an acceleration unit for accelerating high mass ions, separated from the front face of the SEM by a distance of between 100 micrometers and 10 millimeters; the acceleration unit being configured to operate in a mass spectrometer, the acceleration unit comprising at least two electrodes, and each pair of electrodes being separated by an insulating layer for electrical insulation. Optionally, the acceleration unit is separated from the front face of the SEM by a distance between 100 micrometers and 1 millimeter.

[0009] Optionally, the acceleration unit is separated from the front face of the SEM by a distance of between 1 millimeter and 10 millimeters.

[0010] Optionally, the acceleration unit is separated from the front face of the SEM by a distance of 100 micrometers.

[0011] Optionally, the insulating layer of the acceleration unit has a thickness of between 100 micrometers and 10 millimeters.

[0012] Optionally, the insulating layer has a thickness of 2 mm or a thickness of 6 mm.

[0013] Optionally, the insulating layer is composed at least in part of polytetrafluoroethylene (PTFE).

[0014] Optionally, the high mass ion detector comprises two electrodes, wherein a first electrode and a second electrode are separated by a first insulating layer.

[0015] Optionally, the high mass ion detector comprises three electrodes, wherein a first electrode and a second electrode are separated by a first insulating layer, and the second electrode and a third electrode are separated by a second insulating layer.

[0016] Optionally, at least one of the electrodes and / or each of the insulating layers has a circular shape or an annular shape.

[0017] In a second aspect, the invention relates to a mass spectrometer comprising: • an ionization region; • a time-of-flight tube, or TOF tube, mass analyzer, the TOF tube being in communication with the ionization region; and the high mass ion detector of any one of claims 1 to 10, wherein the high mass ion detector is in communication with the TOF tube, and wherein the accelerator unit of the high mass ion detector is between the TOF tube and the SEM of the high mass ion detector.

[0018] In a first aspect, there is provided a high mass ion detector for a mass spectrometer, the high mass ion detector comprising: a secondary electron multiplier, SEM, and an accelerator unit for accelerating the high mass ions. The SEM comprises a front face and a back face where, in use, ions enter through the front face and may exit through the back face. The SEM may have an outer dimension, for example an outer diameter in the case of a ring-shaped SEM, in the range of 20 mm to 60 mm, for example an outer dimension of 30 mm or 40 mm or 50 mm or 60 mm. In some embodiments, the SEM is a PCM. A PCM comprises a plurality of channels, such that ions may enter and exit each channel. The PCM may be used as a detector in conjunction with a TOF tube in a mass spectrometer.The MCP channels can usually be slightly tilted relative to an axis of the TOF tube, such that as many ions as possible hit the MCP walls and induce electrons in the MCP channels, which can in turn excite other electrons. The ions may remain as debris on the surface of the MCPs. This may ultimately lead to a reduction in the reactivity of the MCPs. In some embodiments, the SEM is a dynode, in which case it depends on how the electrodes of the dynodes are arranged, i.e., whether they block the TOF line or are arranged along the TOF line of flight.The accelerator unit faces the front of the SEM and is separated from the front of the SEM by a separation distance of between 100 micrometers and 10 mm, or between 1 millimeter and 10 mm, or between 100 micrometers and 1 millimeter, so that ions exiting the accelerator unit travel the separation distance before contacting the SEM. The SEM, in use, converts ions into electrons and multiplies the number of electrons inside the SEM.

[0019] The acceleration unit for accelerating high mass ions comprises at least two electrodes separated by an insulating layer for electrical insulation. The electrodes allow ions to pass through them, for example through a hole in disc or ring electrodes or in mesh electrodes. In use, ions passing through the electrodes are accelerated due to a voltage potential difference applied to the at least two electrodes. The acceleration unit is configured to operate in a mass spectrometer, which may involve the dimensions of the electrodes, for example an outer dimension of the electrodes, being between 10 mm and 60 mm. For example, the outer diameter of a ring electrode may be between 10 and 60 mm. For example, the outer diameter of an annular insulating layer may be between 10 and 60 mm. In examples, the acceleration unit comprises two electrodes and an insulating layer separating the two electrodes.In examples, the acceleration unit includes 3 or more electrodes. Each pair of electrodes is separated by an insulating layer for electrical insulation; for example, if the acceleration unit includes three electrodes, a first electrode and a second electrode may be separated by a first insulating layer and the second electrode and a third electrode may be separated by a second insulating layer. In use, different voltages may be applied to the 3 or more electrodes, such that different combinations of voltage potential differences accelerate ions passing through the electrodes. For example, a first potential difference between a first electrode and a second electrode may accelerate the ions by a first accelerating force and a second potential difference between the second electrode and a third electrode may further accelerate the ions by a second accelerating force.

[0020] In some examples, the distance defined by the insulating layer between the electrodes may be varied by varying the thickness of the insulating layer. The thickness of the insulating layer may influence the voltage that may be applied to the electrodes to achieve different accelerations for different ions. Ions having specific m / z values ​​may be accelerated by the accelerating unit of the present disclosure by varying the voltages applied to the electrodes. The thickness of the insulating layer may govern the breakdown voltage that may be applied to the electrodes in the accelerating unit of the present disclosure. It may therefore be possible to design the accelerating unit of the present disclosure such that ions having a mass within a specific range of m / z values ​​are accelerated as desired.The design may include different thicknesses of the insulating layer leading to a unit in which different voltages can be applied to the electrodes to provide specific accelerating forces. The higher the m / z values ​​of the ions to be accelerated, the higher the voltages required and by . consequently the greater the thickness of the insulating layer can be.

[0021] The acceleration unit of the present disclosure allows, when used with an SEM, to take measurements and thus detect high mass ions. High mass ions include ions with an m / z value ranging from 50,000 to 1,500,000 daltons, where one dalton refers to the mass of a single hydrogen atom, but the unit is calibrated to 1 / 12 of a C-12 atom. The m / z value, in the present disclosure, refers to the mass in daltons, which is divided by the charge state z of the ion / protein. In a MALDI, values ​​for z = 1, 2, 3 can be found, while in an ESI, z can be in the range of 50 to 100. The value of z can be adjusted. The acceleration unit can be used by establishing zero voltage at the electrodes, which ensures detection equivalent to that of a conventional SEM without an acceleration unit.This usage corresponds to stopping the acceleration unit and can be used to compare a detection with and without an acceleration unit.

[0022] The electrodes may be metallic. The geometry of the electrodes may influence acceleration and subsequent detection by an SEM. In examples, the electrodes have an annular shape such that in operation, a direct line of sight is permitted for ions passing through a hole in the ring toward, for example, an SEM. As may be understood by those skilled in the art, the annular electrode may have a different outer radius and inner radius. A mesh electrode may include holes of a thickness that allows ions to pass therethrough.

[0023] Conventional detectors generally operate by increasing the accelerating voltage in an ion source. An ion source may comprise a sample of molecules illuminated by a laser. A high mass ion detector in accordance with this disclosure delivers an electrical potential supplied by the electrodes to the ions immediately before, i.e., between 100 micrometers and 10 mm before they reach the SEM, electron multiplier or detector, instead of increasing the accelerating voltage in the ion source. A modification of the conventional detector is therefore achieved by the present disclosure.

