How to calibrate a mass spectrometer

Abstract

A method of calibrating a mass spectrometer is provided. The method includes generating calibration ions from an ion source. The calibration ions are transported from the ion source through an ion optical device to a mass analyzer of the mass spectrometer. A characteristic voltage is applied to the ion optical device to control passage of the ions into and / or out of the ion optical device. The applied characteristic voltage results in an unintentional dissociation amount of the calibration ions. An MS1 ​​analysis of the calibration ions is performed using the mass analyzer to obtain an MS1 ​​spectrum of the calibration ions. The unintentional dissociation amount of the calibration ions present in the MS1 spectrum is determined. The characteristic voltage of the ion optical device is calibrated based on the unintentional dissociation amount of the calibration ions present in the MS1 spectrum to provide a target unintentional dissociation amount of the calibration ions during the MS1 analysis.

Description

[Technical field]

[0001] The present disclosure relates to mass spectrometry. In particular, the present disclosure relates to ion optical devices for mass spectrometers. [Background technology]

[0002] Mass spectrometry is a long-established technique for identifying and quantifying often complex mixtures of small and large organic molecules. In recent years, techniques have been developed that allow for the analysis of a wide range of both biological and non-biological materials, with applications across the fields of law enforcement (e.g., identification of drugs and explosives), environment, scientific research, and biology (e.g., in proteomics, the study of simple and complex mixtures of proteins, applications in drug discovery, disease identification, etc.).

[0003] Proteins containing many amino acids typically have a significant molecular weight. Proteins are often digested into several smaller peptides prior to mass analysis. The peptides in a protein digest are of lower molecular weight and therefore easier to mass analyze. Some mass spectrometry techniques involve mass analysis of (unfragmented) analyte ions (MS1 analysis). Other mass spectrometry techniques involve isolation and activation of analyte ions of interest followed by mass analysis of the resulting fragmented ions (MS2 analysis). Often, identification and quantification techniques combine information from the MS1 and MS2 domains to infer information about the analyte ions.

[0004] When performing MS1 analysis, it is important that the analyte ions of interest do not fragment. Unintentional dissociation of analyte ions can lead to errors in the resulting MS1 spectrum. For example, the MS1 spectrum may not contain information about fully dissociated analyte ions. The MS1 spectrum may also contain additional peaks resulting from fragment ions that are not representative of the analytes in the sample, potentially leading to erroneous identification. Also, quantification may be hindered if some of the analyte ion population of interest is dissociated or if the peaks of the analyte ions of interest in the MS1 spectrum overlap with peaks of isobaric fragment ions originating from different analytes.

[0005] Unintentional dissociation of analyte ions is particularly problematic for molecules that have one or more relatively weak chemical bonds (e.g., certain amino acids, peptides, and lipids). Such fragile analyte ions are susceptible to being broken at the weak chemical bonds upon collisional activation. Such collisional activation can undesirably occur during MS1 analysis, for example, when ions are cooled or transported through the mass spectrometer (e.g., entering / cooling in / exiting the ion trap). The extent to which analyte ions may unintentionally dissociate will depend on the chemical nature of the analyte ions, the mass spectrometer settings, as well as variability in conditions and mechanical and electronic tolerances of the hardware. Thus, the impact of unintentional dissociation on MS1 ​​analysis can be difficult to control. Summary of the Invention

[0006] The degree to which ions are unintentionally dissociated will depend on the configuration, conditions, and hardware tolerances of the mass spectrometer, as well as the chemical properties of the ions. In particular, unintentional dissociation of ions may occur in some areas inside the mass spectrometer where the vacuum is not very high or where the magnitude and / or direction of the ion's motion is significantly different from that of the background gas molecules. Thus, in some cases, it may be difficult, if not impossible, to perform an MS1 ​​analysis without some degree of unintentional dissociation of ions. When multiple mass spectrometers are operated at the same nominal settings, different degrees of unintentional dissociation will be observed due to different conditions and hardware tolerances that are not themselves adjustable.

[0007] To reduce or eliminate variability in unintended dissociation, the method of the first aspect provides a way to calibrate a mass spectrometer to provide a target amount of unintended dissociation. Such calibration can be particularly beneficial when multiple mass spectrometers are calibrated to perform similar MS1 analyses.

[0008] There is thus provided, according to a first aspect of the present disclosure, a method of calibrating a mass spectrometer, the method comprising: generating calibration ions from an ion source; transporting calibration ions from an ion source through an ion optical device to a mass analyzer of a mass spectrometer, where a characteristic voltage applied to the ion optical device controls the passage of ions into and / or out of the ion optical device, the applied characteristic voltage also resulting in an amount of unintentional dissociation of the calibration ions; performing MS1 analysis of the calibration ions using a mass analyzer to obtain an MS1 ​​spectrum of the calibration ions; determining the amount of unintentional dissociation of calibration ions present in the MS1 spectrum; A characteristic voltage of the ion optical device is calibrated based on the unintentional dissociation amount of the calibration ion present in the MS1 spectrum to provide a target unintentional dissociation amount of the calibration ion during MS1 analysis.

[0009] It will be appreciated that mass spectrometers include one or more ion optical devices for the purpose of transporting ions from an ion source to a mass analyzer for mass analysis. In general, the ion optical devices are configured to apply various voltages (DC and / or RF bias) to confine and transport ions from the ion source to the mass analyzer. The magnitude of these voltages is typically selected to efficiently transport a wide range of ions (e.g., a wide range of mass-to-charge ratios) from the ion source to the mass analyzer. However, for some relatively fragile ions, such voltages may cause at least some of these ions to unintentionally dissociate during passage. Thus, for a population of relatively fragile ions, variation of one or more voltages of the ion optical devices may affect the probability that the fragile ions will unintentionally dissociate. Thus, by controlling the characteristic voltages of the ion optical devices, the amount of unintentional dissociation that occurs during MS1 analysis can be controlled.

[0010] Relatively fragile ions mean that such ions are more likely to undergo unintentional dissociation than less fragile ions. From a chemical perspective, fragility means that the molecular ion contains one or more weak bonds that are prone to being broken upon collisional activation. Examples of relatively fragile ions include the amino acids isoleucine and phenylalanine, and the (oligo)peptides MRFA, ALELFR, and substance P (RPKPQQFFGLM), among others. Calibration ions may be amino acid ions or peptide ions, e.g., any of the aforementioned species, or lipid ions.

[0011] The method according to the first aspect applies this concept so that the amount of unintentional dissociation occurring in the mass spectrometer can be controlled by performing a calibration process. Thus, known calibration ions (typically relatively fragile ions that are prone to unintentional dissociation) are mass analyzed in the MS1 domain using the mass spectrometer. The amount of unintentional dissociation occurring during the MS1 analysis can be determined based on the MS1 spectrum of the calibration ions. The characteristic voltage of the ion optical device can then be adjusted based on the MS1 mass spectrum to provide a target amount of unintentional dissociation of the calibration ions. Thus, the performance of the mass spectrometer (with respect to unintentional dissociation of ions) can be standardized using the calibration process.

[0012] According to the present disclosure, unintentional dissociation of ions refers to a process in which a parent or precursor ion, i.e., a molecular ion (typically an amino acid ion or a peptide ion), dissociates into two or more mass fragments (at least one of which is an ion) as part of the passage of the molecular ion through one or more ion optical devices, with the intention of mass analyzing the unfragmented molecular ion. Such unintentional dissociation may occur, for example, when a molecular ion moving under the influence of an electric field from an ion optical device interacts with a gas particle present in the ion optical device. It will be understood that such unintentional dissociation events are distinct from processes in which molecular ions are intentionally fragmented. For example, molecular ions may be intentionally fragmented in a fragmentation chamber, followed by mass analysis (i.e., MS2 analysis) on the fragment ions. According to the present disclosure, in some embodiments, the unfragmented molecular ion typically exists in mass spectrometry at a higher abundance than any of its fragment ions resulting from unintentional dissociation, for example at least 1.1 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times higher abundance than any of its fragment ions. As another example, a molecular ion may be intentionally fragmented when exposed to an accelerating potential intended to cause fragmentation of the molecular ion, rather than an accelerating potential intended for the purpose of transporting the molecular ion using one or more ion optical devices. Typically, the accelerating potential used for ion transport in some embodiments of the present disclosure may have a magnitude of about 10V or less, or may have a magnitude of less than 10V. That is, the characteristic voltage calibrated according to the present disclosure may provide an accelerating potential for ions in an ion optical device having a magnitude of about 10V or less. The accelerating potential refers to the total accelerating potential that ions experience to accelerate the ions through the mass analyzer.

