SELF-CALIBRATION OF SPECTRA USING MOLECULAR WEIGHT DIFFERENCES BASED ON KNOWN CHARGE STATES

Abstract

A method for calibrating a mass spectrometer, comprising: Mass analyzing first ions derived from an analyte molecule, wherein the first ions have a first charge state, Determining a first mass-to-charge ratio of the first ions, Mass analyzing second ions derived from the analyte molecule, wherein the second ions have a second charge state and wherein the second ions comprise protonated or adduct variants of the first ions, Determining a second mass-to-charge ratio of the second ions, Calculating a calibration correction based on the first mass-to-charge ratio and the second mass-to-charge ratio, wherein the step of calculating the calibration correction comprises calculating a linear drift α of the mass-to-charge ratio, Determining an uncertainty value for the calibration correction, wherein the step of determining the uncertainty value comprises determining a standard deviation Σ of the calibration correction, and Determine, based on the determined uncertainty value, whether the calibration correction should be applied.

Description

FIELD OF THE PRESENT INVENTION

[0001] The present invention relates to a method for calibrating a mass spectrometer, a mass spectrometry method, and a mass spectrometer. Embodiments relate to methods for calibrating a time-of-flight mass spectrometer. BACKGROUND

[0002] It is known to calibrate the mass-to-charge ratio scale of a mass spectrometer by fitting data from known ion peaks (reference standard) to the underlying sampling law used by the mass spectrometer (e.g., time-of-flight function). This calibration can be performed before, after, and / or during the acquisition of an unknown analyte.

[0003] Internal calibration generally refers to a calibration procedure whereby a known reference standard is added to the analyte sample itself, and the mixture of analyte sample and reference standard is subsequently ionized and mass analyzed. This procedure can be problematic because the reference standard must be carefully selected so that when the reference standard is ionized, the reference standard ions are then generated with a similar intensity to those of the unknown analyte to minimize or avoid saturation effects. Furthermore, the reference standard ions must have mass-to-charge ratios that differ from the analyte ions to avoid interference.

[0004] A more common form of calibration is known and is referred to as external calibration or lock-massing. External calibration, or lock-massing, refers to a process whereby the calibration is corrected at predefined calibration times. This approach relies on the stability of the system between calibration times. However, this can be problematic when short-term disturbances to the mass spectrometer components (e.g., voltage drift) occur. Furthermore, external calibration, or lock-massing, also has the problem of increasing the overall cost of the mass spectrometer, as the approach requires the provision of a separate dedicated ionization source to generate the reference standard, or lock-mass ions. Furthermore, the system must temporarily switch between the analyte and the reference standard, resulting in a loss of analyte data.Yet another problem with known external calibration methods is that the mass spectrometer switches to predefined time points during acquisition to perform a calibration check, and this can sometimes coincide with a time point at which an analyte of interest elutes from, for example, a liquid chromatography separation device, resulting in at least some potential analyte ions of interest not being generated or detected.

[0005] US 2002 / 130259 A1 discloses a calibration method in Fourier transform ion cyclotron resonance mass spectrometry. One embodiment involves identifying a plurality of ions with known mass differences and different charge states and adjusting the calibration parameters to cause the plurality of ions to be shifted to a relative position corresponding to the known mass differences. The measured mass-to-charge signal of analyte ions is then adjusted using the adjusted calibration parameter.

[0006] EP 1672673 A2 discloses a calibration method wherein transformation parameters A and B, determined using multiple peaks of observed mass, are used to transform a measured mass spectrum according to Ax + B, where x is the measured mass. The intercept B can be estimated using mass measurements of a singly charged ion or a doubly charged ion.

[0007] It is therefore desirable to provide an improved method for calibrating a mass spectrometer.

[0008] US 2002 / 0130259 A1 discloses a method for improving the calibration of a Fourier transform ion cyclotron resonance mass spectrometer. Incremental frequency shifts are iteratively added in the frequency domain, and the optimal frequency shift Df is obtained when the observed mass differences in the mass domain for different charge states have been eliminated or correspond to their known differences. SUMMARY

[0009] According to one aspect of the invention, a method for calibrating a mass spectrometer is provided having the features of claim 1

[0010] The accuracy of the calibration correction can be determined by the charge difference between the first charge state and the second charge state.

[0011] The accuracy of the calibration correction can be improved by increasing the charge difference between the first charge state and the second charge state.