[0024] The high mass ion detector of the present disclosure allows amplification of the signal from ionized molecules, or ionized proteins, for example from m / z 50,000 up to 150,000. Ions with other m / z values ​​can also be accelerated by the acceleration unit of the present disclosure by varying the voltages applied to the electrodes. The thickness of the insulating layer can govern the breakdown voltage that can be applied to the electrodes in the acceleration unit of the present disclosure. The electrodes can be assembled before or in front of a conventional SEM detector. By before or in front of the SEM is meant facing the side of the SEM from which, in use, the ions enter. The electrodes can be adjusted to negative electrical voltages to affect positively charged ions directly before they hit the SEM. Electrodes can be set to positive electrical voltages to affect negatively charged ions directly before they hit the SEM. Ions are affected by an increase in velocity. In some examples, ions may be further affected by ion optical effects; ion optical effects should be understood to refer to the guiding of charged particles by electrodes in a manner similar to conventional optics. The geometry and polarization of the electrode configuration can be designed to disperse or collimate a beam of ions, e.g., charged proteins. Different electrode configurations can be implemented to maximize the detection signal.

[0025] The high mass ion detector of the present disclosure may be provided as a ready-to-use solution for connection to a conventional mass spectrometer, as will be explained below.

[0026] In examples of the disclosure, there is provided a mass spectrometer comprising: an ionization region; a time-of-flight, TOF, tube mass analyzer having a predetermined distance d, the TOF tube being in communication with the ionization region; and the high mass ion detector of the present disclosure, wherein the high mass ion detector is in communication with the TOF tube, and wherein the accelerator unit of the high mass ion detector is between the TOF tube and the SEM. The mass spectrometer may include a laser source configured to illuminate the ionization region, to obtain ions from an illuminated sample lying on the ionization region. The laser source may be configured to illuminate a sample to obtain ions from the illuminated sample.The laser source may comprise a UV laser or a nitrogen laser having a wavelength in the nanometer range between 340 nm and 430 nm, for example 337 nm or the like, i.e., suitable for the matrix applied in MALDI. In use, the laser pulses may be directed at matrix crystals mixed with an analyte sample in the form of, for example, dried droplet spots. The matrix crystals and analyte mixture may lie on a plate in the ionization region of the mass spectrometer. The matrix crystals may absorb the laser energy, converting the matrix to an ionized state. Charge is transferred to the analyte where a random collision occurs in the gas phase, and the ionized analyte and matrix molecules are desorbed from the plate.

[0027] The mass spectrometer further comprises the TOF tube which may comprise an inlet area facing or in communication with the ionization region of the mass spectrometer and an outlet area facing or in communication with the high mass ion detector.

[0028] A conventional mass spectrometer may include an existing detector. In examples, the high mass ion detector of the present disclosure may be used to replace the existing detector of the conventional mass spectrometer such that, in use, ions formed in the ionization region pass through the TOF tube with an ion flight path and impinge on the high mass ion detector of the present disclosure. The high mass ion detector of the present disclosure may be placed at the exit region of the TOF tube to act as a detector of the conventional mass spectrometer. Thus, the high mass ion detector of the present disclosure may be provided as a plug-and-play solution for plugging into a conventional mass spectrometer.

[0029] When the mass spectrometer according to the disclosure is in use, the ions are affected by the voltages applied to the electrodes before they hit the SEM, which causes an amplification of the signal. When used with a mass spectrometer, the high mass ion detector influences the analyte ions after they exit the TOF tube and a little before, i.e. between 100 micrometers and 10 mm before they hit the surface of the SEM.

[0030] The accelerator unit and high mass ion detector of the present disclosure may pave the way for increasing the sensitivity of SEM-based detection units for high mass molecules.

[0031] In some examples, the SEM is a PCM. In some examples, the SEM is a dynode. Advantageously, a PCM can be economical to operate and offer fast response times, narrow pulse widths, high gain, and a sufficiently large active size, making PCMs good detectors for TOF mass spectrometers.

[0032] The present disclosure provides a device and system for accelerating and thus influencing analyte ions after they exit a TOF tube and shortly before they hit a SEM by adding the accelerating unit - comprising two electrodes separated by an insulating layer - to the detector. In use, a stronger signal intensity (by 25 times) is generated by the same ion when measured with the high mass ion detector of the present disclosure rather than with the conventional MCP.

[0033] In examples, there is provided a method for accelerating ions, comprising: providing an accelerating unit in accordance with the disclosure; applying a voltage potential difference to the at least two electrodes of the accelerating unit; and providing ions to the accelerating unit; thereby exposing the ions to the voltage potential difference, which ions are affected by an accelerating force given by the voltage potential difference. Providing ions can be performed by ionization of a sample analyte.

[0034] According to examples of the present disclosure, a method for detecting high mass ions may be implemented, the method comprising: • the provision of a mass spectrometer in accordance with the disclosure, the mass spectrometer comprising a sample of analyte; • ionization of the sample by illuminating the sample with the laser source, which thus transfers the sample into a gas phase and releases charged ions from the sample; • after all or part of the charged ions have passed through the TOF tube, exposing the charged ions that have passed through the TOF tube to voltages applied to the at least two electrodes of the acceleration unit, thereby accelerating the exposed charged ions; • converting accelerated charged ions leaving the electrodes into secondary electrons by allowing the accelerated charged ions to strike the SEM; and • detecting the presence of high mass ions based on secondary electrons.

[0035] The charged ions can be positively charged ions or negatively charged ions. The positively charged ions can be exposed to a negative potential difference between the voltages applied to the electrodes while passing through the accelerator unit. The negatively charged ions can be exposed to a positive potential difference between the voltages applied to the electrodes while passing through the accelerator unit. Then, the ions leaving the accelerator unit, also called a multiplier, are converted into secondary electrons when they strike the surface of the SEM.Advantageously, a higher signal intensity is generated by the same ion when measured with a detector after the acceleration unit of the present disclosure than with an existing conventional detector without the acceleration unit due to an ion velocity gain in combination with ion optical effects caused by the electrical multiplier potential provided by the electrodes. An electron shower may accumulate at the anode and a "secondary electron" signal may be measured using an external oscilloscope. In some examples, the SEM is a Chevron PCM. In the Chevron configuration, the secondary electron signal is amplified by the Chevron PCMs and the electron shower that accumulates at the anode is measured using the external oscilloscope.In some examples, voltages can be applied independently to the electrodes, and can for example be between zero and -4 kV. Negative voltages beneficially affect the approach of positive ions emerging from the TOF tube, which improves the intensity of the signal generated by the SEM.

[0036] The advantages arising from these aspects may be similar to those mentioned in as regards the first and second aspects. Brief description of the drawings

[0037] Non-limiting examples of the present disclosure will be described in the following, with reference to the accompanying drawings, in which:

[0038] [Fig-1] [Fig.l] schematically represents an acceleration unit 10 for accelerate high mass ions.