[0013] According to the present disclosure, the target amount of unintentional dissociation may be a predetermined value, a range of acceptable values, or a limit (i.e., the amount of unintentional dissociation may be equal to or less than a predetermined value). For example, in some embodiments, the target amount of unintentional dissociation may be about zero (i.e., below the detection limit of the mass spectrometer).

[0014] According to the present disclosure, a mass spectrometer comprises an ion optical device. The ion optical device is a device used to transport ions from an ion source to a mass analyzer. In some embodiments, the mass spectrometer may comprise multiple ion optical devices. Each ion optical device controls ions through the application of one or more voltages. Depending on the mass spectrometer, one or more of these voltages may affect the amount of unintentional dissociation that occurs when the mass spectrometer is used to perform MS1 analysis. Such voltages are considered to be characteristic voltages of the ion optical device according to the present disclosure. It will be understood that in the case of a mass spectrometer comprising multiple ion optical devices, the method of the first aspect may be repeated to calibrate multiple characteristic voltages of the multiple ion optical devices. Of course, a mass spectrometer may have multiple voltages that may be calibrated according to the method of the first aspect, but it will be understood that in practice some voltages of the ion optical devices will have a more significant effect on unintentional dissociation than other voltages. Thus, the characteristic voltages of ion optical devices according to the present disclosure are preferably those voltages of ion optical devices that have the most significant effect on unintentional dissociation of fragile ions while having little effect on the overall ion transmission and / or m / z transmission window of these devices. In general, such voltages are likely to be found in regions of the mass spectrometer where the pressure is relatively high. Ions in the high pressure regions of the mass spectrometer are more likely to interact with other particles resulting in unintentional dissociation. It will also be understood that some voltages of a mass spectrometer may only have a limited range for calibration without adversely affecting the transmission of ions in the mass spectrometer. Such voltages may not be suitable for use as characteristic voltages according to the present disclosure depending on the range of calibration desired.

[0015] In some embodiments, the amount of unintentional dissociation is determined based on the intensity of a mass spectral peak associated with a fragment ion of the calibration ion relative to the intensity of a mass spectral peak associated with the calibration ion. Thus, the amount of unintentional dissociation can be determined based on the ratio of an amount of one or more fragment ions of the calibration ion to an amount of the calibration ion detected in the MS1 spectrum.

[0016] In some embodiments, the target amount of unintentional dissociation is 50%, 25%, 15%, 10%, 7%, 5%, 3%, 2%, 1%, or 0.5% or less. In some embodiments, the target amount of unintentional dissociation is in the range of 1%-10%, or 2%-8%, or 3%-6%. In some embodiments, it may be preferable to provide a mass spectrometer that is calibrated so that unintentional dissociation does not occur, but for some fragile ions, this may be difficult or require significant operational compromises. In such cases, the mass spectrometer may be calibrated such that the amount of unintentional dissociation is controlled to be consistent and repeatable over the range of the MS1 scan and / or the range of the mass spectrometer.

[0017] In some embodiments, multiple MS1 analyses are performed at different characteristic voltages, and the amount of unintentional dissociation of the calibration ion is determined for each MS1 spectrum, and the characteristic voltage is calibrated based on the amount of unintentional dissociation of the calibration ion present in the MS1 spectrum to provide a target amount of unintentional dissociation of the calibration ion during the MS1 analysis. In this manner, the characteristic voltage may be calibrated based on determining a relationship between the characteristic voltage and the resulting amount of unintentional dissociation of the calibration ion. For example, interpolation may be used to determine a suitable relationship based on calibration measurements.

[0018] In some embodiments, the ion optical device is an ion trap or ion guide. Thus, the mass spectrometer may include an ion trap, the characteristic voltage of which is controlled to control the amount of unintended dissociation that occurs during MS1 analysis.

[0019] For example, in some embodiments, transporting the calibration ions from the ion source to the mass analyzer via the ion optical device includes storing the calibration ions in an ion trap. Ions may tend to unintentionally dissociate when they enter or are stored in the ion trap. This is because the kinetic energy associated with the average motion of the ions entering the ion trap relative to the average motion of background particles in the ion trap may result in particle interactions that result in unintentional dissociation of at least some of the ions. To control for this effect, the kinetic energy of the ions when they enter or are stored in the ion trap may be controlled by a characteristic voltage of the ion trap.

[0020] In some embodiments, the characteristic voltage is at least one DC voltage applied to the ion optical device, in particular the ion trap, to provide a DC offset to the ion optical device / trap. The DC offset of the ion optical device / trap may be used to control the force that accelerates ions into the ion optical device / trap, or to control the force that prevents ions from exiting the ion optical device / trap, or to control the force with which ions exit the ion optical device / trap. In some embodiments, the characteristic voltage is at least one RF voltage applied to the optical device, in particular at least one RF voltage applied to the ion trap configured to confine ions in the ion trap. In some embodiments, the characteristic voltage is used to calibrate a plurality of voltages applied to the ion trap, the plurality of voltages being configured to control one or more of injecting calibration ions into the ion trap, confining calibration ions in the ion trap, and ejecting calibration ions from the ion trap. In this way, by controlling one or more voltages applied to the ion trap, the amount of energy imparted to ions by the ion trap can be controlled. This may affect the degree of unintentional dissociation occurring in the ion trap.

[0021] In some embodiments, the characteristic voltage may include an RF voltage applied to an S-lens, an ion funnel, or a stacked ring ion guide.

[0022] In some embodiments, the method further includes measuring a vacuum pressure associated with the mass spectrometer, and the characteristic voltage of the ion optical device is calibrated based on the amount of unintentional dissociation of the calibration ions present in the MS1 spectrum and the measured vacuum pressure. In some embodiments, the amount of unintentional dissociation occurring in the mass spectrometer may also depend on the pressure of the mass spectrometer, since the pressure of the mass spectrometer may affect the probability that ion-gas particle interactions occur. Over time, the vacuum pressure in the mass spectrometer may vary. By taking into account any variations in pressure, the characteristic voltage of the ion optical device may be further adjusted to maintain a target amount of unintentional dissociation during the MS1 analysis of the calibration ions.

[0023] In some embodiments, transporting the calibration ions from the ion source through the ion optical device to a mass analyzer of the mass spectrometer comprises transporting the calibration ions from the ion source to a mass selector, the mass selector mass filtering the calibration ions; transporting calibration ions to an ion optical device; and transporting the calibration ions from the ion optical device to a mass analyzer.

[0024] As such, the mass spectrometer may mass filter the calibration ions before they enter the ion optical device. By mass filtering the calibration ions, any unwanted ions may be removed, such that only the calibration ions are transported to the ion optical device. This may allow for more specific identification of the effect of the ion optical device (downstream of the mass selector) on unintentional dissociation of the calibration ions. In addition, this method may allow for the calibration ions (i.e., ions of a single species) to be mass selected from a standard sample containing multiple different ion species.

[0025] In some embodiments, the ion optical device may comprise a multipole assembly, preferably a quadrupole assembly. In some embodiments, the mass analyzer may comprise an orbital trap mass analyzer, a time-of-flight mass analyzer, a Fourier transform mass analyzer, an ion trap mass analyzer, a quadrupole mass analyzer, or a magnetic sector mass analyzer. Thus, it will be appreciated that the method of the first aspect may be applied to any mass analyzer. In particular, the method of the first aspect is not limited to any particular arrangement of the ion optical device and / or the mass analyzer.

[0026] In some embodiments, the multiple mass spectrometers are calibrated to provide a target amount of unintentional dissociation of the calibration ion during MS1 analysis. In this way, the method according to the first aspect can be applied to multiple mass spectrometers so that each mass spectrometer provides a consistent target amount of unintentional dissociation during MS1 analysis. Calibrating multiple mass spectrometers in such a way can be particularly advantageous when MS1 ​​analysis of a sample containing fragile ions is to be performed across several mass spectrometers.