[0012] According to a further aspect of the invention, a method for calibrating a mass spectrometer is provided having the features of claim 3

[0013] Typically, in time-of-flight mass analyzers, calibration drift is caused by thermal changes, which can alter the power supply outputs or cause thermal expansion of the flight tube. Calibration drift causes an unknown change in gain or linear drift in the mass axis. If the masses measured after the drift are multiplied by the inverse of the gain error, they can be accurately corrected. Generally, a predominant error in time-of-flight calibrations is caused by a change in gain. Higher-order terms, as well as the time-of-flight offset (intercept), are generally not significantly affected by thermal drift.

[0014] According to these embodiments, a calibration correction can be calculated by measuring two species of analyte ions with two different charge states or two species of analyte ions derived from adducts of two different masses. It is therefore possible to account for calibration drifts, thereby improving the accuracy of mass measurements. Further embodiments are contemplated, wherein the two (or more) species of analyte ions can be derived from adducts of different masses and with different charge states.

[0015] The accuracy of the calibration correction can be determined by the mass difference between the first adductions and the second adductions.

[0016] The accuracy of the calibration correction can be improved by increasing the mass difference between the first adductions and the second adductions.

[0017] The step of calculating the calibration correction involves determining a linear drift α of a mass-to-charge ratio.

[0018] The linear drift α can be calculated using the relationship α=aMa'−bMb'(a−b)H where a is the charge state of the first ions, M a ' is the mass-to-charge ratio of the first ions, b is the charge state of the second ions, M b ' is the mass-to-charge ratio of the second ions and H is the mass of a proton.

[0019] The linear drift α can be calculated using the relationship α=aMa'−bMb'Δa−Δb be determined, where a is the charge state of the first ions, M a ' is the mass-to-charge ratio of the first ions, Δ a is the mass of adducts from which the first ions are derived, b is the charge state of the second ions, M' bis the mass-to-charge ratio of the second ions and Δ b is the mass of adducts from which the second ions are derived.

[0020] The method may further comprise using the calibration correction to correct the mass, mass-to-charge ratio, or time-of-flight scale, or to calibrate the mass spectrometer.

[0021] The method further comprises determining an uncertainty value for the calibration correction.

[0022] The step of determining the uncertainty value comprises determining a standard deviation Σ of the calibration correction, where the standard deviation Σ is calculated using the relationship Σ2=∑a2+∑b2(Δa−Δb)2 can be determined, where Σ a is the standard deviation associated with the measurement of the first ions, Δ a is the mass of adducts from which the first ions are derived, Σ bis the standard deviation associated with the measurement of the second ions, and Δ b is the mass of adducts from which the second ions are derived.

[0023] The procedure may further include: Mass analyzing third ions derived from the analyte molecule, wherein the third ions have a third charge state and wherein the third ions comprise protonated or adduct variants of the first and second ions; Determining a third mass-to-charge ratio of the third ions; and Calculating a calibration correction based on the first mass-to-charge ratio, the second mass or mass-to-charge ratio, and the third mass-to-charge ratio.

[0024] The method may further comprise determining an uncertainty value for the calibration correction by determining a standard deviation Σ of the calibration correction, and wherein the standard deviation Σ is determined using the relationship Σ2=1Σa2+1Σb2+1Σc2(Δb−Δc)2Σb2Σc2+(Δa−Δc)2Σa2Σc2+(Δa−Δb)2Σa2Σb2 is determined, where Δ a is the mass of adducts from which the first ions are derived, Σ a is the standard deviation associated with the measurement of the first ions, Δ b is the mass of adducts from which the second ions are derived, Σ b is the standard deviation associated with the measurement of the second ions, Δ c is the mass of adducts from which the third ions are derived, Σ c is the standard deviation associated with the measurement of the third ions.

[0025] The method may further comprise determining whether the calibration correction is to be applied to correct the mass, mass-to-charge ratio, or time-of-flight scale, or to calibrate the mass spectrometer, based on the determined uncertainty value.

[0026] The calibration correction can be applied if the determined uncertainty value is below a threshold.

[0027] The calibration correction can be determined for each of a plurality of different analyte molecules and the determined calibration corrections are combined to obtain a combined calibration correction.

[0028] The mass spectrometer may comprise a time-of-flight mass spectrometer.

[0029] According to a further aspect, there is provided a mass spectrometry method comprising: a method for calibrating a mass spectrometer as discussed above.

[0030] The method may further comprise calibrating the mass spectrometer without adding a reference standard to an analyte sample to be analyzed.

[0031] The method may further comprise calibrating the mass spectrometer without using an ion source to generate a plurality of lock mass or external calibration ions.

[0032] The method may further comprise performing a recalibration of the instrument if the calibration correction exceeds a predefined threshold.