[0039] [Fig.2] [Fig.2] represents a mesh electrode.

[0040] [Fig.3] [Fig.3] represents an annular electrode.

[0041] [Fig.4] [Fig.4] schematically represents a high mass ion detector 40 in accordance with the present disclosure.

[0042] [Fig.5] [Fig.5] schematically represents a high mass ion detector 40 in accordance with the present disclosure.

[0043] [Fig.6] [Fig.6] represents an example of a conventional mass spectrometer.

[0044] [Fig.7] [Fig.7] represents a mass spectrometer 70 in accordance with the present exposed.

[0045] [Fig.8] [Fig.8] shows the amplification factors, AF, for SAB with three different examples of high mass ion detectors according to the disclosure.

[0046] [Fig.9] [Fig.9] shows the amplification factors, AF, for IgG with three different examples of high mass ion detectors according to the disclosure.

[0047] [Fig. 10] [Fig. 10] shows a direct comparison of the mass spectra obtained with conventional MCPs and with the mass ion detector of the presentation.

[0048] [Fig. 11] [Fig. 11] shows the AF values ​​for the higher charge states of BSA ions and IgG ions. Detailed description of examples

[0049] In these figures, the same reference signs have been used to designate corresponding elements.

[0050] [Fig.l] shows an acceleration unit 10 for accelerating high mass ions, the acceleration unit comprising at least two electrodes 11, 12 separated by an insulating layer 13 for electrical insulation.

[0051] In some examples, the insulating layer has a thickness of between 100 micrometers and 6 millimeters, mm, or more particularly, in some examples, the insulating layer has a thickness of between 2 mm and 6 mm or a thickness of 2 mm or a thickness of 6 mm. The insulating layer may be composed at least in part of an insulating material, for example polytetrafluoroethylene (PTFE), and / or polyether-etherketone (PEEK), and / or Kapton.

[0052] In some examples, the high mass detector of the present disclosure may be referred to as a multiplier SEM, with reference to the acceleration force or "multi plication" that, during use, the high mass ion detector imprints on the ions. In some examples, the SEM is a microchannel plate MCP. In these examples, the multiplier SEM is called BMCP for "booster (multiplier) MCP". The geometry of the electrodes is such that ions can pass through them. For example, [Fig. 1] shows two disk or ring-shaped electrodes. The disk electrodes have a hole through which the ions pass. The electrodes can be ring-shaped or mesh-shaped. [Fig. 2] shows a mesh electrode 20 which, in operation, allows ions to pass through the mesh holes. Other configurations are covered by this disclosure as long as ions can pass through the electrodes, in use.A circular shape, for example disc or ring-shaped, allows, when used and applying a voltage potential difference to the ions passing through the holes of the electrodes, that the ions form a flattened or elongated ion cloud (depending on ionization, acceleration and dispersion during propagation), which is guided and / or modified by the electric field. In the case of disc or ring-shaped electrodes, as shown in [Fig. 3], an electrode may have an external diameter 31 of, for example, between 50 mm and 60 mm or between 2 inches and 3 inches, and an internal diameter of, for example, between 20 mm and 30 mm or between 1 inch and 1.5 inches.

[0053] In use, a disc or ring-shaped electrode provides a direct line of sight to the detector, which in the examples may be an SEM or an MCP. This advantage becomes evident in the examples that follow in accordance with this disclosure. The electrodes in the accelerator unit may have different shapes: for example, a first electrode may be a mesh electrode and a second electrode may be a ring electrode. A third electrode may be a square electrode having a hole through which ions may pass. The holes through which ions pass may not be aligned from one electrode to the other. The holes through which ions pass may not be aligned from one electrode to the other, in which case the ions may divert the path from one electrode to the next.

[0054] [Fig. 4] schematically represents a high mass ion detector 40 in accordance with the present disclosure. The high mass ion detector comprises, as shown in [Fig. 4], a secondary electron multiplier, SEM 47, for converting ions into electrons and for multiplying the number of electrons inside the SEM, where the SEM comprises a front face 45 and a rear face 46 where, in use, ions enter from the front face; and the accelerator unit 41 separated by a distance 48, between 100 micrometers and 10 mm, from the front face of the SEM. The accelerator unit 41 of [Fig. 4] comprises a first electrode 42, a layer insulating electrode 43 and a second electrode 44. In operation, as shown in [Fig. 5], ions moving in the direction of the arrow enter the high mass ion detector 40 via the first electrode 42 and can exit through the rear face. Ions exiting the accelerator unit travel a distance of between 100 micrometers and 10 mm before striking the SEM 47. As shown in [Fig. 5], electrons 53 exiting the SEM can accumulate in an anode 52 and the electron shower can be detected.

[0055] The example shown in Figures 4 and 5 shows two circular electrodes or multiplier electrodes. The electrodes may be separated by polytetrafluoroethylene (PTFE) plates for electrical insulation. The SEM shown in Figures 4 and 5 is an MCP microchannel plate. In the examples, two MCPs assembled in a chevron configuration are used. [Fig. 5] shows a metal anode 52. The elements may be fastened together by polyether ether ketone (PEEK) screws and nuts and may rest on an electrically insulated base, for example a PTFE base for electrical insulation from a vacuum flange located below.

[0056] [Fig. 6] shows an example of a conventional mass spectrometer comprising a laser source 61, a matrix with analyte 62, an ionization region 63, a TOF tube 64 in which the ions move in the direction of the arrow and are separated according to their mass or m / z values, and an MCP detector 65.

[0057] [Fig. 7] shows a mass spectrometer 70 in accordance with the present disclosure, comprising an ionization region 73; a laser source 71 configured to illuminate the ionization region, so that ions 75 are obtained from an illuminated sample 72 lying on the ionization region; a time-of-flight, TOF, tube mass analyzer 74, the TOF tube being in communication with the ionization region 73 so that ions can pass from the ionization region to the TOF tube; and the high mass ion detector 40 in accordance with this disclosure, wherein the high mass ion detector is in communication with the TOF tube, and wherein the accelerator unit 41 of the high mass ion detector is located between the TOF tube 74 and the SEM 47. As may become apparent, the MCP detector 65 of [Fig.6] can be replaced by the high mass ion detector 40 in accordance with the present disclosure to obtain a mass spectrometer 70 in accordance with the present disclosure. As can be seen, despite the extension of the acceleration unit, the ions still have direct access to the surface of the SEM.

[0058] In operation, one power supply, or two or more power supplies may be used. Two separate first power supplies may be used to apply negative voltages to each of the electrodes, for example up to -4 kV. A third power supply may be coupled to a voltage divider circuit to ground one input of the SEM. In examples where the SEM includes two microchannel plates MCPs assembled in a Chevron configuration, the third power supply may be coupled to a voltage divider circuit to ground one input of the first MCP and the second MCP to approximately +1.8 kV and the anode to +2 kV for positive ion detection. High potentials may be achieved by combining more than 2 electrodes. The configuration may be flexible depending on the application needs.