[0027] According to a second aspect, there is provided a method of mass spectrometry for a mass spectrometer, the method comprising: generating sample ions from an ion source; Transporting sample ions from an ion source to a mass analyzer of a mass spectrometer through an ion optical device, where a characteristic voltage applied to the ion optical device controls passage of ions into and / or out of the ion optical device. performing MS1 analysis of the sample ions using a mass analyzer to obtain an MS1 ​​spectrum of the sample ions; The characteristic voltage of the ion optical device is calibrated according to the method of the first aspect so as to provide the amount of sample ions unintentionally dissociated during MS1 analysis.

[0028] In this way, the method of the second aspect is performed using a mass spectrometer calibrated according to the first aspect of the present disclosure. Therefore, the MS1 scan performed according to the second aspect may have a consistent amount of unintentional dissociation, which may be particularly advantageous when MS1 ​​analysis of relatively fragile ions is to be performed. In some embodiments, the sample ions analyzed may be the same ions, or the same type of ions (e.g., peptides), as the calibration ions used to calibrate the mass spectrometer, but in some embodiments, the sample ions analyzed may be different from the calibration ions used to calibrate the mass spectrometer. By calibrating the characteristic voltage of the ion optical device, the unintentional dissociation of the sample ions resulting from the calibrated ion optical device may be controlled via the characteristic voltage. Thus, by calibrating the mass spectrometer using the calibration ions, the amount of unintentional dissociation occurring when performing MS1 scans on the sample ions may be controlled.

[0029] In some embodiments, the method further includes determining the amount of unintentional dissociation based on the intensity of one or more mass spectral peaks associated with fragment ions (i.e., fragment ion species) of the sample ions relative to the intensity of one or more mass spectral peaks associated with the sample ions (i.e., unfragmented ions).

[0030] In some embodiments, the target amount of unintentional dissociation is less than or equal to 50%, 25%, 15%, 10%, 7%, 5%, 3%, 2%, 1%, or 0.5% of the sample ions transported through the ion optical device to the mass analyzer, hi some embodiments, the target amount of unintentional dissociation is within the range of 1%-10%, or 2%-8%, or 3%-6% of the sample ions transported through the ion optical device to the mass analyzer.

[0031] In some embodiments, the method further includes measuring a vacuum pressure associated with the mass analyzer. A characteristic voltage of the ion optical device is then updated based on the vacuum pressure of the mass analyzer. By updating the characteristic voltage based on the vacuum pressure associated with the mass analyzer, the method of mass analysis can account for changes in operating conditions of the mass analyzer, which may affect the amount of unintentional dissociation occurring within the mass analyzer.

[0032] According to a third aspect of the present disclosure, there is provided a mass spectrometer comprising an ion source, an ion optical device, a mass analyzer, and a controller. The ion source is configured to generate calibration ions. The ion optical device is configured to receive the calibration ions from the ion source. The mass analyzer is configured to receive the calibration ions from the ion optical device. The controller is configured to: Producing calibration ions in an ion source; causing a mass spectrometer to transport calibration ions from an ion source through an ion optical device to a mass analyzer of the mass spectrometer, wherein a characteristic voltage is applied to the ion optical device to control passage of the ions into and / or out of the ion optical device, the applied characteristic voltage resulting in an unintentional dissociation amount of the calibration ions; causing a mass analyzer to perform MS1 analysis of the calibration ions to obtain an MS1 ​​spectrum of the calibration ions; determining the amount of unintentional dissociation of calibration ions present in the MS1 spectrum; and calibrating a characteristic voltage of the ion optical device based on the unintentional dissociation amount of the calibration ion present in the MS1 spectrum to provide a target unintentional dissociation amount of the calibration ion during the MS1 analysis.

[0033] The mass spectrometer of the third aspect is thereby configured to perform the calibration method according to the first aspect of the present disclosure. It will be appreciated that the mass spectrometer of the third aspect may also be configured to perform the method of mass spectrometry according to the second aspect of the present disclosure. Embodiments of the present invention will now be described with reference to the following figures. [Brief description of the drawings]

[0034] [Figure 1] 1 shows a schematic diagram of a mass spectrometer according to an embodiment of the present disclosure. [Diagram 2] 1 is a flow chart of a method for calibrating a mass spectrometer according to an embodiment of the present disclosure. [Diagram 3] 1 is a graph showing the effect of changing the characteristic voltage on the unintentional dissociation of the MRFA2+ ion. [Figure 4] 1 is a flow chart of a method of mass spectrometry according to an embodiment of the present disclosure. [Diagram 5] 1 is a graph showing the effect of varying the fore-vacuum pressure and transfer line temperature of a mass spectrometer on the calibrated characteristic voltages. [Figure 6] 13 is a graph showing the effect of independently varying the fore-vacuum pressure of a mass spectrometer on calibrated characteristic voltages. [Figure 7] 13 is a graph showing the effect of independently varying transfer tube temperature on calibrated characteristic voltages. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] According to a first embodiment of the present disclosure, there is provided a mass spectrometer 10. A schematic diagram of the mass spectrometer is shown in Figure 1. The arrangement of Figure 1 generally represents the configuration of a Thermo Fisher Scientific, Inc. Orbitrap Exploris (RTM) 240 mass spectrometer.

[0036] 1, molecules to be analyzed (e.g., sample molecules or calibration molecules) are provided to a mass spectrometer 10. For some molecules, particularly calibration molecules, the molecules may be provided to the mass spectrometer 10 by direct injection. In this manner, the molecules to be analyzed may be provided directly to an ion source for ionization.

[0037] As an alternative to direct injection, the molecules to be analyzed (e.g., sample molecules or calibration molecules) may be delivered to the mass spectrometer 10 (e.g., from an autosampler) via a chromatographic device such as a liquid chromatography (LC) column (not shown in FIG. 1). One such example of an LC column is Thermo Fisher Scientific, Inc.'s ProSwift (RTM) monolithic column, which provides high performance liquid chromatography (HPLC) by forcing the sample with a mobile phase under high pressure through a stationary phase of irregularly shaped or spherical particles. In an HPLC column, sample molecules elute at different rates depending on the extent of their interaction with the stationary phase.

[0038] The sample molecules (or calibration molecules) thus separated via liquid chromatography or provided by direct injection are then ionized using an ion source. In the embodiment of FIG. 1, the ion source is a (heated) electrospray ionization ((H)ESI) source 20 at atmospheric pressure. The sample ions (or calibration ions) then enter the vacuum chamber of the mass spectrometer 10 and are directed to the mass analyzer via multiple ion optical devices. In the embodiment of FIG. 1, the sample ions (or calibration ions) are directed by a capillary 25 into a stacked ring (RF) ion guide, an RF-only S-lens 30, at low to medium vacuum pressure. The ions are focused by the S-lens 30 into an injection flatapole 40, which injects the ions into a bent flatapole 50 with an axial field. The bent flatapole 50 guides the (charged) ions along a curved path through it, whereas undesired neutral molecules such as entrained solvent molecules are not guided along the curved path and are lost.

[0039] An ion gate (TK lens) 60 is disposed at the distal end of the bent flatapole 50 and controls the passage of ions from the bent flatapole 50 to a downstream quadrupole mass filter 70. The quadrupole mass filter 70 is typically, but not necessarily, segmented and acts as a bandpass filter, allowing the passage of selected mass numbers or a limited mass range while rejecting ions of other mass to charge ratios (m / z). The quadrupole mass filter 70 can also be operated in a broadband transmission mode. The quadrupole mass filter 70 can be configured to have a filter size of, for example, 1×10 -4 mbar or less, or 5×10 -5 It is located within the high vacuum region of the spectrometer, which has a pressure of up to 10 ...

[0040] The ions then pass through a quadrupole exit lens / splitting lens arrangement 80 and enter a transfer multipole 90. The transfer multipole 90 directs the mass-filtered ions from the quadrupole mass filter 70 into a curved linear ion trap (C-trap) 100. Like the quadrupole mass filter 70, the C-trap 100 is also located in the high vacuum region of the spectrometer. Typically, the cavity of the C-trap 100 in which the ions are trapped has a volume of approximately 5×10 due to a supply of nitrogen gas coming from the adjacent ion routing multipole 120 (which is at a higher pressure). -3 The C-trap 100 is at a pressure of 1000 psi or less. The C-trap 100 has longitudinally extending curved electrodes that are supplied with an RF voltage and end caps that are supplied with a DC voltage. The result is a potential well that extends along the curved longitudinal axis of the C-trap 100. In a first mode of operation, a DC end cap voltage is set on the C-trap so that ions arriving from the transfer multipole 90 are accumulated in the potential well of the C-trap 100 and cooled there. The cooled ions exit in a cloud towards the bottom of the potential well and are ejected orthogonally from the C-trap 100 towards the mass analyzer 110.

