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.

Description

[Technical Field]

[0001] The present disclosure relates to a high mass ion detector for a mass spectrometer, and mass spectrometer Place Regarding. [Background technology]

[0002] Top-down proteomics is a method of protein identification that can use an ion trap mass spectrometer to store isolated protein ions for mass measurement and tandem mass spectrometry (MS / MS) analysis, or can use other protein purification methods such as two-dimensional gel electrophoresis in conjunction with MS / MS. Top-down proteomics deals with the characterization of intact biomolecules, reducing sample complexity and facilitating the detection of modifications at the protein level. The combination of matrix-assisted laser desorption / ionization (MALDI) technology with a time-of-flight (TOF) mass analyzer generates gaseous ions of low charge state from high-mass biomolecules, allowing for their subsequent mass-to-charge ratio (m / z) separation, as high-mass ions drift through the flight tube more slowly than lighter ones. Matrix-assisted laser desorption / ionization-time-of-flight (MALDI-TOF) instruments are widely used for the mass analysis of macromolecules. To determine the m / z values ​​of analytes using a MALDI-TOF instrument, the sample is mixed with a matrix that absorbs and transfers the energy of a pulsed laser beam to the analyte molecules. In this way, ions are usually obtained, which are first accelerated and then pass through a flight tube of known length. Signal amplification is performed using a secondary electron multiplier SEM, such as a discrete dynode or microchannel plate MCP, from which the analyte ions emit electrons upon impact. These electrons are multiplied by further dynodes or in individual channels of the MCP unit. Using an anode behind the SEM, the electron shower can be detected.

[0003] For example, MCP detectors may be incorporated into MALDI-TOF instruments because 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 of different mass and charge within the flight tube of a MALDI-TOF device, all ions are first brought to the same kinetic energy E in the ion source by an accelerating voltage U applied between the MALDI sample plate and the counter electrode before entering the field-free TOF mass analyzer. kin Based on the formula,

number

[0005] Because ion velocity is inversely proportional to the square root of the ion mass, high-mass molecules exhibit relatively slow velocities. It is known that the probability of secondary electron generation due to ion collisions decreases with decreasing ion velocity, resulting in a significant decrease in SEM detection efficiency with increasing ion mass. This means that ion-to-electron conversion decreases, and instead, more secondary ions are generated by the impact of analyte ions on the MCP. Therefore, signal intensity decreases significantly for higher-mass ions, posing a significant limitation in MALDI-TOF applications. This effect is further enhanced for complex samples, as even small ions that first reach the detector can lead to oversaturation of individual MCP channels.

[0006] Some solutions to address the aforementioned inefficiencies of MCP detectors include cryogenic detectors operating at temperatures below 100 mK. However, these designs have not been accepted for routine use due to their complex and expensive cooling systems and small active detector area. Some approaches to extend the accessible mass range are based on modifying conventional MCP detectors, either by changing the detection mechanism for the electron signal emerging from the rear of the MCP or by converting slow-moving, high-mass ions into electrons or smaller secondary ions before impinging on the front of the MCP. Generally, signal intensity from MCP detectors can be improved by increasing the velocity of the arriving charged particles, which can be achieved by accelerating them at large potentials. Ions can be accelerated by voltages up to 30 kV in the ion sources of commercial mass analyzers. Several electrode configuration combinations can be found to enable further spatial focusing of the ion beam. Higher accelerating voltages in the ion source pose a risk of discharge and can lead to shorter flight times, necessitating very fast and expensive measurement equipment to achieve sufficient mass resolution. Summary of the Invention

[0007] The present disclosure provides a device that at least partially overcomes some of the aforementioned drawbacks. S Provide an example.

[0008] In a first aspect, a high-mass ion detector for a mass spectrometer is provided, comprising a secondary electron multiplier, a SEM, and an acceleration unit for accelerating high-mass ions. The SEM includes a front side and a rear side through which ions can enter and exit the front side during use. The SEM can have an outer dimension ranging from 20 mm to 60 mm, e.g., an outer diameter of 30 mm, 40 mm, 50 mm, or 60 mm for a ring-shaped SEM. In some embodiments, the SEM is an MCP. The MCP includes multiple channels so that ions can enter and exit each channel. The MCP can be used as a detector together with a TOF tube of a mass spectrometer. The channels of the MCP may typically be slightly tilted relative to the axis of the TOF tube, allowing the maximum possible number of ions to collide with the walls of the MCP and induce electrons into the MCP channels, thereby exciting additional electrons. Ions may remain as debris on the surface of the MCP, which can ultimately reduce the responsivity of the MCP. In some embodiments, the SEM is a dynode, depending on how the dynode's electrodes are positioned, i.e., whether they block the TOF line or whether they are positioned along the TOF line of flight. The acceleration unit faces the front of the SEM and is separated from the front of the SEM by a separation distance between 100 micrometers and 10 mm, or 1 millimeter and 10 mm, or 100 micrometers and 1 millimeter, such that ions exiting the acceleration unit travel the separation distance before impacting the SEM. The SEM, in use, converts ions to electrons, increasing the number of electrons within the SEM.

[0009] An acceleration unit for accelerating high-mass ions includes at least two electrodes separated by an insulating layer for electrical insulation. The electrodes pass ions through holes in, for example, a disk or ring electrode or a mesh electrode. During use, ions passing through the electrodes are accelerated by a voltage potential difference applied to the at least two electrodes. The acceleration unit is configured to operate with a mass spectrometer, which may include an electrode dimension, e.g., an outer electrode dimension, ranging from 10 mm to 60 mm. For example, the outer diameter of the ring electrode may be ranging from 10 mm to 60 mm. For example, the outer diameter of the ring insulating layer may be ranging from 10 mm to 60 mm. In an example, the acceleration unit includes two electrodes and an insulating layer separating the two electrodes. In an embodiment, the acceleration unit includes three 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, the first and second electrodes may be separated by a first insulating layer, and the second and third electrodes may be separated by a second insulating layer. In use, different voltages may be applied to the three or more electrodes such that different combinations of voltage potential differences accelerate ions passing through the electrodes. For example, a first voltage potential difference between the first and second electrodes may accelerate ions with a first accelerating force, and a second voltage potential difference between the second and third electrodes may further accelerate ions with a second accelerating force.

[0010] In some examples, the distance defined by the insulating layer between the electrodes can be varied by changing the thickness of the insulating layer. The thickness of the insulating layer can affect the voltage that can be applied to the electrodes to obtain different accelerations for different ions. Ions exhibiting specific m / z values ​​can be accelerated by the acceleration unit of the present disclosure by changing the voltage 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. Thus, it may be possible to design the acceleration unit of the present disclosure so that ions having masses falling within a specific range of m / z values ​​are accelerated as desired. The design can include different thicknesses of the insulating layer leading to a unit that can apply different voltages to the electrodes to provide a specific acceleration force. Higher m / z values ​​of ions to be accelerated require higher voltages, and therefore a higher thickness of the insulating layer can be designed.