[0059] Compared to the conventional SEM detector, the signal intensity provided by the high mass ion detector of the present disclosure is amplified by a factor of 24.3 for a charged ion of bovine serum albumin, BSA (BSA, >98%, molecular weight 66,400 u), BSA ion (m / z 66,400) and 10.7 for a charged ion of immunoglobulin G, IgG, from human serum (IgG, >95%, molecular weight 150,000 u), IgG ion (m / z 150,000) and higher up to the MDa range.

[0060] In examples, different voltages may be applied independently to the electrodes, for example between zero and -4 kV in 1 kV steps, and different distances defined by the insulation layer, for example a PTFE spacer, between the electrodes are constructed. The different combinations of configurations may give different results. For example, this allows testing for a configuration that results in the greatest signal intensification for a particular application and with a particular analyte sample. In one example, a 6 mm thick insulation layer was used, referred to as a "long distance configuration". In a second example, referred to as a "medium distance configuration", a 2 mm thick insulation layer was used.In a third example, called the "short-distance configuration," a 10-micrometer insulating layer was used, which can function as a "zero-gap" accelerator unit—or a short-distance configuration, which, combined with the establishment of zero voltage across the electrodes, corresponds to the stopping of the accelerator unit. Sample preparation

[0061] The experiments performed included the use of the standard proteins bovine serum albumin (BSA, >98%, molecular weight 66,400 u) and immunoglobulin G from human serum (IgG, >95%, molecular weight 150,000 u), acetone, acetonitrile, acetonitrile with 0.1% trifluoroacetic acid (TFA) and water with 0.1% TFA (all LC-MS grade), the two MALDI matrices a-cyano-4-hydroxycinnamic acid (a-CHCA) and 2,5-dihydroxybenzoic acid (DHB), formic acid (>99%, LC-MS grade), water purified using an on-site purification system). Various protocols were initially evaluated for MALDI sample preparation, all using manual pipetting for matrix deposition onto the target plate. First, both SAB and IgG proteins were dissolved in 0.1% TFA in water and adjusted to a concentration of 3.0 pmol / l and 1.3 pmol / l, respectively. For the MALDI matrix, 7.0 mg of α-CHCA was added to 100 μl of acetone to prepare a saturated solution. Using a 10 μl pipette tip, which had been briefly wetted with this solution, a thin layer was applied to the MALDI target (ground steel) and allowed to dry. In addition, 20 mg of α-CHCA was dissolved in 1 ml of a mixture of acetonitrile and 5% formic acid in water (70 / 30, v / v). Also, 20 mg of DHB was taken up in 1 ml of a mixture of acetonitrile and 0.1% TFA in water (70 / 30, v / v). These two solutions were mixed in a ratio of 1 / 1 (v / v). Then, 2 μl of the BSA solution or 2 μl of the IgG protein solution were briefly mixed with 2 μl of the a-CHCA and DHB solution. 0.5 μl of this new solution obtained was added to the thin layer of a-CHCA.The spots were allowed to dry under ambient conditions and were then measured directly. Measures

[0062] The measurements were performed on a modified ultrafleXtreme TOF / TOF MALDI system in linear positive ion mode. The instrument is equipped with a Smartbeam 2 laser. The MALDI source control was performed via flexControl software (Bruker Daltonics). The method used was optimized for the measurement of m / z values ​​in the range 30,000 to 210,000 with the discrete dynode detector of the mass spectrometer and was provided by the instrument manufacturer (accelerating voltage U = 25 kV, pulsed ion extraction at 460 ns). The measurements were performed in positive polarity mode and 100 laser shots at a frequency of 20 Hz were averaged to obtain a mass spectrum. The detector side of the mass spectrometer was modified by removing the discrete dynode detector and replacing it with the high mass ion detector 40 in accordance with this disclosure. A charge lock chamber was installed for convenient exchange of detectors.Calibration measurements with the conventional MCP detector revealed an adjusted flight path length of approximately d = 2.21 m. The high-mass ion detector 40 according to this disclosure comprised: two circular multiplication electrodes, referred to as BEs, separated by polytetrafluoroethylene (PTFE) plates for electrical isolation, two MCP microchannel plates assembled in a Chevron configuration (F1552-01, Hamamatsu Photonics, Hamamatsu City, Japan), and a metal anode. The assembly was secured by polyether ether ketone (PEEK) screws and nuts and rested on a PTFE base for electrical isolation from the vacuum flange below. Each initial measurement was made at least 24 hours after mounting the high mass ion detector 40 in accordance with this disclosure under a high vacuum (less than 2 105 Pa) to allow adequate degassing and thus reduce the risk of electrical discharge. in the MCPs. To provide the electrical voltages to the individual components of the high-mass ion detector as described, three external high-voltage power supplies were used. Separate power supplies were used to apply negative voltages to each of the two BE multiplication electrodes (down to -4 kV). The third device was coupled to a voltage divider circuit to ground the input of the first MCP, the second MCP to approximately +1.8 kV, and the anode to +2 kV. A capacitor was assembled between the anode and an external oscilloscope (DSO9254A, Keysight Technologies, Santa Rosa, CA, USA) to suppress any DC noise and to acquire only the AC signal produced by the ion impact. The oscilloscope input was set to 1 MΩ to match the impedance between the oscilloscope and the rest of the circuit to provide a better signal response.An external oscilloscope was used to record the signal amplitude in volts as a function of time of flight, and the trigger signal from the mass spectrometer was fed to one channel of the oscilloscope to mark time of flight zero. Then, the signal was acquired for 500 ps (sampling rate 500 MSa s1, sample size 250 kSa). To obtain a single mass spectrum, the signal generated by 100 laser shots was averaged using the oscilloscope's averaging function. The voltages applied to the MCP-anode assembly were kept constant, but the voltages supplied to the two electrodes were varied independently between zero and -4 kV to maximize the intensity of the signal generated by the arriving ions. Data processing

[0063] Before further evaluation of the measured data, the time of flight can be converted into m / z value using the following equation: [Math 2] f =^ü.(t / d) 2

[0064] with an accelerating voltage U, for example U = 25 kV; a determined length of the flight path d, for example d = 2.21 m; an electronic charge e = 1.602*1019 C; and an atomic mass unit u, for example u = 1.661*1027 kg.