[0041] The number of ions accumulated in the C-trap 100 (i.e., the ion population) determines the number of ions that are subsequently ejected from the C-trap 100 into the mass analyzer 110. The C-trap 100 can eject ions as packets of ions into the mass analyzer 110. The C-trap 100 can also eject ions axially into an ion routing multipole 120 located at the opposite end of the C-trap 100 relative to the quadrupole mass filter 70. In another mode, the DC endcap voltages of the C-trap 100 can be set such that no potential well is formed for ions arriving from the transfer multipole 90, and ions instead transmit through the C-trap 100 and into the ion routing multipole 120 without being accumulated therein.

[0042] Thus, ions in the mass spectrometer 10 may travel from the bent flatapole 50, through the ion gate 60, the quadrupole mass filter 70, the lens 80, the transfer multipole 90, and the C-trap 100 into the ion routing multipole. Along this ion trajectory, ions in the mass spectrometer 10 may be subjected to an accelerating potential for the purpose of transporting ions from the bent flatapole 50 to the ion routing multipole. It will be appreciated that the accelerating potentials applied to these ion optical devices are significantly lower than accelerating potentials that may be applied to intentionally fragment ions in, for example, a fragmentation chamber. For example, in some embodiments, the accelerating potential applied between the bent flatapole 50 and the ion routing multipole may be about 10V or less.

[0043] The ion routing multipole in use is filled with a collision gas (e.g., helium or nitrogen) and thus has a higher pressure than the C-trap 100. The ion routing multipole, in normal operation, has a collision pressure of at least 3×10 -3 mbar ~ approx. 2 × 10 -2 The ion routing multipole is filled with collision gas at a pressure of 1.1×10 mbar. Typical operating pressures for the multipole are approximately 1.1×10 -2mbar. Because the ion routing multipole 120 is at a relatively higher pressure than other ion optical components of the mass spectrometer 10 (e.g., the C-trap 100), the kinetic energy of ions entering the ion routing multipole 120 may be sufficient to cause unintentional dissociation of some of the ions if these ions are fragile. Thus, the ion routing multiple 120, and specifically the passage of ions through the ion routing multipole, represents a portion of the mass spectrometer where unintentional dissociation may occur. Thus, control and calibration of the accelerating potentials that ions experience as they enter the ion routing multipole provides a mechanism by which one or more characteristic voltages can be used to control the amount of unintentional dissociation that occurs within the ion optical devices of the mass spectrometer 10.

[0044] The ion routing multipole 120, in some embodiments, may be configured to store ions for mass analysis in the mass analyzer 110. As such, the ion routing multipole 120 is a form of ion trap that acts as an ion optical device along the ion path between the ESI source 20 and the mass analyzer 110. Ions may be stored and / or cooled in the ion routing multipole 120 for purposes of performing MS1 or MS2 analysis.

[0045] The ion routing multipole 120 typically comprises a set of multipole rods extending along the ion routing multipole 120 and arranged around a central axis of the ion routing multipole. The multipole rods may be, for example, quadrupoles, hexapoles, or octopoles. The ion routing multipole 120 may also include pairs of end electrodes arranged at opposite ends of the set of multipole rods. An RF potential is applied to the set of multipole rods to form a pseudo-potential field to confine ions along the central axis of the ion routing multipole 120. A DC potential may also be applied to the multipole rods such that the RF potential is superimposed on the DC potential. In some embodiments, the ion routing multipole may include additional axial DC electrodes along the length of the ion routing multipole. The axial DC electrodes are configured to provide multiple DC voltages along the length of the multipole rods. The axial DC electrodes may be configured to provide an axially increasing, axially flat, or axially decreasing DC voltage profile along the length of the ion routing multipole. Thus, the ion routing multipole includes a number of different voltages that can be controlled by the controller 130 to control how ions are injected into, cooled within, and ejected from the ion routing multipole. For example, the DC potential of the axial DC electrodes and / or the DC potential of the multipole rods can determine the acceleration potential that the ions experience as they move from the bent flatterpole 50 and enter the ion routing multipole 120. The amount of acceleration that the ions experience affects the kinetic energy of the ions as they enter the ion routing multipole.

[0046] Ions may be stored in the ion routing multipole 120 by application of a DC voltage (known as a trapping voltage) applied to the axial ends of the ion routing multipole 120, which DC voltage is also applied to the set of multipole rods. Application of the trapping voltage prevents ions from escaping from the ion routing multipole 120 into the C-trap when not desired. Thus, the trapping potential controls not only when ions are released from the ion trap, but also the entry of ions into the ion routing multipole. For MS1 analysis, ions stored in the ion routing multipole 120 are (intentionally) moved back into the C-trap 100, and then the ions are ejected into the mass analyzer 110.

[0047] The controller 130 may calibrate one or more of the above-mentioned voltages of the ion routing multipole 120 according to a method for calibrating a characteristic voltage of an ion optical device. In some embodiments, the characteristic voltage may be one of the above-mentioned voltages (e.g., a DC voltage applied to the axial end of the ion routing multipole, a DC voltage applied to the multipole rods, or a DC or RF voltage applied to the multipole rods). In some embodiments, the various voltages of the ion routing multipole 120 (or indeed any other ion optical device having multiple voltages) may be related to each other by one or more relationships. In this manner, the determined characteristic voltage may be used to calibrate the multiple voltages of the ion optical device. For example, in some embodiments, the DC voltage applied to the entrance electrode of the ion routing multipole may be about 30% of the DC potential applied to the multipole rods. Thus, the characteristic voltage (e.g., the DC voltages of the rods) may be used to calibrate the multiple voltages of the ion optical device based on one or more voltage relationships.

[0048] 1, the mass analyzer 110 is an orbital trap mass analyzer 110, such as the Orbitrap (RTM) mass analyzer sold by Thermo Fisher Scientific, Inc. The orbital trap mass analyzer 110 has an off-center injection aperture through which ions are injected as coherent packets into the orbital trap device 110. The ions are then trapped within the orbital trap mass analyzer 110 by a hyperlogarithmic electric field and undergo a back and forth longitudinal (z) motion as they orbit around an inner electrode.

[0049] The axial (z) component of motion of an ion packet in the orbital trap mass analyzer 110 is defined as (more or less) simple harmonic motion, with the angular frequency in the z direction related to the square root of the mass-to-charge ratio of a given ion species, which causes ions to separate over time according to their mass-to-charge ratio (m / z).

[0050] Ions in the orbital trap mass analyzer 110 are detected using an imaging current detector (not shown in FIG. 1), which produces a "transient" in the time domain that contains information about all ion species as they pass through the imaging detector. The transient is then subjected to a Fast Fourier Transform (FFT), which produces a series of peaks in the frequency domain. From these peaks, a mass spectrum can be produced that represents the presence / intensity of ions versus m / z.

[0051] In the above configuration, sample ions (more specifically, a subset of sample ions within a mass range of interest selected by the quadrupole mass filter) are analyzed without intentional fragmentation by the orbital trap mass analyzer 110. The resulting mass spectrum is denoted MS1.

[0052] MS / MS (or, more commonly, MS n) may also be performed by the mass spectrometer 10 of FIG. 1. To achieve this, precursor sample ions are generated and transported to the quadrupole mass filter 70 where an auxiliary mass range is selected. The ions leaving the quadrupole mass filter 70 are injected through the C-trap 100 into the ion routing multipole 120. The ion routing multipole 120 may also be configured to function as a fragmentation chamber configured to fragment the precursor ions into fragment ions. For example, in the mass spectrometer 10 of FIG. 1, the ion routing multipole may comprise a higher energy collisional dissociation (HCD) device to which a collision gas is supplied. The precursor ions arriving at the ion routing multipole 120 may collide with gas molecules at high energy, resulting in the fragmentation of the precursor sample ions into fragment ions. The fragment ions are then ejected from the ion routing multipole 120 back towards the C-trap 100, where the fragment ions are trapped and cooled again in a potential well. Finally, the fragment ions trapped in the C-trap are ejected orthogonally towards the orbital trapping device 110 for analysis and detection. The resulting mass spectrum of the fragment ions is denoted MS2.