[0011] When used with an SEM, the acceleration unit of the present disclosure allows measurements to be made and thereby high-mass ions to be detected. High-mass ions include ions with m / z values ​​from 50,000 to 1,500,000 Daltons, where 1 Dalton refers to the mass of a single hydrogen atom, but the units are calibrated to 1 / 12 of a C-12 atom. In this disclosure, the m / z value refers to the mass in Daltons divided by the charge state z of the ion / protein. In MALDI, values ​​for z = 1, 2, or 3 can be found, while in ESI, z can be within the order of 50 to 100. z may be adjusted by a factor. The acceleration unit may be used by setting zero voltage on the electrodes, which provides detection equivalent to that achieved by a conventional SEM without an acceleration unit. This use corresponds to the acceleration unit being turned off and can be used to compare detection with and without the acceleration unit.

[0012] The electrodes may be metallic. The shape of the electrodes may affect acceleration and subsequent detection by the SEM. In an embodiment, the electrodes are ring-shaped to allow a direct line of sight during operation, allowing ions to pass through holes in the ring, for example, towards the SEM. As will be appreciated by those skilled in the art, ring electrodes may have an outer radius and a different inner radius. Mesh electrodes may include holes of a thickness that allow ions to pass through.

[0013] Conventional detectors typically operate by increasing the accelerating voltage of an ion source. The ion source may contain a molecular sample irradiated by a laser. Instead of increasing the accelerating voltage of the ion source, the high mass ion detector of the present disclosure provides a potential applied to the ions by an electrode just before the ions reach the SEM, electron multiplier, or detector, i.e., between 100 micrometers and 10 mm. Thus, a modification of conventional detectors is provided by the present disclosure.

[0014] The high mass ion detector of the present disclosure detects ionized molecules or ionized proteins, e.g., m / z This allows for signal amplification up to 50,000-150,000. Ions exhibiting other m / z values ​​can also be accelerated by the acceleration unit of the present disclosure by varying the voltage 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. Electrodes can be assembled in front of or in front of a conventional detector SEM. The front or front of the SEM should be understood to refer to the side of the SEM where ions enter during use. Electrodes may be set to a negative voltage to affect positively charged ions just before they are applied to the SEM. Electrodes may be set to a positive voltage to affect negatively charged ions just before they are applied to the SEM. Ions are subject to a velocity boost. In some instances, ions may also be subject to ion-optical effects. Ion-optical effects should be understood to refer to the guidance of charged particles by electrodes, similar to conventional optical systems. The geometry and bias of the electrode configuration can be designed to disperse or collimate the ion beam, e.g., charged proteins. Different electrode configurations can be implemented to maximize the detection signal.

[0015] The high mass ion detector of the present disclosure may be provided as a plug-and-play solution that plugs into a conventional mass spectrometer, as described below.

[0016] In a second aspect of the present disclosure, there is provided a mass spectrometer comprising an ionization region, a time of flight tube (TOF tube), a mass analyzer at a predetermined distance, a TOF tube in communication with the ionization region, and a high-mass ion detector in communication with the TOF tube, the high-mass ion detector being between the TOF tube and the SEM. The mass spectrometer may also include a laser source configured to irradiate the ionization region to obtain ions from an irradiated sample placed in the ionization region. The laser source may be configured to irradiate the sample to obtain ions from the irradiated sample. The laser source may include a UV laser or a nitrogen laser light having a wavelength in the nanometer range between 340 nm and 430 nm, e.g., 337 nm, i.e., suitable for the matrix used in MALDI. During use, laser pulses may be fired at matrix crystals mixed with an analyte sample, for example, in the form of dried droplet spots. The matrix crystals and analyte mixture may be placed on a plate in the ionization region of the mass spectrometer. The matrix crystals can absorb laser energy, which converts the matrix to an ionized state. Charge is transferred to the analyte, where random collisions occur in the gas phase, causing the ionized analyte and matrix molecules to desorb from the plate.

[0017] The mass analyzer further comprises a TOF tube which may comprise an entrance zone facing or in communication with the ionization region of the mass analyzer, and an exit zone facing or in communication with the high mass ion detector.

[0018] A conventional mass analyzer may be equipped with an existing detector. In an example, the high mass ion detector of the present disclosure may be used to replace the existing detector of a conventional mass analyzer such that, in use, ions formed in the ionization region pass through a TOF tube having 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 disposed in the exit zone of the TOF tube and function as a detector for the conventional mass analyzer. As can be seen, the high mass ion detector of the present disclosure may be provided as a plug-and-play solution that plugs into a conventional mass analyzer.

[0019] When a mass spectrometer according to the present disclosure is used, the ions are subjected to a voltage applied to the electrodes before impinging on the SEM, causing signal amplification. When used in a mass spectrometer, the high mass ion detector acts on the analyte ions after they exit the TOF tube and just before they impact the SEM surface, i.e., between 100 micrometers and 10 mm.

[0020] The acceleration unit and high mass ion detector of the present disclosure can pave the way for increasing the sensitivity of SEM-based detector units to high mass molecules.

[0021] In some instances, the SEM is an MCP. In some instances, the SEM is a dynode. Advantageously, an MCP can be economical to operate and provides a fast response time, narrow pulse width, high gain, and a sufficiently large active size to make it a good detector for a TOF mass analyzer.

[0022] The present disclosure provides devices and systems that accelerate and thereby affect analyte ions after they exit the TOF tube and just before they collide with the SEM by adding an acceleration unit to the detector that includes two electrodes separated by an insulating layer. In use, the same ions produce a greater signal intensity (25 times) when measured with the disclosed high mass ion detector rather than a conventional MCP.

[0023] In an embodiment, there is provided a method of accelerating ions, comprising providing an acceleration unit according to the present disclosure, applying a voltage potential difference to at least two electrodes of the acceleration unit, and supplying ions to the acceleration unit, thereby exposing the ions to the voltage potential difference, wherein the ions are subjected to an accelerating force imparted by the voltage potential difference. The supplying of ions may be performed by ionizing a sample analyte.