[0065] Then, the baseline of the mass spectrum was corrected using the "msbackadj" function of MATLAB (MathWorks, Natick, MA, USA). Then the peak intensities in the mass spectrum were extracted using the "mspeaks" function for the determination of the signal amplification caused by the different voltage and distance configurations tested for the high mass ion detector according to the disclosure in comparison with the conventional MCP assembly. The variations in the intensity of the signal generated by a single bovine serum albumin ion (BSA +1, at about m / z 66,400) and by doubly charged BSA ions (BSA +2, at about m / z 33,200) were studied as a function of the change in accelerating electrode voltages, BE, for the three high-mass ion detector distance configurations—long-distance configuration, medium-distance configuration, and zero-space or short-space configuration. It should be noted that this discussion may refer to the "zero-space distance" or "short-space configuration" when using a 10-micrometer insulating layer, which can function as a "zero-space" accelerating unit—or to the short-space configuration, which, combined with zeroing the voltage across the electrodes, corresponds to the switching off of the accelerating unit. Five mass spectra were obtained for each voltage combination.Mean intensity values ​​and standard deviations were derived from the five voltage-combining mass spectra for singly and doubly charged BSA ions to account for changes in signal intensity that are usually related to local deviations in MALDI sample quality ("sweet spots"). To enable direct comparison of the acquired mass spectra, amplification factors (AFs) were determined by normalizing the peak intensities measured with the high-mass ion detector of the presentation, also called BMCP for "booster MCP", relative to the value obtained with the BEs switched off, i.e., with the conventional MCP detector. Errors in the AFs were derived by Gaussian error propagation using the standard deviations of the peak intensities for the calculation.

[0066] [Fig. 8] shows the amplification factors, AF, for SAB with three different examples of high-mass ion detectors according to the discussion. The AFs found for the large electrode separation are shown in [Fig. 8](a), for the medium separation in [Fig. 8](b), and for measurements with zero electrode spacing in [Fig. 8](c). The AFs for doubly charged and singly charged SAB ions are shown in the top and bottom rows of the image, respectively. For the large and medium electrode separation, all combinations of electrode voltages between zero and -4 kV were tested in 1 kV steps. It should be noted that, for the accelerator configuration with zero electrode separation—resulting in a single electrode—a single high-voltage power supply was used to apply the voltages between zero and -4 kV in 1 kV steps.In the following, the results for the doubly charged SAB ion will first be compared for the different interelectrode distances. Then, the AFs derived for the singly charged SAB ion will be examined. For the large BE distance shown in [Fig.8](a), a maximum amplification factor, AF, of 3.4 ± 1.0 was found for -3 kV at the lower BE and for -4 kV at the upper BE, as well as for the reverse voltage combination. A reduced AF, of 2.0 ± 0.7, was obtained for -4 kV at both electrodes. The largest AF, of 13.4 ± 7.4, for SAB +2, was found for the distance . average between electrodes with -3 kV at the lower BE and -4 kV at the upper BE, as shown in [Fig.8](b). In a similar manner to the results obtained for the large interelectrode separation, a comparable AF was derived for the reverse voltage combination and a slightly lower AF was found when the same voltage of -4 kV was applied to both BEs. Furthermore, a local maximum of 9.1 ± 5.3 was observed for a voltage of -2 kV at the lower BE and -1 kV at the upper BE. For zero interelectrode distance, the AF varied between 3.9 ± 2.1 and 4.4 ± 3.7 with changing electrode voltage. It should be noted that the signal intensity for -4 kV was not sufficient to derive a value for the AF. Similar observations to those for the doubly charged SAB ion were made for the singly charged SAB ion, as shown in the bottom row of [Fig.8].Again, the largest AF of 24.3 ± 8.9 was obtained for the medium interelectrode spacing ( [Fig.8](b)) for a combination of -3 kV at the lower BE and -4 kV at the upper BE. Slightly lower AFs were found for the reverse voltage combination and for -4 kV applied to both BEs. In addition, a local maximum was also found with the voltage combination of -2 kV and -1 kV at the lower and upper electrodes respectively, resulting in an AF of 15.0 ± 8.6. Significantly lower AF values ​​were observed for the large interelectrode separation ([Fig.8](a)) as well as for the zero interelectrode separation ([Fig.8] (c)). The largest AF of 13.4 ± 14.7, obtained for the large electrode spacing with -3 kV at the lower electrode and with the upper electrode switched off, can possibly be considered an outlier.In general, greater signal amplification was obtained with a higher negative voltage applied to the BEs, regardless of the detector configuration used. The observed behavior is assumed to have been generated by increased acceleration of positively charged ions with increasing negative multiplication voltage, by ion optical effects, or by a combination of these two effects.

[0067] On the one hand, a higher negative voltage will likely result in a stronger acceleration of the positively charged ions before they hit the MCP surface. Thus, the intensification of AF may be related to the well-known increase in MCP detection efficiency with increasing ion velocity. The strongest signal intensification for singly and doubly charged BSA ions was observed for the medium electrode separation ([Fig.8](b)). On the other hand, one could identify two well-known, but opposing, ion-optical mechanisms that could have led to the observed signal amplification: First, the used electrode configuration may cause - in addition to the expected ion acceleration - some spatial focusing of the ion beam, as has been noticed for other electrode configurations before. Second, the Negative electrode multiplication potential may cause ion beam divergence. Thus, the positively charged ion cloud would cover a larger area of ​​the MCP active surface, which could be advantageous with respect to the relatively long dead time (in the order of milliseconds) of single channels. It should be noted that only one of these two opposing ion optical effects may have contributed to the observed increase in signal intensity.

[0068] Compared to the findings for the medium spacing, the AFs were reduced for the large electrode separation. In this configuration, the BEs may be too far apart to effectively accelerate and concentrate the ions. It is possible that the positively charged ions are attracted to the negative BEs and therefore strike them instead of reaching the MCPs due to the elongated flight path in the multiplier. Thus, fewer ions would have reached the MCP surface for the large electrode distance, which could have led to the observed reduction in signal intensity compared to the medium electrode separation.

[0069] The AFs found for zero electrode separation are generally higher than the values ​​observed for large electrode spacing. However, the signal amplification is significantly lower than for the average electrode configuration. Therefore, it can be expected that BE alone (zero electrode spacing) leads to an increase in ion velocity, but has no distinct influence on the spatial distribution of the ion beam. In general, assemblies that are designed for focusing an ion beam consist of several electrostatic lenses, such as the three-element Einzel lens, which is a combination of tubular accelerating lenses of opposite polarities.

[0070] The strongest signal amplification was not observed for the highest voltage (-4 kV) applied simultaneously to both multiplication electrodes, but for a multiplication voltage combination of -3 kV / 4 kV (lower / upper BE). It is possible that this voltage combination creates an electric field gradient that has an effect on the spatial extent of the ion beam and turns out to lead to an amplified detection efficiency at the MCPs. Furthermore, local maxima were detected at voltages lower than -4 kV for the large as well as the average inter-electrode separation. Therefore, the maximum voltages at the BEs are not necessary for obtaining a significant signal amplification by the high mass ion detector according to the disclosure.Furthermore, a shift of the local maximum was observed for the AF measured for singly and doubly charged SAB ions when the detector configuration was changed from medium to large electrode separation. This observation may indicate an effect of the length of the insulating layer between the two BEs on the . ion beam focusing properties of the multiplier.