[0053] Although an ion routing multiplex 120 comprising an HCD device is shown in FIG. 1, other fragmentation devices using methods such as collision induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), photodissociation, etc. may alternatively be used for MS2 analysis.

[0054] The “dead-end” configuration of ion routing multipole 120 of FIG. 1 is described in further detail in WO 2006 / 103412(A), in which precursor ions are ejected axially from C-trap 100 in a first direction towards fragmentation chamber 120 and the resulting fragment ions are returned to C-trap 100 in the opposite direction.

[0055] The mass spectrometer 10 is under the control of a controller 130 which is configured to, for example, control the timing of ejection and trapping voltages, set appropriate potentials on electrodes such as the S-lens, quadrupole, etc. to focus and filter ions, acquire mass spectral data from the orbital trapping device 110, control sequences of MS1 and MS2 scans, etc. It will be appreciated that the controller 130 may comprise a computer capable of operating according to a computer program which includes instructions to cause the mass spectrometer to carry out steps of a method according to the present invention.

[0056] It should be understood that the particular arrangement of components shown in Figure 1 is not required for the methods described subsequently, and indeed the methods described in this disclosure may be implemented in any controller for controlling the injection of ions into a Fourier transform mass analyzer, a TOF mass analyzer, or an ion trap mass analyzer.

[0057] Further, one skilled in the art will appreciate that the mass spectrometer 10 of Figure 1 is one example of an instrument in which ions are transported from an ion source (ESI source 20) through one or more ion optical devices to a mass analyzer (110). Thus, in the embodiment of Figure 1, the capillary 25, S-lens 30, injection filter 40, bent flatapole 50, ion gate 60, quadrupole mass filter 70, exit lens / split lens arrangement 80, transfer multipole 90, C-trap 100, and ion routing multipole 120 are examples of ion optical devices. Each of the ion optical devices is configured to transport sample ions (or calibrant ions) from the ESI ion source 20 to the mass analyzer 110. In other embodiments, other configurations of ion optical devices may be used to transport ions from the ion source to the mass analyzer.

[0058] According to an embodiment of the present invention, there is provided a method 200 for calibrating a mass spectrometer 10. A flow chart of the method 200 is shown in Figure 3. For example, the controller 130 may be configured to cause the mass spectrometer 10 to perform the calibration method 200 using the mass spectrometer 10 of Figure 1.

[0059] As shown in step 201, mass spectrometer 10 generates calibration ions. The calibration ions are generated using ESI source 20. The sample used to generate the calibration ions may be, for example, a calibration solution containing molecules known to form suitably fragile ions. One example of a suitable calibration solution is Pierce (RTM) FlexMix (RTM) calibration solution from Thermo Scientific (RTM). This calibration solution, when ionized, produces peptide ions [MRFA+H2O] 2+ (M.R.F.A. 2+ This ion is known to be a fragile ion under normal operating conditions. In particular, at least some of the [MRFA+H2O] 2+ The calibration ions are primarily fragment ions [MRFA-MeSH] during transport through one or more ion optical devices. 2+(m / z238.634Th) and [RFA+H2O] + (m / z 393.224).

[0060] In step 202, the mass analyzer 10 transports the calibration ions through the capillary 25, S-lens 30, injection filter 40, bent flatapole 50, ion gate 60, quadrupole mass filter 70, exit lens / split lens arrangement 80, transfer multipole 90, C-trap 100, and ion routing multipole 120 to the mass analyzer 110. The accelerating potential applied to the ions between the bent flatapole 50 and the ion routing multipole affects the kinetic energy of the calibration ions as they enter the ion routing multipole and are cooled. The kinetic energy of the calibration ions affects the amount of unintentional dissociation that occurs as the calibration ions pass through the mass analyzer.

[0061] As part of the ion transmission process, the calibrant ions may be mass filtered by a quadrupole mass filter with a narrow mass window centered around m / z of 262.636 (i.e., MRFA). 2+ (A suitable narrow mass window may be a mass window of about 2-10 m / z. Following mass selection, the mass selected calibration ions are then stored briefly in the ion routing multipole 120 prior to mass analysis. To store the calibration ions in the ion routing multipole, a trapping voltage is applied to the ion routing multipole 120 (relative to the voltage at which the C-trap 100 may be held). For example, under normal operation in MS1 ​​mode, the trapping voltage applied to the ion routing multipole to store non-fragile (positively charged) ions may be -2.0 V relative to the C-trap 100.

[0062] In step 203, the mass analyzer 110 mass analyzes the calibration ions and outputs a mass spectrum of the calibration ions.

[0063] The controller 130 may then determine the amount of unintentional dissociation based on the mass spectral peaks present in the MS1 mass spectrum in step 204. It will be appreciated that the methods and systems of the present disclosure may be applied to a wide range of calibration ions and various types of mass spectrometers used. With this in mind, there are several different ways in which the amount of unintentional dissociation occurring in the MS1 spectrum may be determined.

[0064] The present disclosure refers to the intensity of the mass spectrum peak of the MS1 spectrum.When analyzing the MS1 spectrum, it will be understood that in some embodiments, the intensity of the mass spectrum peak can be represented by the absolute value of the intensity provided in the MS1 spectrum.In some embodiments, the intensity of the mass spectrum peak of the mass spectrum can also be the signal-to-noise ratio of each of the mass spectrum peaks.

[0065] One such method is to measure the intensities of mass spectral peaks associated with the fragment ions of the calibrant ions (M fragment ) and the intensity of the mass spectral peaks associated with the calibration ions (M calib ) (i.e., the relative amount of unintended fragments), or the sum of the intensities of the mass spectral peaks associated with the fragment ions and the calibration ions (M fragment +M calib ) (i.e., the extent to which unintended fragmentation occurs). For example, in some embodiments, the relative amount of unintended dissociation (C frag ) is M calib and M fragment Based on this, it can be calculated according to the following formula: C frag =M fragment / M calib

[0066] In some embodiments, the intensities of mass spectral peaks associated with fragment ions of the calibration ions (M fragment) may be replaced by the sum of the intensities of mass spectral peaks associated with multiple different fragment ions of the calibration ion.

[0067] In some embodiments, the amount of unintentional dissociation can be determined by the intensity of the mass spectral peak associated with the first calibration ion (M calib1 ) and the intensity of the mass spectral peak associated with the second calibration ion (M calib2 ) can be determined based on the ratio of C analyte_ratio =M calib1 / M calib2

[0068] This method may be suitable when the sample used to calibrate the mass spectrometer contains two different calibration molecules with a known concentration ratio, particularly when one of the calibration molecules forms a relatively weak ion, i.e., one of the calibration ions is weaker than the other.

[0069] Thus, there are various ways according to the present disclosure that the amount of unintentional dissociation occurring may be characterized. It will also be appreciated that each of the above examples may be mathematically expressed in different ways, but still provide a value for the amount of unintentional dissociation. Once the amount of unintentional dissociation occurring has been determined, the mass spectrometer proceeds to calibrate a characteristic voltage of one or more ion optical devices in step 205. To determine whether the characteristic voltage should be adjusted, the controller 130 determines the amount of unintentional dissociation occurring (e.g., C frag , C analyte_ratio ) may be compared to a target amount of unintentional dissociation (T). Calibration of the characteristic voltage is discussed in more detail below.

[0070] 3 is a graph showing how the amount of unintentional dissociation in an MS1 ​​scan varies with the variation of the characteristic voltage applied to the ion routing multipole 120. In the graph of FIG. 3, a series of MS1 scans are performed at different trapping voltages, with [MRFA+H2O] 2+ (M.R.F.A. 2+, m / z 262.636Th) calibrant ions were trapped in the ion routing multipole 120 prior to MS1 analysis in the mass analyzer 110.