[0024] A third aspect of the present disclosure is a method for detecting high mass ions, comprising: providing a mass spectrometer according to the present disclosure, the mass spectrometer including an analyte sample; ionizing the sample by irradiating the sample with a laser source, thereby transferring the sample to a gas phase and releasing charged ions from the sample; After all or a portion of the charged ions have passed through the TOF tube, exposing the charged ions that have passed through the TOF tube to a voltage applied to at least two electrodes of an acceleration unit, thereby accelerating the exposed charged ions; converting the accelerated charged ions exiting the electrode into secondary electrons by causing the accelerated charged ions to impinge on the SEM; detecting the presence of high mass ions based on secondary electrons; The present invention provides a method comprising:

[0025] The charged ions may be positively or negatively charged. Positively charged ions may be exposed to a negative potential difference between the voltages applied to the electrodes by passing through the acceleration unit. Negatively charged ions may be exposed to a positive potential difference between the voltages applied to the electrodes by passing through the acceleration unit. Subsequently, ions exiting the acceleration unit, also called a booster, are converted to secondary electrons upon impacting the SEM surface. Advantageously, due to the ion velocity gain combined with the ion optical effect caused by the electric booster potential provided by the electrodes, the same ions produce a greater signal intensity when measured with a detector after the acceleration unit of the present disclosure than with existing conventional detectors without an acceleration unit. An electron shower may accumulate at the anode, and a "secondary electron" signal may be measured with an external oscilloscope. In some examples, the SEM is a Chevron-configured MCP. In the Chevron configuration, the secondary electron signal is amplified by the Chevron-configured MCP, and the electron shower accumulated at the anode is measured with an external oscilloscope. In some examples, voltages may be applied independently to the electrodes, for example, in a range of zero to -4 kV. The negative voltage advantageously influences oncoming positive ions emerging from the TOF tube, improving the signal intensity produced by the SEM.

[0026] The advantages resulting from these aspects may be similar to those described with respect to the first and second aspects.

[0027] Non-limiting examples of the present disclosure are described below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows a schematic representation of an acceleration unit 10 for accelerating high-mass ions.

[0029] [Figure 2] 1 shows a mesh electrode.

[0030] [Figure 3]A ring electrode is shown.

[0031] [Figure 4] 1 shows a schematic representation of a high mass ion detector 40 according to the present disclosure.

[0032] [Figure 5] 1 shows a schematic representation of a high mass ion detector 40 according to the present disclosure.

[0033] [Figure 6] 1 shows an example of a conventional mass spectrometer.

[0034] [Figure 7] 1 depicts a mass spectrometer 70 according to the present disclosure.

[0035] [Figure 8] 1 shows the amplification factor AF of BSA with three different examples of high mass ion detectors according to the present disclosure.

[0036] [Figure 9] 1 shows the amplification factor AF of IgG with three different examples of high mass ion detectors according to the present disclosure.

[0037] [Figure 10] 1 shows a direct comparison of mass spectra obtained with a conventional MCP and the disclosed high mass ion detector.

[0038] [Figure 11] AF values ​​for the higher charge states of BSA and IgG ions are shown. DETAILED DESCRIPTION OF THE INVENTION

[0039] In these figures, the same reference numbers are used to indicate corresponding elements.

[0040] FIG. 1 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.

[0041] In some examples, the insulating layer exhibits a thickness between 100 micrometers and 6 millimeters, mm, or more specifically, in some examples, the insulating layer exhibits a thickness between 2 mm and 6 mm, or a thickness of 2 mm, or a thickness of 6 mm. The insulating layer may be at least partially composed of an insulating material, such as polytetrafluoroethylene (PTFE), and / or polyetheretherketone (PEEK), and / or Kapton.

[0042] In some examples, the high-mass detector of the present disclosure may be referred to as a booster SEM, referring to the acceleration or "boost" force that the high-mass ion detector exerts on ions during use. In some examples, the SEM is a microchannel plate (MCP). In these examples, the booster SEM is referred to as a BMCP for "booster MCP." The electrode shape is such that ions can pass through it. For example, FIG. 1 shows two disk or ring electrodes. The disk electrode provides holes through which ions pass. The electrodes may also have a ring or mesh shape. FIG. 2 shows a mesh electrode 20 that passes ions through the holes in the mesh during operation. Other configurations are also encompassed by the present disclosure, so long as ions can pass through the electrode during use. A circular shape, such as a disk or ring shape, allows ions to form a flattened or flattened cloud of ions (depending on ionization, acceleration, and dispersion during propagation) during use and when a voltage potential difference is applied to ions passing through the holes in the electrode, which can be guided and / or modified by an electric field. In the case of a disk or ring shaped electrode as shown in FIG. 3, the electrode may have an outer diameter 31, for example, between 50 mm and 60 mm or between 2 inches and 3 inches, and an inner diameter, for example, between 20 mm and 30 mm or between 1 inch and 1.5 inches.

[0043] In use, the disk- or ring-shaped electrode provides a direct line of sight to the detector, which may be, for example, an SEM or MCP. This advantage may become apparent in the following examples of the present disclosure. The electrodes in the acceleration unit may have different shapes; for example, the first electrode may be a mesh electrode and the second electrode may be a ring electrode. The third electrode may be a square electrode with holes that allow ions to pass through. The holes through which ions pass through the electrode may be aligned from one electrode to the other. The holes through which ions pass through the electrode may not be aligned from one electrode to the other, allowing ions to bypass the path they took from one electrode to the next.

[0044] FIG. 4 is a schematic representation of a high mass ion detector 40 according to the present disclosure. The high mass ion detector includes a secondary electron multiplier (SEM) 47 for converting ions to electrons and multiplying the number of electrons within the SEM, as shown in FIG. 4. The SEM includes a front side 45 and a back side 46 through which ions enter the front side during use. The acceleration unit 41 is spaced a distance 48 between 100 micrometers and 10 mm from the front side of the SEM. The acceleration unit 41 in FIG. 4 includes a first electrode 42, an insulating layer 43, and a second electrode 44. During operation, as shown in FIG. 5, ions traveling in the direction of the arrows can enter the high mass ion detector 40 through the first electrode 42 and exit through the back side. Ions exiting the acceleration unit travel a distance between 100 micrometers and 10 mm before colliding with the SEM 47. As shown in FIG. 5, electrons 53 exiting the SEM can accumulate at the anode 52, allowing an electron shower to be detected.

[0045] The example shown in Figures 4 and 5 shows two circular electrodes or booster electrodes. The electrodes may be separated by a polytetrafluoroethylene (PTFE) plate for electrical insulation. The SEM shown in Figures 4 and 5 is a microchannel plate MCP. In the example, two MCPs assembled in a Chevron configuration are used. Figure 5 shows a metal anode 52. The elements may be secured together with polyetheretherketone (PEEK) screws and nuts and may rest on an electrically insulating base, such as a PTFE pedestal for electrical insulation from the underlying vacuum flange.

[0046] FIG. 6 shows an example of a conventional mass spectrometer showing a laser source 61, a matrix with analytes 62, an ionization region 63, a TOF tube 64 in which ions travel in the direction of the arrows and are separated according to their mass or m / z value, and an MCP detector 65.

[0047] 7 illustrates a mass analyzer 70 according to the present disclosure, including an ionization region 73; a laser source 71 configured to illuminate the ionization region to obtain ions 75 from an irradiated sample 72 resting in the ionization region; a time-of-flight (TOF) mass analyzer 74, the TOF tube in communication with the ionization region 73 such that ions can pass from the ionization region to the TOF tube; a high mass ion detector 40 according to the present disclosure, the high mass ion detector in communication with the TOF tube, and an acceleration unit 41 for the high mass ion detector between the TOF tube 74 and the SEM 47. As can be seen, the MCP detector 65 of FIG. 6 can be replaced with a high mass ion detector 40 according to the present disclosure to obtain a mass analyzer 70 according to the present disclosure. As can be seen, despite the extension of the acceleration unit, ions still have direct access to the surface of the SEM.