[0071] In conclusion, [Fig.8] shows the signal amplification of singly and doubly charged SAB ions for three multiplication electrode configurations. The separation between the top and bottom electrodes, provided by an insulating layer, was changed from (a) a large distance to (b) a medium distance and finally to (c) zero distance between the two electrodes. The strongest amplification was observed for the medium electrode spacing. The peak intensities found for SAB +2 and SAB +1 were normalized to the measurements with the electrode voltages switched off for signal amplification extraction. The dark gray spaces indicate the absence of detectable signal. In addition, the different amplitudes in the color-coded 3D plots should be noted.

[0072] [Fig. 9] shows the amplification factors, AF, for IgG with three different examples of high mass ion detectors according to the disclosure. The AFs for IgG were derived from measurements made with BMCP in the three different configurations, as previously explained for SAB. The results for doubly charged (IgG +2, at about m / z 75,000) and singly charged (IgG +1, at about m / z 150,000) IgG ions are summarized in the top and bottom rows of [Fig. 9], respectively. The AFs are given in [Fig. 9](a) for the large inter-electrode separation, in [Fig. 9](b) for the medium distance, and in [Fig. 9](c) for the zero inter-electrode separation.

[0073] First, the results for the doubly charged IgG ion and then the observations for the singly charged IgG ion are discussed in the following. For the large electrode separation ([Fig.9](a)), the largest AF of 2.4 ± 2.0 for IgG +2 was found for a lower / upper electrode voltage combination of -2 kV7-4 kV. However, the AFs found for -4 kV at both electrodes as well as for both variants of -3 kV7-4 kV, vary within this standard deviation of the AF for -2 kV / -4 kV. For the medium electrode separation ([Fig.9](b)), a much larger AF of 23.6 ± 14.6 was found for -3 kV / -4 kV at the lower / upper BEs. Slightly lower AF values ​​were obtained for the reverse voltage combination and for -4 kV applied to both BEs. In addition, a local maximum of 11.1 ± 6.6 was measured for 2 kV / -1 kV at the lower / upper BEs. For zero separation between electrodes ([Fig.9](c)), significant variations in AF between 1.7 ± 1.3 and 6.8 ± 6.0 were observed for IgG +2. .

[0074] For the singly charged IgG ion, a maximum AF of 7.2 ± 0.2 was observed at -4 kV at the lower BE to -3 kV at the upper BE by use of the large interelectrode separation, as shown in the bottom row of [Fig.9](a). The AF was found to be smaller for the reverse voltage combination and for -4 kV at both BEs. A local maximum of 5.8 was found for -2 kV / -1 kV at the lower / upper BEs. Since the signal intensity measured with the multiplier switched off was not sufficient for detection and thus for calculating an AF, the peak intensities were normalized to the value found for -1 kV at the lower electrode and zero volts at the upper electrode. For the average electrode configuration in [Fig.9](b), a maximum AF of 10.7 ± 5.2 was found for -4 kV / - 3 kV at the lower / upper electrodes. For the other BE voltage combinations, comparable trends were observed, just as for the previously described results of IgG +2 measured with an average interelectrode distance. For zero separation between electrodes ([Fig.9](c)), no reliable signal amplification was observed because the AFs vary between 0.2 ± 0.2 and 1.2 + 2.1.

[0075] Similar to the observations for SAB in [Fig.8], the AFs generally increased as the voltage applied to the BEs increased, which could be related to the amplification of the detection efficiency of the MCP with the increase in ion velocity and ion focusing effects. In addition, the medium electrode separation also produced the strongest signal intensification for IgG compared to the other two BE configurations. The AF decreased only slightly between SAB +1 at about m / z 66,400 and IgG +2 at about m / z 75,000, namely from 24.3 to 23.7.

[0076] However, a significantly lower AF of 10.7 was found for IgG +1 which appears at a much larger m / z value of approximately 150,000. This decrease in AF with increasing m / z value is expected behavior, given that the accelerating voltages at BEs are the same for all approaching ions. Thus, higher mass ions are always slower than lighter ions when striking the MCP surface, which explains the mass-dependent detection efficiency of MCPs. It is noteworthy that the largest AF of the doubly charged IgG ion (at approximately m / z 75,000) was found for a combination of -3 kV at the lower BE and -4 kV at the upper BE, which is similar to the observations made for the doubly charged BSA ion (at approximately m / z 33,200) and the singly charged BSA ion (at approximately m / z 66,400).However, the strongest amplification for the singly charged IgG ion (at about m / z 150,000) was obtained for the reverse BE voltage combination of -4 kV / -3 kV (lower / upper BE). It is known that the expansion of the ion cloud in vacuum is expected to increase with the mass of the ions in the TOF mass analyzer, leading to a loss of control of the spatial distribution of the ions for higher mass ions. Therefore, the effect of the multiplier electric potential on the spatial extension of the ions is probably mass dependent, since ion clouds of different masses can approach the multiplier with different degrees of lateral expansion.

[0077] Furthermore, the local maxima for the average separation between electrodes were observed for -2 kV at the lower BE and -1 kV at the upper BE for SAB +2, SAB +1, as well as for IgG +2. Only for the singly charged IgG ion was another local maximum observed for a voltage combination of -3 kV / -1 kV at the lower / upper BE, which may be related to a mass-dependent expansion of the ion beam.

[0078] In conclusion, [Fig.9] shows the signal amplification of singly and doubly charged IgG ions for three multiplication electrode configurations. The separation between the top and bottom electrodes, provided by an insulating layer, changed from (a) a large distance to (b) a medium distance and finally to (c) zero distance between the two electrodes. The strongest amplification was again observed for the medium electrode spacing. The peak intensities observed for IgG +2 and IgG +1 were normalized to measurements made with the multiplication voltages turned off for signal amplification extraction, and the gray spaces indicate the absence of a detectable signal.However, the peak intensities measured with the large interelectrode distance for IgG +1 were normalized to the signal for -1 kV at the lower electrode and zero voltage at the upper electrode, since no sufficient peak intensity was detected for both electrode voltages turned off.

[0079] Among the three electrode configurations tested, the medium spacing between BEs produced the greatest amplification for the signal generated by the multiplied ions upon impact on the PCM surface. This configuration appears to cause efficient acceleration of the ions, and possibly also convenient focusing of the ion beam, which results in an amplified signal intensity compared to the conventional PCM detector. Since the detection mechanism relies on the conversion of analyte ions into secondary electrons using PCMs, the speed of the ions always determines the detection efficiency. Therefore, the AF is expected to decrease with increasing ion mass, since higher mass ions will always be slower than lighter ions, given that all ions experience the same electrical potential when passing through the BEs. [Fig.10] shows a direct comparison of the mass spectra obtained with conventional MCPs, plotted as a continuous line at the bottom of the graphs, and with BMCP with medium electrode spacing, represented by a line above the previous line, showing the strong signal enhancement obtained. The detected signal was most amplified for a voltage of -3 kV at the lower BE and -4 kV at the upper BE for SAB ([Fig.l0](a), plotted on the upper line) and for the reverse voltage combination for IgG ([Fig.l0](b), plotted on the upper line). In addition, a local maximum of the AF was observed for both proteins with the combination of -2 kV at the lower BE and -1 kV at the upper BE.