[0071] The left side of Figure 3 is [MRFA] 2+ Figure 3 shows a plot of the ratio of the intensity of a known fragment ion peak to the intensity of the mass spectrum peak, expressed as a percentage. 2+ (m / z 238.634Th) and fragment ion [RFA+H2O] + 3 shows a plot of (m / z 393.224). As previously mentioned, a typical MS1 trapping potential used for the ion routing multipole 120 is about 2.0 V (negative for positive ions and vice versa) relative to the voltage of the C-trap 100. By lowering the trapping potential, the amount of unintentional dissociation that occurs is reduced. By increasing the trapping potential, the amount of unintentional dissociation is increased. This allows the controller 130 to adjust the trapping potential applied to the ion routing multipole 120 to control the amount of unintentional dissociation that occurs. It will be appreciated that for the mass spectrometer 10 of FIG. 1, the magnitude of the trapping potential can vary between about 0.5 V and 3.0 V. In the example of FIG. 3, the variation in trapping potential is a function of the magnitude of the [MRFA-MeSH] 2+ (m / z238.634Th) fragment ion, about 3% to 13%, [RFA+H2O] + The (m / z 393.224) fragment ion results in unintentional dissociation fluctuations of approximately 3% to 15%.

[0072] The right axis of Fig. 3 shows the [MRFA] for different trapping potentials. 2+ The total ion transmission percentage of the calibration ions plus their unintentional MS1 fragments is shown. The total ion transmission percentage is expressed as a percentage of the total transmission at a trapping potential magnitude of 2.0 V. Figure 3 shows that for trapping voltage magnitudes between 0.5 V and 3.0 V, the reference transmission varies between approximately 104% and 96% of the total transmission at a "normal" trapping potential magnitude of 2.0 V.

[0073] In some embodiments, the mass spectrometer 10 may be calibrated by making multiple measurements of unintentional dissociation at different characteristic voltages. For example, as shown in FIG. 3, making multiple measurements may allow a relationship between the characteristic voltage and the amount of unintentional dissociation to be determined. For example, the controller 130 may interpolate data points to determine the relationship between the amount of unintentional dissociation and the characteristic voltage. Suitable interpolation methods include, for example, polynomial interpolation. The characteristic voltage may then be calibrated based on the target amount of unintentional dissociation using the determined relationship.

[0074] In some embodiments, the mass spectrometer 10 detects a target amount of unintended dissociation T equal to a target amount of unintended dissociation T (e.g., C frag =T). For example, in some embodiments, the target amount of unintended dissociation may be about 2%, 4%, 6%, 8%, or 10%.

[0075] In some embodiments, the mass spectrometer detects that the amount of unintended dissociation does not exceed the target amount of unintended dissociation (C frag For example, the amount of unintentional dissociation may be less than or equal to 10%, 8%, 6%, 4%, or 2%.

[0076] In some embodiments, the mass spectrometer is configured to measure the amount of unintended dissociation so that the amount of unintended dissociation falls within a range of a target amount of unintended dissociation (T1, T2) (T1≦C frag ≦T2), which can be calibrated. frag , C analyte_ratio It will be understood that similar limitations may apply to any of the methods for characterizing the amount of unintended dissociation that actually occurs, or any other suitable method for characterizing the amount of unintended dissociation that actually occurs. For example, the target range may be about 1% to 10%, or 2% to 8%, or 3% to 6%.

[0077] In some embodiments, the controller 130 may calibrate the characteristic voltage (e.g., trapping potential) using an iterative process in which the mass spectrometer 10 may repeatedly perform MS1 analysis and characteristic voltage calibration until the mass spectrometer meets a desired criterion for the amount of unintentional dissociation of the calibration ions generated (a target amount of unintentional dissociation).

[0078] It will be appreciated that the process for arriving at the calibrated voltage will depend on the criteria for the target amount of unintentional dissociation. For example, in some embodiments, a general relationship between the characteristic voltage and the amount of unintentional dissociation may be known a priori by the controller. In other embodiments, the controller 130 may calibrate the characteristic voltage using a predefined relationship between the characteristic voltage and the resulting change in the amount of unintentional dissociation.

[0079] In some embodiments, the controller may calibrate a characteristic voltage of an ion optical device (e.g., ion routing multipole 120), although it will be understood that the calibration method may be repeated for multiple voltages of an ion optical device of a mass analyzer. For example, if an ion optical device includes multiple voltages, each of the voltages may be calibrated in turn. In some embodiments, the calibration method may be applied in turn to each of multiple ion optical devices. Calibrating multiple ion optical devices in turn may be particularly advantageous when it is desirable to minimize or reduce unintentional dissociation to a relatively low level.

[0080] Thus, according to the above-mentioned method 200, the mass spectrometer 10 can be calibrated to provide a target amount of unintentional dissociation. The method 200 according to the present disclosure can be of particular interest when applied to multiple mass spectrometers 10. When multiple mass spectrometers 10 are used to perform similar MS1 analysis, slight differences in the setup of each mass spectrometer can result in variations in the performance of each mass spectrometer 10. In particular, the degree of unintentional dissociation of ions when performing MS1 analysis can vary from one mass spectrometer to another depending on hardware tolerances (mechanical and electronic). The method 200 can be applied to achieve very close performance across spectrometers. According to the method of calibrating the mass spectrometer 200, multiple mass spectrometers 10 can be calibrated to control possible variations in the amount of unintentional dissociation between mass spectrometers 10.

[0081] When multiple mass spectrometers 10 are calibrated according to the embodiments of the present disclosure, the multiple mass spectrometers 10 may be calibrated such that the unintentional dissociation amount of the calibration ions across the multiple mass spectrometers has a relative standard deviation of 10% or 5% or less. In this way, the mass spectrometer 10 may perform MS1 measurements with reduced variation in the unintentional dissociation amount of the calibration ions. In some embodiments, the mass spectrometer 10 may also be calibrated based on the calibration ions such that the unintentional dissociation amount of different sample ions is controlled. That is, in some embodiments, the mass spectrometer 10 may be calibrated using the calibration ions such that the relative standard deviation of the unintentional dissociation amount of the sample ions (different from the calibration ions) has a relative standard deviation within a specified range. For example, the relative standard deviation of the unintentional dissociation amount of the sample ions may be 20%, 15%, 10%, or 5% or less.

[0082] 4 shows a flow chart of a method 300 of mass spectrometry according to an embodiment of the present disclosure. The method 300 may be performed by the mass spectrometer 10 shown in FIG. 1, calibrated according to the calibration method 200 described above.

[0083] According to step 301 of method 300, sample ions are generated by ESI source 20. Sample ions may include any ions suitable for MS1 analysis. Thus, the sample ions analyzed may be different (e.g., have a different mass-to-charge ratio) than the calibration ions.

[0084] In step 302, the sample ions are transported to the mass analyzer 110 via the ion optical device. When transporting the sample ions, the ion optical device utilizes a calibrated characteristic voltage set by the calibration method 200. In this manner, the effect of the voltage applied to the ion optical device can be controlled to control any unintentional dissociation that occurs during ion transport. For example, according to the calibration method 200 described above, the trapping voltage applied to the ion routing multipole 120 can be a calibrated voltage such that the amount of unintentional dissociation that occurs when the sample ions are cooled within the ion routing multipole 120 can be controlled.

[0085] In step 303, mass analyzer 110 performs the MS1 analysis and outputs the resulting mass spectrum to controller 130 for analysis.

[0086] In some embodiments, the sample being analyzed may include a known vulnerable ion, or the sample may include an internal standard that includes a known calibration ion. This allows the resulting MS1 mass spectrum generated in step 303 of method 300 to include information regarding the amount of unintentional dissociation occurring in the mass spectrometer, which can be used to update the calibration of mass spectrometer 10. For example, the MS1 spectrum may include mass spectral peaks representing the known vulnerable ion and mass spectral peaks of known mass fragments (fragment ions) of the vulnerable ion. This allows, in some embodiments, the mass spectrometer to effectively perform method 200 of calibrating the characteristic voltage of mass spectrometer 10 in real time based on the data in the MS1 spectrum acquired in step 303.

[0087] If the mass spectrometer 10 is configured to update the characteristic voltage in real time, the mass spectrometer may average data over several MS1 scans to determine whether the characteristic voltage should be updated. In some embodiments, the mass spectrometer may filter data from one or more MS1 scans as to whether the amount of unintentional dissociation occurring varies enough that the characteristic voltage should be updated. For example, if the change in the amount of unintentional dissociation is within a specified range of a target value or is within a target value, the mass spectrometer may choose not to update the characteristic voltage. By averaging or filtering data related to unintentional dissociation, jitter associated with repeated updates to the characteristic voltage may be reduced or eliminated.