[0048] During operation, one power supply or two or more power supplies can be used. Two separate power supplies can be used to apply a negative voltage, e.g., up to -4 kV, to each electrode. A third power supply can be coupled to a voltage divider circuit to set the input of the SEM to ground. In an example where the SEM includes two microchannel plate MCPs assembled in a Chevron configuration, the third power supply can be coupled to a voltage divider circuit to set the input of the first MCP to ground, the second MCP to approximately +1.8 kV, and the anode to +2 kV for positive ion detection. Strong potentials can be achieved by combining three or more electrodes. The configuration can be flexible depending on the needs of the application.

[0049] Compared to conventional SEM detectors, the signal intensity provided by the high mass ion detector of the present disclosure is amplified by a factor of 24.3 for charged bovine serum albumin, BSA (BSA, ≥ 98%, molecular weight 66 400 u) BSA ions (m / z 66 400) and by a factor of 10.7 for charged immunoglobulin G, IgG from human serum (IgG, ≥ 95%, molecular weight 150 000 u) IgG ions (m / z 150 000) up to the MDa range.

[0050] In one example, different voltages can be applied independently to the electrodes, e.g., between zero and -4 kV in 1 kV steps, and different distances are established, defined by insulating layers, e.g., PTFE spacers, between the electrodes. Different combinations of configurations can provide different results. For example, this allows testing of configurations that provide the greatest signal enhancement for specific applications and specific sample analytes. In one example, a 6 mm thick insulating layer was used, referred to as the "long-distance configuration" example. In a second example, referred to as the "medium-distance configuration" example, a 2 mm thick insulating layer was used. In a third example, referred to as the "short-distance configuration" example, a 10 micron insulating layer was used, which can function as a "zero-spacing" acceleration unit or short-distance configuration, and combined with setting zero voltage to the electrodes, corresponds to the acceleration unit being turned off.

[0051] Sample Preparation: Experiments performed included the use of standard proteins bovine serum albumin (BSA, ≥98%, molecular weight 66,400 μm) and human serum-derived immunoglobulin G (IgG, ≥95%, molecular weight 150,000 μm), acetone, acetonitrile, acetonitrile with 0.1% trifluoroacetic acid (TFA), and water with 0.1% TFA (all LC-MS grade), both MALDI matrices α-cyano-4-hydroxycinnamic acid (α-CHCA) and 2,5-dihydroxybenzoic acid (DHB), formic acid (≥99%, LC-MS grade), and water purified using an on-site purification system. Initially, various protocols for MALDI sample preparation were evaluated, all of which use manual pipetting for matrix deposition onto the target plate. First, the two proteins, BSA and IgG, were dissolved in 0.1% TFA in water to concentrations of 3.0 μmol / L and 1.3 μmol / L, respectively. The MALDI matrix was prepared by adding 7.0 mg of α-CHCA to 100 μL of acetone to form a saturated solution. A thin layer was applied to a MALDI target (ground steel) using a 10 μL pipette tip briefly wetted with this solution and allowed to dry. An additional 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). 20 mg of DHB was also dissolved 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 1:1 (v / v) ratio. Subsequently, either 2 μL of BSA or 2 μL of IgG protein solution was briefly mixed with 2 μL of the α-CHCA and DHB solution. 0.5 μL of the resulting new solution was added to the thin layer of α-CHCA. The spots were dried at ambient conditions and then directly measured.

[0052] Measurements were performed on a modified UltrafleXtreme MALDI TOF / TOF system in linear positive ion mode. The instrument was equipped with a SmartBeam 2 laser. The MALDI source was controlled via the flexControl software (Bruker Daltonics). The method used was optimized for measuring m / z values ​​in the range of 30,000 to 210,000 using the mass spectrometer's discrete dynode detector and was provided by the instrument manufacturer (U = 25 kV accelerating voltage, 460 ns pulsed ion extraction). Measurements were performed in positive polarity mode, and 100 laser shots at a frequency of 20 Hz were averaged to obtain one mass spectrum. The detector side of the mass spectrometer was modified by removing the discrete dynode detector and replacing it with a high-mass ion detector 40 according to the present disclosure. A load-lock chamber was installed to facilitate detector replacement. Calibration measurements using a conventional MCP detector revealed a calibrated flight path length of approximately d = 2.21 m. The high-mass ion detector 40 according to the present disclosure comprised two circular booster electrodes, designated BE, separated by a polytetrafluoroethylene (PTFE) plate for electrical insulation, two microchannel plates MCP assembled in a Chevron configuration (F1552-01, Hamamatsu Photonics, Hamamatsu, Japan), and a metal anode. The assembly was secured with polyetheretherketone (PEEK) screws and nuts and seated on a PTFE pedestal for electrical insulation from the lower vacuum flange. The high-mass ion detector 40 according to the present disclosure was placed under high vacuum (2×10 -5Each initial measurement was performed at least 24 hours after installation at a pressure of less than 100 kJ / s (less than 100 kJ / s) to allow for proper degassing and thus reduce the risk of MCP discharge. Three external high-voltage power supplies were used to supply voltage to the individual components of the high-mass ion detector according to the present disclosure. Separate power supplies were used to apply a negative voltage (maximum -4 kV) to each of the two booster electrodes BE. A third device was coupled to a voltage divider circuit, setting the input of the first MCP to ground, 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 DC current noise and capture only the AC signal generated by the ion action. The oscilloscope input was set to 1 MΩ for impedance matching between the oscilloscope and the rest of the circuit, providing better signal response. An external oscilloscope was used to record the signal amplitude in volts as a function of time of flight. A trigger signal from the mass analyzer was sent to one channel of the oscilloscope to mark zero time of flight. The signal was then acquired for 500 μs (500 MSa s-1 sample rate, 250 kSa sample size). To obtain a single mass spectrum, the signal generated by 100 laser shots was averaged using the averaging function of the oscilloscope. The voltage applied to the MCP-anode assembly was held constant, while the voltages provided to the two electrodes were independently varied between zero and -4 kV to maximize the signal intensity generated by the arriving ions.

[0053] Data Processing Before further evaluation of the measured data, the time of flight can be converted to m / z values ​​using the following formula:

number

[0054] The accelerating voltage U is, for example, U = 25 kV, and the determined flight path length d is, for example, d = 2.21 m; the electron charge e = 1.602 · 10-19 C; and the atomic mass unit u is, for example, u = 1.661 · 10-27 kg.