[0080] However, using the same voltage at both BEs did not result in the greatest signal enhancement, for -3 kV and for -4 kV. In the following, the discussion will focus on the mass spectra obtained for the multiplier settings that resulted in the greatest signal amplification for BSA and IgG in comparison with the results obtained with the multiplier part switched off. The signal for the doubly and singly charged BSA ion, see [Fig.l0](a), was amplified by a factor of 15.5 and 27.0, respectively, when detected with the BMCP, plotted on the upper line, rather than with the conventional MCP, plotted on the lower line. The combination of voltages of -3 kV at the lower BE and -4 kV at the upper BE generated the largest AFs for BSA.Additionally, the average peak intensity and corresponding standard deviation are presented for SAB +2 and SAB +1 indicating the variation among the five mass spectra obtained for this voltage combination. For the doubly charged SAB ion, the average intensity increased from 0.0016 ± 0.0007 to 0.0218 ± 0.0072, and for the singly charged SAB ion, the intensity increased from 0.0009 ± 0.0002 to 0.0210 ± 0.0057 when using BMCP. Thus, the absolute variation in signal intensity for successively measured mass spectra was found to increase with AF values ​​and, consequently, with BE voltages. Furthermore, the full width at half maximum (FWHM) was derived for the peaks attributed to singly and doubly charged SAB ions in [Fig.l0](a) as a measure of the change in mass resolving power when using the BMCP instead of the MCP detector.The FWHM increased from 1300 u to 1900 u for the signal at m / z 33,200 (SAB 2+) and decreased from 4300 u to 2900 u for the signal at m / z 66,400 (SAB 1+) when the BMCP was used instead of the conventional MCP detector. A laser turn-on frequency of 20 Hz can be used to average the signal produced by 100 single laser shots. It is possible that modifications to the BMCP detector measurement, such as improvements to the data acquisition system, may allow a direct comparison between the conventional system and the MCP / BMCP detector as discussed.

[0081] [Fig.10](b) compares a single IgG mass spectrum measured with the conventional MCP (lower line) with a spectrum acquired with the high mass ion detector according to the disclosure (upper line). For the mass spectra shown, AF values ​​of 14.3 for IgG +2 and 9.2 for IgG +1 were found with -4 kV at the lower BE and -3 kV at the upper BE. The average signal intensity of the five mass spectra at this voltage combination increased for the doubly charged IgG ion from 0.0009 ± 0.0003 to 0.0141 ± 0.0049, and for the singly charged IgG ion from 0.0009 to 0.0099 ± 0.0048. Again, an increase in signal intensity variation is observed with increasing AF value and, therefore, with increasing BE voltages. The FWHM increased from 3,700 u to 4,200 u for the signal at m / z 75,000 (IgG 2+), and it was reduced from 10,800 u to 3,500 u for the signal at m / z 150,000 (IgGl+) when using the high mass ion detector as discussed. The variation of FWHM with BE adjustment is illustrated for both proteins. No significant variation in m / z values ​​was observed for the different ions depending on the BE voltage combinations.

[0082] Increasing the accelerating voltage in the ion source may be the obvious way to further improve the detection efficiency of the PCM. However, the applied voltage is in practice limited by the increase in ion decay in the source with increasing accelerating voltage. Thus, the increase in intensity variations with increasing BE voltage may be related to an increased probability of fragmentation of ions as they pass through the multiplication electrodes for acceleration purposes. Furthermore, the variation in peak width with changing BE voltage may be related to the spatial distribution of the ion beam. For example, a reduction in ion mass resolution has been reported elsewhere for the combined use of a conversion dynode and a post-acceleration stage compared to the conventional PCM detector.The observed reduction in resolution was attributed to the influence of the post-acceleration stage on the ion beam. The overall reproducibility of measurements performed with the high-mass ion detector as discussed can be improved by applying matrix deposition methods other than the manual pipetting used here. Other matrix deposition techniques, such as sublimation and nebulization, are known to generate MALDI samples with better homogeneity and purity of the deposited matrix, which could reduce the observed variation in signal intensity. Comparison of peak intensities for BSA and IgG revealed a mass-dependent decrease in signal intensity. The average AF derived for the singly charged BSA ion (24.3) was reduced to approximately 56% of the singly charged IgG ion (10.7).Thus, the higher mass IgG ion will always arrive at the PCM surface with a lower velocity than for the lower mass BSA ion, which explains the mass-dependent detection efficiency of PCM. Furthermore, it cannot be excluded that the decrease in signal intensity for IgG is also related to the spatial expansion of the ion cloud when approaching the multiplier extension at the PCM detector. Since the expansion of the ion cloud in vacuum is assumed to increase with ion mass, the decrease in signal intensity could also be associated with the loss of higher mass ions when they hit the BEs instead of reaching the PCM. Furthermore, several peaks below the m / z values ​​of the doubly charged BSA ion (< m / z . 33,200) and the doubly charged IgG ion (< m / z 75,000) were observed in both mass spectra of [Fig. 10], which are assumed to correspond to higher charge states of BSA and IgG. A similar observation was already made by Choi et al, where a strong correlation with the MALDI matrix used was observed. Since multiply charged ions were observed for both MCP and BMCP measurements, their occurrence is likely related to other influences, such as the properties of the matrix type used. When the AF found for the doubly charged BSA ion (m / z 33,200) is compared with the value found for the doubly charged IgG ion (m / z 75,000), an increase from 13.4 to 23.7 is observed, which indicates a dependence of the AF on the m / z value.

[0083] [Fig. 11] shows the AF values ​​for higher charge states of the ions of SAB and IgG ions. The figure can be divided into the following three m / z regions: First, the signal amplification generated by the multiplier appears to be most effective for ions in the m / z range of approximately 50,000–75,000, and second, the AF decreases for ions with an m / z value above approximately 75,000 due to the mass-dependent detection efficiency of MCP. Third, for m / z values ​​below approximately 50,000, the AF decreases as the m / z value decreases. Ions with lower m / z values ​​have a greater velocity than ions with higher m / z values, since the velocity of ions is inversely proportional to the square root of their m / z value. Therefore, the acceleration effect of the multiplier on ions with lower m / z values ​​may be smaller, because they pass through the multiplier portion faster.It is possible that the reduction in relative AF with increasing ion velocity (smaller m / z values) is due to less efficient acceleration generated by the multiplier than for slower moving ions that have larger m / z values. It should be noted that the study of other ionized molecules in the future may reveal further details of the observed dependence of relative signal amplification on m / z value.