[0088] Thus, a method 300 of mass spectrometry may be provided in accordance with an embodiment of the present disclosure.

[0089] Although the above embodiment discusses the calibration method in terms of the characteristic voltage being a trapping potential applied to the ion routing multipole 120, it will be appreciated that in other embodiments, a different voltage of the mass spectrometer may be calibrated (i.e., a different voltage may be the characteristic voltage to be calibrated).

[0090] In some embodiments, the characteristic voltage may be an RF voltage applied to the ion trap (or similar ion confinement device that utilizes an RF voltage). Alternatively, the RF voltage supplied to the S-lens 30 may be calibrated. The voltage or voltages that are calibrated will depend on the geometry of the mass spectrometer being calibrated.

[0091] According to the above-described embodiments of the present disclosure, the characteristic voltage of the ion optical device may be calibrated based on the amount of unintentional dissociation of the calibration ions determined in the MS1 mass spectrum. In addition to the information in the MS1 spectrum, in some embodiments the mass spectrometer may also consider further information regarding the operating conditions of the mass spectrometer when performing the calibration.

[0092] In some embodiments, the amount of unintentional dissociation that occurs for a given ion optical characteristic voltage may also depend on the pressure of the mass analyzer. Variations in the pressure of the mass analyzer, for example in the region of one or more ion optical devices, may affect the amount of unintentional dissociation that occurs. For example, a reduction in the pressure of the mass analyzer may result in a reduction in the efficiency of ion cooling within the mass analyzer, leading to ions having higher kinetic energy when passing through the ion optical device. When ions have higher kinetic energy, unintentional dissociation may be more likely to occur.

[0093] In some embodiments, it is desirable to provide a mass spectrometer that has a relatively consistent amount of unintentional dissociation so that the characteristic voltage can be further calibrated and controlled to account for any variations in pressure and the resulting effect that may have on unintentional dissociation.

[0094] Thus, in some embodiments, the method of calibrating a mass spectrometer may further include measuring a vacuum pressure associated with the mass spectrometer. The vacuum pressure may be measured at various locations within the mass spectrometer. For example, in the mass spectrometer 10 of FIG. 1, the mass spectrometer vacuum pressure may be measured at the injector filter 40. In this region, gas passing through the injector filter 40 and entering the bent flatterpole 50 provides ion cooling within the bent flatterpole 50. In this manner, the vacuum pressure in the region of the injector filter 40 reflects the efficiency with which ions may be cooled within the ion optical device of the mass spectrometer 10. Alternatively, the vacuum pressure may be measured elsewhere, for example, at the mass spectrometer fore-vacuum pressure (mass spectrometer 10 fore-vacuum pressure) in the region of the S-lens 30. The pressure measurement may be performed using any suitable pressure sensor (not shown in FIG. 1).

[0095] For example, [MRFA] 2+For a calibration ion such as , the mass spectrometer of Figure 1 may measure the fore-vacuum pressure while calibrating the trapping potential. Table 1 below shows the relationship between calibrated trapping potential (TP) and fore-vacuum pressure that provides a target unintentional dissociation amount of 3%.

[0096] [Table 1]

[0097] Table 1 shows the mass distribution of the two fragments ([MRFA-MeSH]) at a trapping potential of 2.0 V for different fore-vacuum pressures. 2+ (m / z238.634Th) and [RFA+H2O] + 1 shows the value of the amount of unintentional dissociation of the calibration ion (m / z 393.224). The characteristic voltage (trapping potential of the ion routing multiple 120) is then calibrated according to the method 200 described above so that the amount of unintentional dissociation that occurs is about 3%. The calibrated trapping potential for each fore-vacuum pressure is shown in Table 1. It will be appreciated that as the fore-vacuum pressure is reduced, the characteristic voltage can also be reduced in order to maintain a similar amount of unintentional dissociation of the calibration ion.

[0098] By measuring the pressure when performing the calibration of the characteristic voltage, the characteristic voltage can then be calibrated based on the measured vacuum pressure and the amount of unintentional dissociation of the calibration ions present in the MS1 spectrum. In this way, the calibration method can take into account the pressure of the mass spectrometer / ion optical device when performing the calibration.

[0099] Thus, when the mass spectrometer is used to perform MS1 analysis using sample ions, the mass spectrometer 10 may take an additional pressure measurement using the same pressure sensor. The mass spectrometer 10 may then update the previously calibrated characteristic voltage based on the measured pressure. In this manner, if the pressure of the mass spectrometer differs from the pressure at which the calibration was performed, the mass spectrometer may update the characteristic voltage to account for changes in ion cooling efficiency.

[0100] In some embodiments, the mass spectrometer may update the characteristic voltage based on a predetermined relationship between pressure, the characteristic voltage, and the resulting amount of unintentional dissociation. This relationship may be, for example, a linear or non-linear adjustment of the characteristic voltage in response to pressure variations. The desired linear or non-linear relationship may be provided by the controller 130 to update the characteristic voltage based on the measured pressure. In some embodiments, this relationship may be determined during a calibration process by performing a series of MS1 analyses at different characteristic voltages and operating pressures of the mass spectrometer.

[0101] For example, Figure 5 shows the fore-vacuum pressure of the mass spectrometer in Figure 1 and the [MRFA] of 3%. 2+ 5 shows a graph of the relationship between the calibrated trapping potential and the calibrated characteristic voltage that provides a target amount of unintentional dissociation of calibration ions. In the graph of FIG. 5, the fore-vacuum pressure was intentionally varied by varying the temperature of the transfer tube 25 and by closing or opening the gas ballast of the fore-vacuum pump. It will be appreciated that the mass spectrometer 10 may be configured to perform a series of MS1 experiments to establish an empirical relationship between vacuum pressure and the calibrated characteristic voltage. Such experiments may be performed as part of a method of calibrating the mass spectrometer 10.

[0102] Figure 6 shows a further graph of the relationship between the fore-vacuum pressure of the mass spectrometer of Figure 1 and the resulting calibrated trapping potential (trapping potential for 3% unintentional dissociation). Figure 6 also shows the [MRFA] at a trapping potential of 2.0 V for different fore-vacuum pressures. 2+ [RFA+H2O] +6 shows a graph of the amount of unintentional dissociation to (m / z 393). The measurements (and associated calibrated trapping potentials) shown in FIG. 6 were taken at a constant transfer tube 25 temperature of 320° C. It will be appreciated that the mass spectrometer 10 may be configured to perform a series of MS1 experiments to establish an empirical relationship between vacuum pressure and calibrated characteristic voltage. The measurements may be performed at a number of different transfer tube 25 temperatures, for example as shown in FIGS. 5 and 6. Such experiments may be performed as part of a method for calibrating the mass spectrometer 10.

[0103] Figure 7 shows a further graph of the relationship between the temperature of the transfer tube 25 and the resulting calibrated trapping potential (trapping potential for 3% unintentional dissociation). Figure 7 also shows the [MRFA] at a trapping potential of 2.0 V for different transfer tube 25 temperatures. 2+ [RFA+H2O] + (m / z 393) and [MRFA-MeSH] 2+ 7 shows a graph of the amount of unintentional dissociation to (m / z 238.634Th). The measurements (and associated calibrated trapping potentials) shown in FIG. 7 were taken at a constant fore-vacuum pressure of 1.94 mbar. It will be appreciated that the mass spectrometer 10 may then be configured to perform a series of MS1 experiments to establish an empirical relationship between the transfer tube 25 temperature (or indeed the temperature of any ion optical device) and a calibrated characteristic voltage. It will be appreciated that the measurements may be performed at a number of different vacuum pressures. Such experiments may be performed as part of a method for calibrating the mass spectrometer 10.

[0104] During operation of the mass spectrometer 10 performing a series of MS1 and / or MS2 analyses, the characteristic voltage may be repeatedly updated based on the mass spectrometer pressure. For example, the mass spectrometer 10 may update the characteristic voltage approximately every 15 minutes, or whenever the measured pressure changes by more than a threshold amount (e.g., by 0.01 mbar). For example, the mass spectrometer 10 may update the characteristic voltage based on a relationship between the fore-vacuum pressure and the characteristic voltage (e.g., based on the relationships shown in Figures 5, 6, and 7) obtained as part of a method for calibrating the mass spectrometer 10.