[0055] The baseline of the mass spectrum was then corrected using the "msbackadj" function in MATLAB (MathWorks, Natick, MA, USA). The intensities of the mass spectral peaks were then extracted using the "mspeaks" function to determine the signal enhancement caused by different voltage and distance configurations tested for the high-mass ion detector according to the present disclosure compared to a conventional MCP assembly. The intensity variations of the signals generated by singly charged bovine serum albumin (BSA+1, at approximately m / z 66,400) and doubly charged (BSA+2, at approximately m / z 33,200) BSA ions were investigated in response to changes in the voltage of the booster electrode BE for three high-mass ion detector distance configurations: the long-distance configuration, the medium-distance configuration, and the zero-spacing or short-distance configuration. Note that the present disclosure may refer to the "zero-spacing" or "short-distance configuration" when using a 10-micron insulator layer, which can function as a "zero-spacing" acceleration unit or short-distance configuration, and which, combined with setting zero voltage to the electrode, corresponds to turning off the acceleration unit. Five mass spectra were acquired for each voltage combination. Mean intensity values ​​and standard deviations were derived from the five mass spectra for each voltage combination for singly and doubly charged BSA ions to account for variations in signal intensity typically associated with local variations in MALDI sample quality ("sweet spots"). To allow for direct comparison of acquired mass spectra, amplification factors (AFs) were determined by normalizing the peak intensities measured with the disclosed high-mass ion detector, also referred to as the "booster MCP" BMCP, to values ​​obtained with a conventional MCP detector with the BE turned off. The error in AF was derived by Gaussian error propagation using the standard deviation of the peak intensities for the calculation.

[0056] Figure 8 shows the amplification factor (AF) of BSA with three different examples of high-mass ion detectors according to the present disclosure. The AF found for large electrode separation is shown in Figure 8(a), for medium electrode separation in Figure 8(b), and for measurements with zero electrode separation in Figure 8(c). The AFs for doubly charged and singly charged BSA ions are shown in the top and bottom rows of the image, respectively. For large and medium electrode separations, all combinations of electrode voltages between zero and -4 kV were tested in 1 kV steps. Note that for the booster configuration with zero electrode separation resulting in one electrode, only one high-voltage power supply was used to apply voltages between zero and -4 kV in 1 kV steps. Below, the results for doubly charged BSA ions are first compared for different electrode distances. Subsequently, the AF induced for singly charged BSA ions is considered. For the large BE distance shown in Figure 8(a), a maximum amplification factor (AF) of 3.4 ± 1.0 was observed for the lower BE of -3 kV and the upper BE of -4 kV, as well as for the reverse voltage combination. A reduced AF of 2.0 ± 0.7 was obtained at -4 kV for both electrodes. A maximum AF of 13.4 ± 7.4 for BSA+2 was observed at intermediate electrode distances, with the lower BE at -3 kV and the upper BE at -4 kV, as shown in Figure 8(b). Similar to the results for large electrode separation, comparable AFs were derived for the reverse voltage combinations, with a slightly lower AF observed when the same voltage of -4 kV was applied to both BEs. Furthermore, a maximum value of 9.1 ± 5.3 was observed at the lower BE at -2 kV and the upper BE at -1 kV. At zero electrode distance, the AF varied between 3.9 ± 2.1 and 4.4 ± 3.7 with varying electrode voltages. Note that the signal intensity at -4 kV was insufficient to derive an AF value. As shown in the bottom panel of Figure 8, similar observations were made for singly charged BSA ions as for doubly charged BSA ions. Again, the maximum AF of 24.3 ± 8.9 was obtained for the medium electrode spacing (Figure 8(b)) for the combination of -3 kV on the bottom BE and -4 kV on the top BE. Slightly lower AF was observed for the combination of reverse voltages and -4 kV applied to both BEs. Furthermore, a local maximum was also found for the voltage combination of -2 kV and -1 kV on the bottom and top electrodes, respectively, resulting in an AF of 15.0 ± 8.6.Significantly lower AF values ​​were observed for large electrode separations (Figure 8(a)) as well as for zero electrode separations (Figure 8(c)). The maximum AF of 13.4 ± 14.7 obtained for large electrode spacing with -3 kV applied to the bottom electrode and the top electrode off may be treated as an outlier. In general, greater signal amplification was achieved when a higher negative voltage was applied to the BE, regardless of the detector configuration used. The observed behavior may be caused by increased acceleration of positively charged ions due to increasing the negative boost voltage, ion optical effects, or a combination of both effects.

[0057] On the one hand, a higher negative voltage likely causes stronger acceleration of positively charged ions before they collide with the MCP surface. Therefore, the AF enhancement may be related to the well-known increase in MCP detection efficiency with increasing ion velocity. The strongest signal enhancement for singly and doubly charged BSA ions was observed for the medium electrode separation (Figure 8(b)). On the other hand, we were able to identify two well-known but opposing ion-optical mechanisms that may have led to the observed signal amplification: first, the electrode configuration used could cause a specific spatial focusing of the ion beam, apart from the intended ion acceleration, as previously noted for other electrode configurations. Second, the negative electrode booster potential could lead to divergence of the ion beam. Thus, the positively charged ion cloud covers a larger area of ​​the active MCP surface, which may be advantageous in terms of the relatively long dead time (millisecond range) of a single channel. Note that only one of these two opposing ion-optical effects could have contributed to the observed increase in signal intensity.

[0058] Compared to the findings at medium separation, the AF was reduced at large electrode separation. In this configuration, the BEs may be too far from each other to efficiently accelerate and focus the ions. Possibly, positively charged ions are attracted to the negative BE instead of reaching the MCP due to their longer flight path through the booster, resulting in collision with the negative BE. Therefore, at large electrode distances, fewer ions reach the MCP surface, which may have resulted in the observed decrease in signal intensity compared to medium electrode separation.

[0059] The AF found for zero electrode separation is generally larger than that observed for large electrode spacing. However, the signal amplification is significantly smaller than that for medium electrode configurations. Therefore, it is expected that a single BE (zero electrode spacing) may cause ion velocity enhancement but may not have a clear effect on the spatial distribution of the ion beam. Typically, assemblies designed for ion beam focusing consist of several electrostatic lenses, such as a three-element Einzel lens, which is a combination of tubular acceleration lenses with opposite polarities.

[0060] The strongest signal amplification was not observed at the highest voltage (-4 kV) applied simultaneously to both booster electrodes, but was observed at a booster voltage combination of -3 kV / 4 kV (lower / upper BE). This voltage combination can create an electric field gradient that appears to affect the spatial extent of the ion beam and lead to improved detection efficiency at the MCP. Furthermore, a local maximum was detected at voltages below -4 kV for the large and medium electrode separation. Therefore, the maximum voltage at the BE is not necessary to achieve significant signal amplification with the high-mass ion detector disclosed herein. Furthermore, when the detector configuration was changed from medium to large electrode separation, a shift in the local maximum was observed for the AF measured for singly and doubly charged BSA ions. This observation may indicate the effect of the length of the insulating layer between the two BEs on the ion beam focusing properties of the booster.