[0084] Conclusions: An extension of the conventional MCP detector was used for signal amplification of standard protein ions up to at least m / z 150,000. Three configurations of the high mass ion detector according to the disclosure were tested to maximize the amplification factor. The medium electrode separation provided the greatest signal amplification by generating a practical combination of acceleration of positively charged ions by the negative multiplying electrodes and ion optical effects. It is likely that the spatial distribution of the ion beam is affected by the electrical multiplication potential generating the increased signal intensity observed for this BMCP configuration, unlike the other two designs. In general, the signal amplification is became stronger for higher applied multiplication voltages. However, the largest amplification factors were found for combinations of -3 kV and -4 kV at the multiplication electrodes. In addition, a maximum local amplification factor was detected for the voltage combination of -2 kV at the lower electrode and -1 kV at the upper multiplier electrode, which offers the possibility of operating the high-mass ion detector according to the disclosure with increased sensitivity already at electrical potentials of up to 2 kV. The absolute signal intensity was found to decrease with increasing mass. This behavior is expected since the same multiplication voltage was used for all masses, and therefore higher-mass ions will remain slower than lighter ions after passing the multiplication stage.Nevertheless, the influence of the multiplier part proved sufficient to enable reliable detection of m / z values ​​up to 150,000. The accessible m / z range of BMCP can be extended by applying higher electrical voltages to the multiplication electrodes. It should be noted that the concept presented here is not limited to proteins, but could also be applied to the analysis of high-mass substances, e.g., polymers and dendrimers.

[0085] In addition, a ground grid may be used in combination with the high mass ion detector in accordance with the disclosure to avoid saturation of the MCP channels by the pulsed deflection of the first arriving low mass ions, which could lead to further amplification of the signal generated by the high mass ions. Such an experiment may require rapid switching (on the order of microseconds) between zero (or low) and high applied voltages.

[0086] For the sake of completeness, various aspects of this disclosure are set forth in the following numbered clauses: One aspect relates to a high mass ion detector for a mass spectrometer, the high mass ion detector comprising: - a secondary electron multiplier, SEM, for converting ions into electrons and for multiplying a number of electrons within the SEM, which SEM comprises a front face and a back face where, in use, ions enter through the front face; and - an accelerator unit separated from the front face of the SEM by a distance of between 100 micrometers and 10 millimeters; which accelerator unit comprises at least two electrodes, each pair of electrodes being separated by an insulating layer for electrical insulation; and - in which the accelerator unit is separated by a distance of between 100 micrometers and 10 millimeters from the front face of the SEM. Clause 2. The high mass ion detector of clause 1, wherein the insulating layer has a thickness of between 100 micrometers and 10 millimeters. The insulating layer may have a thickness of 2 mm or a thickness of 6 mm. At least one of the electrodes and / or one of the insulating layers may have a circular shape or an annular shape. A first electrode and a second electrode may be separated by a first insulating layer and the second electrode and a third electrode are separated by a second insulating layer. One aspect relates to a mass spectrometer comprising - an ionization region; - a laser source configured to illuminate the ionization region, so that ions are obtained from an illuminated sample resting on the ionization region; - a time-of-flight tube, or TOF tube, mass analyzer, the TOF tube being in communication with the ionization region; and - the high mass ion detector as defined above, which high mass ion detector is in communication with the TOF tube, and wherein the acceleration unit of the high mass ion detector is located between the TOF tube and the SEM of the high mass ion detector. One aspect relates to a method for accelerating ions, comprising - providing an acceleration unit which comprises at least two electrodes, which acceleration unit is configured to operate in a mass spectrometer, the acceleration unit comprising at least two electrodes, and wherein each pair of electrodes is separated by an insulating layer for electrical insulation - applying a voltage potential difference to the at least two electrodes of the acceleration unit; and - supplying ions to the accelerating unit; thereby exposing the ions to the voltage potential difference, which ions are affected by an accelerating force given by the voltage potential difference. Ion delivery can be accomplished by ionization of a sample analyte. One aspect relates to a method of detecting high mass ions, the method comprising: - the provision of a mass spectrometer as defined above, the mass spectrometer comprising a sample of analyte; - ionization of the analyte sample and release of charged ions from the analyte sample; - after all or part of the charged ions have passed through the TOF tube, exposing the charged ions that have passed through the TOF tube to voltages applied to the at least two electrodes of the accelerator unit, thereby accelerating the exposed charged ions; - converting the accelerated charged ions leaving the electrodes into secondary electrons by letting the accelerated charged ions strike the SEM; and - detection of the presence of high mass ions based on secondary electrons. Ionization of the analyte sample can be achieved by illuminating the sample with a laser source, thereby transferring the sample into a gas phase.

[0087] Although only a few examples have been set forth herein, other alternatives, modifications, uses and / or equivalents are possible. Furthermore, all possible combinations of the described examples are also covered. Thus, the scope of the present disclosure should not be limited by particular examples, but should be determined solely by a fair reading of the claims that follow.

Claims

Claims

1. A high mass ion detector for a mass spectrometer, the high mass ion detector comprising: • a secondary electron multiplier, SEM (47), for converting ions into electrons and for multiplying a number of electrons within the SEM, which SEM comprises a front face (45) and a back face (46) where, in use, the ions enter from the front face; and • an accelerator unit (41) for accelerating the high mass ions, separated from the front face of the SEM by a distance of between 100 micrometers and 10 millimeters; the accelerator unit being configured to operate in a mass spectrometer, the accelerator unit comprising at least two electrodes (42, 44), and each pair of electrodes being separated by an insulating layer (43) for electrical insulation.

2. A high mass ion detector according to claim 1, wherein the accelerator unit (41) is separated from the front face of the SEM (47) by a distance of between 100 micrometers and 1 millimeter.

3. A high mass ion detector according to claim 1, wherein the accelerator unit (41) is separated from the front face of the SEM (47) by a distance of between 1 millimeter and 10 millimeters.

4. A high mass ion detector according to claim 1, wherein the accelerator unit (41) is separated from the front face of the SEM (47) by a distance of 100 micrometers.

5. A high mass ion detector according to any one of claims 1 to 4, wherein the insulating layer (43) of the accelerator unit (10) has a thickness of between 100 micrometers and 10 millimeters.

6. The high mass ion detector of claim 5, wherein the insulating layer (43) has a thickness of 2 mm or a thickness of 6 mm.

7. A high mass ion detector according to any one of claims 1 to 6, wherein the insulating layer (43) is composed at least in part of polytetrafluoroethylene (PTFE).

8. A high mass ion detector according to any one of claims 1 to 7, comprising two electrodes (42, 44), wherein a first electrode and a second electrode are separated by a first insulating layer (43).

9. A high mass ion detector according to any one of claims 1 to 8, comprising three electrodes, wherein a first electrode and a second electrode are separated by a first insulating layer, and the second electrode and a third electrode are separated by a second insulating layer.

10. A high mass ion detector according to any one of claims 1 to 9, wherein at least one of the electrodes (42, 44) and / or each of the insulating layers (43) has a circular shape or an annular shape.

11. A mass spectrometer (70) comprising: • an ionization region (73); • a time-of-flight tube, or TOF tube, mass analyzer (74), the TOF tube being in communication with the ionization region; and • the high mass ion detector (40) of any one of claims 1 to 10, wherein the high mass ion detector is in communication with the TOF tube, and wherein the acceleration unit (41) of the high mass ion detector is located between the TOF tube (74) and the SEM (47) of the high mass ion detector.