[0105] According to the embodiment described above, the characteristic voltage to be updated may be, for example, the trapping potential of the ion routing multipole 120. In another embodiment, the characteristic voltage to be updated may be, for example, the RF potential applied to the S-lens 30. The pressure of the mass spectrometer 10 in the region of the S-lens 30 may also affect the characteristics of the ion transport through the S-lens 30. In this manner, the characteristic voltage of the S-lens 30 may also be calibrated based on the amount of unintentional dissociation and the pressure of the mass spectrometer 10 according to the method described above. Furthermore, the RF voltage applied to the S-lens 30 may be controlled to improve the ion transport characteristics, as further described in GB 2569639(A).

[0106] While the above method involves updating the characteristic voltage based on the pressure of the mass spectrometer 10, it will be appreciated that the mass spectrometer may also update the characteristic voltage based on other parameters of the mass spectrometer 10 that may affect the amount of unintentional dissociation occurring within the mass spectrometer 10. In this manner, the characteristic voltage may be controlled via feedback from one or more sensors of the mass spectrometer 10 to reduce or eliminate variations in the amount of unintentional dissociation occurring during operation of the mass spectrometer 10. For example, the temperature of one or more of the ion optical devices may also affect the amount of unintentional dissociation occurring during, for example, an MS1 ​​analysis. For example, as shown in FIG. 7, a relationship may be established between the temperature of the transfer tube 25 and the calibrated trapping potential. Thus, the characteristic voltage may be controlled based on the temperature of the transfer tube 25.

[0107] Therefore, a mass spectrometer 10 is provided that can be calibrated to provide the amount of unintentional dissociation of ions. That is, for example, when performing MS1 analysis, the amount of unintentional dissociation of sample ions can be controlled so that MS1 analyses performed at different times or on different mass spectrometers can be comparable in terms of unintentional dissociation of sample ions. Control of unintentional dissociation of sample ions is particularly relevant when the sample ions analyzed are relatively fragile ions, such as ions of amino acids (e.g., isoleucine, phenylalanine), ions of (oligo)peptides (e.g., MRFA, ALELFR, substance P (RPKPQQFFGLM)), and ions of lipids.

[0108] Although preferred embodiments of the present invention have been described in detail herein, those skilled in the art will recognize that changes may be made to the embodiments of the invention without departing from the scope of the invention or the appended claims.

Claims

1. 1. A method of calibrating a mass spectrometer, comprising the steps of: generating calibration ions from an ion source; transporting the calibration ions from the ion source to a mass analyzer of the mass spectrometer through an ion optical device, wherein a characteristic voltage applied to the ion optical device controls the passage of ions into and / or out of the ion optical device, the applied characteristic voltage also resulting in an amount of unintentional dissociation of the calibration ions; performing MS1 analysis of the calibration ions using the mass analyzer to obtain an MS1 ​​spectrum of the calibration ions; determining the amount of unintentional dissociation of the calibration ions present in the MS1 spectrum; A method in which the characteristic voltage of the ion optical device is calibrated based on the unintentional dissociation amount of the calibration ion present in the MS1 spectrum to provide a target unintentional dissociation amount of the calibration ion during MS1 analysis.

2. 2. The method of claim 1, wherein the amount of unintentional dissociation is determined based on an intensity of a mass spectral peak associated with a fragment ion of the calibration ion relative to an intensity of a mass spectral peak associated with the calibration ion.

3. 3. The method of claim 2, wherein the target amount of unintentional dissociation is less than or equal to 15%, 10%, 7%, 5%, 3%, 2%, 1%, or 0.5%.

4. 3. The method of claim 2, wherein the target amount of unintended dissociation is within a range of 1% to 10%, or 2% to 8%, or 3% to 6%.

5. a plurality of MS1 analyses are performed at different characteristic voltages, and the amount of unintentional dissociation of the calibration ion is determined for each MS1 spectrum; 5. The method of claim 1, wherein the characteristic voltage is calibrated based on the amount of unintentional dissociation of the calibration ion present in the MS1 spectrum to provide a target amount of unintentional dissociation of the calibration ion during MS1 analysis.

6. the characteristic voltage is at least one DC voltage applied to the ion optical device to provide a DC offset to the ion optical device; or The method of any one of claims 1 to 4, wherein the characteristic voltage is at least one RF voltage applied to the ion optical device configured to confine ions within the ion optical device.

7. The method of any one of claims 1 to 4, wherein the ion optical device is an ion trap.

8. The method of claim 7 , wherein transporting the calibration ions from the ion source to the mass analyzer through the ion optical device comprises storing the calibration ions in the ion trap.

9. 8. The method of claim 7, wherein the characteristic voltage is used to calibrate a plurality of voltages applied to the ion trap, the plurality of voltages being configured to control one or more of: injecting calibration ions into the ion trap; confining calibration ions in the ion trap; and ejecting calibration ions from the ion trap.

10. measuring a vacuum pressure associated with the mass spectrometer; 5. The method of claim 1, wherein the characteristic voltage of the ion optical device is calibrated based on the amount of unintentional dissociation of the calibration ions present in the MS1 spectrum and the measured vacuum pressure.

11. The method of any one of claims 1 to 4, wherein the ion optical device comprises a multipole assembly.

12. Transporting the calibration ions from the ion source to a mass analyzer of the mass spectrometer through an ion optical device comprises: transporting the calibration ions from the ion source to a mass selector that mass filters the calibration ions; transporting the calibration ions to the ion optical device; and transporting the calibration ions from the ion optical device to the mass analyzer.

13. The method of any one of claims 1 to 4, wherein the mass analyser comprises an orbital trap mass analyser, a time-of-flight mass analyser, a Fourier transform mass analyser, an ion trap mass analyser, a quadrupole mass analyser, or a magnetic sector mass analyser.

14. The method of any one of claims 1 to 4, wherein a plurality of mass spectrometers are calibrated to provide a targeted amount of unintentional dissociation of calibration ions during MS1 analysis.

15. 1. A method of mass spectrometry for a mass spectrometer, comprising: generating sample ions from an ion source; transporting the sample ions from the ion source to a mass analyzer of the mass spectrometer through an ion optical device, a characteristic voltage applied to the ion optical device controlling the passage of ions into and / or out of the ion optical device; performing MS1 analysis of the sample ions using the mass analyzer to obtain an MS1 ​​spectrum of the sample ions; A method, wherein the characteristic voltage of the ion optical device is calibrated according to the method of any one of claims 1 to 4 to provide an amount of unintentional dissociation of sample ions during the MS1 analysis.

16. 16. The method of claim 15, further comprising determining an amount of unintentional dissociation based on an intensity of one or more mass spectral peaks associated with fragment ions of the sample ions relative to an intensity of one or more mass spectral peaks associated with the sample ions.

17. 17. The method of claim 16, wherein the amount of unintentional dissociation is less than or equal to 15%, 10%, 7%, 5%, 3%, 2%, 1%, or 0.5% of the sample ions transported through the ion optical device to the mass analyzer.

18. 16. The method of claim 15, wherein the amount of unintentional dissociation is within the range of 1% to 10%, or 2% to 8%, or 3% to 6% of the sample ions transported through the ion optical device to the mass analyzer.

19. measuring a vacuum pressure associated with the mass spectrometer; 16. The method of claim 15, further comprising: updating the characteristic voltage of the ion optical device based on the vacuum pressure of the mass spectrometer.

20. 1. A mass spectrometer comprising: an ion source configured to generate calibration ions; an ion optic device configured to receive calibration ions from the ion source; a mass analyzer configured to receive calibration ions from the ion optical device; A controller, The controller: generating calibration ions in the ion source; causing the mass spectrometer to transport the calibration ions from the ion source through the ion optical device to the mass analyzer of the mass spectrometer, wherein a characteristic voltage is applied to the ion optical device to control passage of ions into and / or out of the ion optical device, the applied characteristic voltage resulting in an amount of unintentional dissociation of the calibration ions; causing the mass analyzer to perform MS1 analysis of the calibration ions to obtain an MS1 ​​spectrum of the calibration ions; determining the amount of unintentional dissociation of the calibration ions present in the MS1 spectrum; calibrating the characteristic voltage of the ion optical device based on the unintentional dissociation amount of the calibration ion present in the MS1 spectrum to provide a target unintentional dissociation amount of the calibration ion during MS1 analysis; 16. A mass spectrometer configured to calibrate the mass spectrometer, comprising:

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