[0061] In conclusion, Figure 8 shows the signal amplification of singly and doubly charged BSA ions for three booster electrode configurations. The separation between the top and bottom electrodes, provided by the insulating layer, was varied from (a) a large distance to (b) a medium distance, and finally (c) the distance between the two electrodes was changed to zero. The strongest amplification was observed for the medium electrode spacing. The peak intensities found for BSA+2 and BSA+1 were normalized to measurements with the electrode voltage turned off to extract the signal amplification. Dark gray spaces indicate the absence of detectable signal. Furthermore, note the different amplitudes in the color-coded 3D plots.

[0062] Figure 9 shows the amplification factor, AF, for IgG for three different exemplary high-mass ion detectors according to the present disclosure. The AF for IgG was obtained from measurements using three different BMCP configurations, as described above for BSA. Results for doubly charged IgG ions (IgG+2, approximately m / z 75,000) and singly charged IgG ions (IgG+1, approximately m / z 150,000) are summarized in the top and bottom panels of Figure 9, respectively. The AF is shown for large electrode distances in Figure 9(a), for medium electrode distances in Figure 9(b), and for zero electrode separation in Figure 9(c).

[0063] First, the results for doubly charged IgG ions and second, the observations for singly charged IgG ions are discussed below. At large electrode separation (Figure 9(a)), a maximum AF of 2.4 ± 2.0 was observed for IgG+2 at the bottom / top electrode voltage combination of -2 kV / -4 kV. However, the AFs found at both -4 kV and -3 kV / -4 kV variations at both electrodes fluctuate within this standard deviation of the AF at -2 kV / -4 kV. At medium electrode separation (Figure 9(b)), a much larger AF of 23.6 ± 14.6 was observed at the bottom / top BE for -3 kV / -4 kV. Slightly lower AF values ​​were obtained for the reverse voltage combination and -4 kV applied to both BEs. Additionally, a local maximum of 11.1 ± 6.6 was measured at the bottom / top BE for 2 kV / -1 kV. At zero electrode separation (Figure 9(c)), a large variation in AF was observed between 1.7 ± 1.3 and 6.8 ± 6.0 for IgG+2.

[0064] For singly charged IgG ions, a maximum AF of 7.2 ± 0.2 was observed at -4 kV on the bottom electrode and -3 kV on the top electrode using the large electrode separation shown in the bottom panel of Figure 9(a). The AF was found to be smaller at both reverse voltage combinations and at -4 kV. A local maximum of 5.8 was observed for -2 kV / -1 kV on the bottom / top electrode. Because the signal intensity measured with the booster off was not strong enough for detection and therefore calculation of the AF, the peak intensity was normalized to the value found for -1 kV on the bottom electrode and zero volts on the top electrode. For the medium electrode configuration in Figure 9(b), a maximum AF of 10.7 ± 5.2 was observed for -4 kV / -3 kV on the bottom / top electrode. For other BE voltage combinations, a similar trend to the previously described results for IgG+2 measured at medium electrode separation was observed. At zero electrode separation (Figure 9(c)), no reliable signal amplification was observed, as AF varied between 0.2 ± 0.2 and 1.2 ± 2.1.

[0065] Similar to the observation for BSA in Figure 8, the AF generally increased with increasing applied voltage in the BE, which may be related to the increased ion velocity and improved MCP detection efficiency due to the ion focusing effect. Furthermore, the medium electrode separation also resulted in the strongest signal enhancement for IgG compared to the two other BE configurations. The AF only slightly decreased from BSA+1 at approximately m / z 66,400 to IgG+2 at approximately m / z 75,000, i.e., from 24.3 to 23.7.

[0066] However, a significantly lower AF of 10.7 was found for IgG+1, which appears at a much higher m / z of approximately 150,000. This decrease in AF with increasing m / z value is expected behavior, since the acceleration voltage in the BE is the same for all approaching ions. Thus, higher-mass ions still impinge on the MCP surface slower than lighter-mass ions, leading to the mass-dependent detection efficiency of the MCP. Note that the largest AF for doubly charged IgG was found (at approximately m / z 75,000) for a combination of -3 kV lower BE and -4 kV upper BE, similar to the observations made for doubly charged BSA ions (approximately m / z 33,200) and singly charged BSA ions (approximately m / z 66,400). However, the strongest amplification for singly charged IgG ions (approximately 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 increases with ion mass in a TOF mass analyzer, possibly leading to a loss of control over the spatial distribution of ions for higher mass ions. Therefore, the effect of the electric booster potential on the spatial elongation of ions is likely mass-dependent, as ion clouds with different masses may approach the booster with different degrees of lateral expansion.

[0067] Furthermore, a local maximum in the medium electrode separation was observed for BSA+2, BSA+1, and IgG+2 at −2 kV lower and −1 kV upper BE. For singly charged IgG ions only, another local maximum was observed at the voltage combination of −3 kV / −1 kV lower / upper BE, which may be related to mass-dependent ion beam expansion.

[0068] In conclusion, Figure 9 shows the signal amplification of singly and doubly charged IgG ions for three booster electrode configurations. The separation between the top and bottom electrodes, provided by the insulating layer, was varied from (a) a large distance to (b) a medium distance, and finally (c) zero distance between the two electrodes. The strongest amplification was again observed for the medium electrode spacing. The peak intensities found for IgG+2 and IgG+1 were normalized to measurements with the booster voltage turned off to extract the signal amplification; gray spaces indicate the absence of detectable signal. However, the peak intensity measured at the large electrode distance for IgG+1 was normalized to the signal at -1 kV below and zero voltage for the top electrode, as no sufficient peak intensity was detected even when both electrode voltages were turned off.

[0069] Of the three tested electrode configurations, the medium BE spacing provided the greatest amplification for the signal generated by boosted ions upon impact with the MCP surface. This configuration appears to result in efficient ion acceleration and, in some cases, simple focusing of the ion beam, resulting in improved signal intensity compared to conventional MCP detectors. Because the detection mechanism relies on the conversion of analyte ions to secondary electrons using the MCP, ion velocity still determines detection efficiency. Therefore, since all ions experience the same potential as they pass through the BE, the AF is expected to decrease with increasing ion mass, as high-mass ions are still slower than lighter-mass ions. Figure 10 shows a direct comparison of mass spectra obtained with a conventional MCP, plotted as a solid line below the graphic, and a BMCP with medium electrode spacing, plotted as a line above the previous line, demonstrating the strong signal enhancement achieved. The detected signal was most enhanced at -3 kV and -4 kV reverse voltage combinations for BSA (plotted as the bottom line in Figure 10(a)) and IgG (plotted as the top line in Figure 10(b)). Furthermore, a local maximum in AF was observed for both proteins at the combination of −2 kV at the bottom and −1 kV at the top BE.

[0070] However, using the same voltage in both BEs did not result in the strongest signal enhancement at -3 kV and -4 kV. Below, we focus on the mass spectra obtained for the booster settings that provided the greatest signal amplification for BSA and IgG, compared to results when the booster was turned off. The signals of doubly and singly charged BSA ions (see Figure 10(a)) were amplified by 15.5 and 27.0 times, respectively, when detected with the BMCP plotted on the upper line instead of the conventional MCP plotted on the lower line. The voltage combination of -3 kV in the lower BE and -4 kV in the upper BE provided the greatest AF for BSA. Furthermore, the mean peak intensities and corresponding standard deviations for BSA+2 and BSA+1 demonstrate the variation among the five mass spectra obtained for this voltage combination. For doubly charged BSA ions, the average intensity increased from 0.0016 ± 0.0007 to 0.0218 ± 0.0072, and for singly charged BSA ions, the intensity increased from 0.0009 ± 0.0002 to 0.0210 ± 0.0057 when BMCP was used. Thus, the absolute change in signal intensity of consecutively measured mass spectra was found to increase with the AF value, and therefore with the BE voltage. Furthermore, the full width at half maximum (FWHM) of the peaks assigned to the singly and doubly charged BSA ions in Figure 10(a) was derived as a measure of the change in mass resolution when BMCP was used instead of the MCP detector. When a BMCP was used instead of a conventional MCP detector, the FWHM increased from 1,300 u to 1,900 u for the signal at m / z 33,200 (BSA 2+) and decreased from 4,300 u to 2,900 u for the signal at m / z 66,400 (BSA 1+). Using a 20 Hz frequency for firing the laser, the signals generated by 100 single laser shots can be averaged. In some cases, modifications to the BMCP detector measurement, such as upgrading the data acquisition system, can enable a direct comparison between conventional systems and the MCP / BMCP detector disclosed herein.

[0071] Figure 10(b) compares a single mass spectrum of IgG measured with a conventional MCP (bottom line) with one acquired with a high-mass ion detector according to the present disclosure (top line). In the mass spectra shown, AF values ​​of 14.3 for IgG+2 and 9.2 for +1 IgG were observed at -4 kV (bottom) and -3 kV (top) BEs. The average signal intensity of the five mass spectra at this voltage combination increased from 0.0009 ± 0.0003 to 0.0141 ± 0.0049 for doubly charged IgG ions and from 0.0009 to 0.0099 ± 0.0048 for singly charged IgG ions. Again, an increase in signal intensity variation is observed with increasing AF values, and therefore with increasing BE voltages. Using the high mass ion detector according to the present disclosure, the FWHM increased from 3,700 u to 4,200 u for the signal at m / z 75,000 (IgG2+) and decreased from 10,800 u to 3,500 u for the signal at m / z 150,000 (IgG1+). The change in FWHM with BE setting is shown for both proteins. No significant variation in m / z values ​​was observed for different ions with varying BE voltage combinations.

[0072] An increase in the acceleration voltage in the ion source could be an obvious way to further improve the MCP detection efficiency. However, the applied voltage is actually limited by an increase in ion attenuation within the source associated with the increase in the acceleration voltage. Therefore, the increase in intensity variation associated with the increase in the BE voltage may be related to an improvement in the probability of ion fragmentation when passing through the booster electrode for acceleration. Furthermore, the variation in peak width associated with the change in the BE voltage may be related to the spatial distribution of the ion beam. For example, when using a combination of a conversion dynode and a post-acceleration stage compared to a conventional MCP detector, a decrease in ion mass resolution has been reported elsewhere. The observed decrease in resolution was due to the effect of the post-acceleration stage on the ion beam. The overall reproducibility of measurements using the high-mass ion detector according to the present disclosure can be improved by applying matrix deposition methods other than manual pipetting used herein. Other matrix deposition techniques such as sublimation and spraying are known to produce MALDI samples with improved uniformity and increased purity of the deposited matrix, thereby reducing the observed signal intensity variation. Comparison of the peak intensities of BSA and IgG revealed a mass-dependent decrease in signal intensity. The average AF derived for single-charged BSA ions (24.3) decreased by approximately 56% for single-charged IgG ions (10.7). Therefore, the higher-mass IgG ions still reach the MCP surface at a slower rate than the lower-mass BSA ions, causing a mass-dependent MCP detection efficiency. Furthermore, it cannot be excluded that the decrease in the signal intensity of IgG is also related to the spatial expansion of the ion cloud when approaching the booster expansion in the MCP detector. Since the expansion of the ion cloud in vacuum is thought to increase with ion mass, the decrease in signal intensity may also be related to the loss of higher-mass ions when colliding with the BE rather than reaching the MCP. Furthermore, several peaks below the m / z values of the double-charged BSA ions (<m / z 33 200) and double-charged IgG ions (<m / z 75 000) are observed in the two mass spectra of FIG. 10, and these are thought to correspond to the higher charge states of BSA and IgG.Similar observations have already been made by Choi et al., who observed a strong correlation with the MALDI matrix used. Because multiply charged ions were observed in MCP and BMCP measurements, their occurrence is likely related to other influences, such as the properties of the matrix type used. When comparing the AF found for the doubly charged BSA ion (m / z 33,200) with that found for the doubly charged IgG ion (m / z 75,000), an increase from 13.4 to 23.7 was observed, indicating a dependence of the AF on the m / z value.

[0073] Figure 11 shows AF values ​​for higher charge states of BSA and IgG ions. The figure can be separated into three m / z regions: first, the signal amplification generated by the booster appears to work most efficiently for ions in the m / z range of approximately 50,000 to 75,000; second, due to mass-dependent MCP detection efficiency, AF decreases for ions with m / z values ​​above approximately 75,000. Third, for m / z values ​​below approximately 50,000, AF decreases as the m / z value decreases. Because the velocity of ions is inversely proportional to the square root of their m / z value, ions with lower m / z values ​​have greater velocities than ions with higher m / z values. Therefore, the acceleration effect of the booster on ions with lower m / z values ​​may be smaller because they pass through the booster section more quickly. Presumably, the decrease in relative AF with increasing ion velocity (smaller m / z values) is caused by less effective acceleration produced by the booster than for slower-moving ions with larger m / z values. Note that future investigations of other ionized molecules may reveal further details regarding the observed dependence of relative signal amplification on m / z value.

[0074] Conclusion: An extension of the conventional MCP detector was used to amplify the signals of standard protein ions up to m / z 150,000. To maximize the amplification factor, three configurations of the disclosed high-mass ion detector were tested. Medium electrode separation provided the greatest signal enhancement by creating a convenient combination of acceleration of positively charged ions by the negative booster electrode with an ion-optical effect. The spatial ion beam distribution was influenced by the electrical booster potential, which may be responsible for the enhanced signal intensity observed with this BMCP configuration, in contrast to the two other designs. Generally, the higher the applied booster voltage, the stronger the signal amplification. However, the greatest amplification factor was observed with the combination of -3 kV and -4 kV at the booster electrode. Furthermore, the maximum local amplification factor was detected with the combination of -2 kV at the bottom and -1 kV at the top booster electrode, offering the possibility of operating the disclosed high-mass ion detector at potentials up to 2 kV with increased sensitivity. The absolute signal intensity was found to decrease with increasing mass. This behavior is to be expected because the same booster voltage is used for all masses, and therefore higher mass ions remain slower than lighter mass ions after passing through the booster stage. Nevertheless, the impact of the booster section is significant, with a maximum of 150 It was found that this is sufficient to allow reliable detection of m / z values ​​of 000. The accessible m / z range of BMCP can be extended by application of larger voltages to the booster electrode. It should be noted that the concepts presented here are not limited to proteins but can also be applied to the analysis of high-mass materials such as polymers and dendrimers.

[0075] Furthermore, mass gates can be used in combination with high-mass ion detectors according to the present disclosure to avoid saturation of the MCP channel due to pulsed deflection of the first-arriving low-mass ions, which may result in further enhancement of the signal produced by the high-mass ions. Such experiments may require fast switching (on the order of a few microseconds) between zero (or low) applied voltage and a high voltage.

[0076] For completeness, various aspects of the disclosure are set forth in the following numbered clauses. Item 1. A high mass ion detector for a mass spectrometer, comprising: a secondary electron multiplier SEM for converting ions to electrons and increasing the number of electrons in the SEM, the SEM including a front side and a back side through which ions enter the front side in use; an acceleration unit spaced from the front side of the SEM by a distance between 100 micrometers and 10 millimeters, the acceleration unit comprising at least two electrodes, each pair of electrodes being separated by an insulating layer for electrical insulation; Equipped with The acceleration unit is spaced from the front of the SEM at a distance between 100 micrometers and 10 millimeters. High mass ion detector. Item 2. The high mass ion detector according to item 1, wherein the insulating layer has a thickness between 100 micrometers and 10 millimeters. Item 3. The high mass ion detector according to Item 1 or 2, wherein the insulating layer has a thickness of 2 mm or 6 mm. Item 4. The high mass ion detector according to any one of Items 1 to 3, wherein at least one of the electrodes and / or one of the insulating layers has a circular or ring shape. Item 5. The high mass ion detector according to any one of Items 1 to 4, wherein the first electrode and the second electrode are separated by a first insulating layer, and the second electrode and the third electrode are separated by a second insulating layer. Item 6. A mass spectrometer, an ionization region; a laser source configured to illuminate the ionization region to obtain ions from the illuminated sample resting in the ionization region; a time-of-flight tube, a TOF tube, a mass analyzer, a TOF tube in communication with an ionization region; Item 6. The high mass ion detector according to any one of items 1 to 5, wherein the high mass ion detector is in communication with the TOF tube and an acceleration unit of the high mass ion detector is between the TOF tube and the SEM of the high mass ion detector; A mass spectrometer comprising: Item 7. A method for accelerating ions, comprising: providing an acceleration unit comprising at least two electrodes, the acceleration unit being configured to operate with a mass spectrometer, the acceleration unit comprising at least two electrodes, each pair of electrodes being separated by an insulating layer for electrical insulation; applying a voltage potential difference to at least two electrodes of the acceleration unit; providing ions to an acceleration unit, thereby exposing the ions to a voltage potential difference, whereby the ions are subjected to an accelerating force imparted by the voltage potential difference; A method comprising: Item 8. A method for accelerating ions according to Item 7, wherein the supply of ions is achieved by ionizing a sample analyte. Item 9. A method for detecting high mass ions, providing a mass spectrometer according to item 6 containing an analyte sample; ionizing the analyte sample to release charged ions from the analyte sample; After all or a portion of the charged ions have passed through the TOF tube, exposing the charged ions that have passed through the TOF tube to a voltage applied to at least two electrodes of an acceleration unit, thereby accelerating the exposed charged ions; converting the accelerated charged ions exiting the electrode into secondary electrons by causing the accelerated charged ions to impinge on the SEM; detecting the presence of high mass ions based on secondary electrons; A method comprising: Clause 10. The method of clause 9, wherein ionizing the analyte sample is performed by irradiating the sample with a laser source, thereby transferring the sample into the gas phase.

[0077] While only a few examples are disclosed herein, other alternatives, modifications, uses, and / or equivalents are possible. Moreover, all possible combinations of the examples described are also covered. Therefore, the scope of the present disclosure should not be limited by the specific examples, but should be determined solely by a fair reading of the appended claims.

Claims

1. 1. A high mass ion detector for a mass spectrometer, the high mass ion detector comprising: a secondary electron multiplier SEM for converting ions to electrons and increasing the number of electrons in the SEM, said SEM comprising a front side and a back side through which, in use, said ions enter the front side; an acceleration unit for accelerating high-mass ions at a distance between 100 micrometers and 10 millimeters from the front side of the SEM, the acceleration unit configured to operate with a mass analyzer, the acceleration unit comprising at least two electrodes, each pair of electrodes being separated by an insulating layer for electrical insulation; A high mass ion detector comprising:

2. 2. The high mass ion detector of claim 1, wherein the acceleration unit is spaced from the front side of the SEM by a distance between 100 micrometers and 1 millimeter.

3. 2. The high mass ion detector of claim 1, wherein the acceleration unit is spaced from the front side of the SEM by a distance between 1 millimeter and 10 millimeters.

4. The high mass ion detector of claim 1 , wherein the acceleration unit is spaced a distance of 100 micrometers from the front side of the SEM.

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

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

7. 7. The high mass ion detector according to claim 1, wherein the insulating layer is at least partially made of polytetrafluoroethylene (PTFE).

8. 8. A high mass ion detector according to any one of claims 1 to 7, comprising two electrodes, the first electrode and the second electrode being separated by a first insulating layer.

9. 9. The high mass ion detector of claim 1, comprising three electrodes, a first electrode and a second electrode separated by a first insulating layer, and the second electrode and a third electrode 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 and / or each of the insulating layers has a circular or ring shape.

11. A mass spectrometer, comprising: an ionization region; a time-of-flight tube, a TOF tube, a mass analyzer, the TOF tube in communication with the ionization region; a high mass ion detector according to any one of claims 1 to 10, wherein the high mass ion detector is in communication with the TOF tube and the acceleration unit of the high mass ion detector is between the TOF tube and the SEM of the high mass ion detector; A mass spectrometer comprising:

12. 1. A method for detecting high mass ions, comprising: Providing a mass spectrometer according to claim 11 containing an analyte sample; ionizing the analyte sample to release charged ions from the analyte sample; After all or a part of the charged ions have passed through the TOF tube, exposing the charged ions that have passed through the TOF tube to a voltage applied to at least two electrodes of the acceleration unit, thereby accelerating the exposed charged ions; converting the accelerated charged ions exiting the electrode into secondary electrons by causing the accelerated charged ions to impact the SEM; detecting the presence of high mass ions based on the secondary electrons; A method comprising:

13. The method of claim 12 , wherein exposing the charged ions to a voltage applied to the at least two electrodes comprises applying a voltage to at least one of the electrodes.

14. 14. The method of claim 13, wherein the voltage applied to the at least one of the electrodes ranges from 0 KV to −4 KV.

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