[0033] According to further aspects of the invention, mass spectrometers are provided with the features of claims 15 and 16, respectively

[0034] According to one embodiment, the mass spectrometer may further comprise: (a) an ion source selected from the group consisting of: (i) an electrospray ionization (ESI) ion source; (ii) an atmospheric pressure photoionization (APPI) ion source; (iii) an atmospheric pressure chemical ionization (APCI) ion source; (iv) a matrix-assisted laser desorption ionization (MALDI) ion source; (v) a laser desorption ionization (LDI) ion source; (vi) an atmospheric pressure ionization (API) ion source; (vii) a desorption ionization on silicon (DIOS) ion source; (viii) an electron impact (EI) ion source; (ix) a chemical ionization (Cl) ion source; (x) a field ionization (FI) ion source; (xi) a field desorption (FD) ion source; (xii) an inductively coupled plasma (ICP) ion source; (xiii) a fast atom bombardment (FAB) ion source; (xiv) a liquid secondary ion mass spectrometry (LSIMS) ion source; (xv) a desorption electrospray ionization (DESI) ion source;(xvi) a radioactive nickel-63 ion source; (xvii) an atmospheric pressure matrix-assisted laser desorption ionization ion source; (xviii) a thermal spray ion source; (xix) an atmospheric sampling mica discharge ionization (ASGDI) ion source; (xx) a mica discharge (GD) ion source; (xxi) an impactor ion source; (xxii) a direct analysis in real-time (DART) ion source; (xxiii) a laser spray ionization (LSI) ion source; (xxiv) a sonic spray ionization (SSI) ion source; (xxv) a matrix-assisted inlet ionization (MAII) ion source; and (xxvi) a solvent-assisted inlet ionization (SAII) ion source; and / or; (b) one or more continuous or pulsed ion sources; and / or (c) one or more ion guides; and / or (d) one or more ion mobility separation devices and / or one or more field asymmetric ion mobility spectrometer devices; and / or (e) one or more ion traps or one or more ion capture regions; and / or (f) one or more collision, fragmentation, or reaction cells selected from the group consisting of: (i) a collision-induced dissociation (CID) fragmentation device; (ii) a surface-induced dissociation (SID) fragmentation device; (iii) an electron-transfer dissociation (ETD) fragmentation device; (iv) an electron-capture dissociation (ECD) fragmentation device; (v) an electron collision or electron impact dissociation fragmentation device; (vi) a photo-induced dissociation (PID) fragmentation device; (vii) a laser-induced dissociation fragmentation device; (viii) an infrared-ray-induced dissociation device; (ix) a UV-radiation-induced dissociation device; (x) a nozzle-skimmer interface fragmentation device; (xi) a fragmentation device in the source;(xii) a collision-induced dissociation fragmentation device in the source; (xiii) a heat or temperature source fragmentation device; (xiv) an electric field induction fragmentation device; (xv) a magnetic field induction fragmentation device; (xvi) an enzyme digestion or enzyme degradation fragmentation device; (xvii) an ion-ion reaction fragmentation device; (xviii) an ion-molecule reaction fragmentation device; (xix) an ion-atom reaction fragmentation device; (xx) an ion-metastable ion reaction fragmentation device; (xxi) an ion-metastable molecule reaction fragmentation device; (xxii) an ion-metastable atom reaction fragmentation device; (xxiii) an ion-ion reaction device for reacting ions to form adducts or product ions; (xxiv) an ion-molecule reaction device for reacting ions to form adducts or product ions;(xxv) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxvi) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxvii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; (xxviii) an ion-metastable atom reaction device for reacting ions to form adduct or product ions; and (xxix) an electron ionization dissociation (EID) fragmentation device; and / or; (g) a mass analyzer selected from the group consisting of: (i) a quadrupole mass analyzer; (ii) a 2D or linear quadrupole mass analyzer; (iii) a Paul or 3D quadrupole mass analyzer; (iv) a Penning trap mass analyzer; (v) an ion trap mass analyzer; (vi) a magnetic sector mass analyzer; (vii) an ion cyclotron resonance (ICR) mass analyzer; (viii) a Fourier transform ion cyclotron resonance (FTICR) mass analyzer; (ix) an electrostatic mass analyzer configured to generate an electrostatic field having a quadrolologarithmic potential distribution; (x) a Fourier transform electrostatic mass analyzer; (xi) a Fourier transform mass analyzer; (xii) a time-of-flight mass analyzer; (xiii) an orthogonal acceleration-time-of-flight mass analyzer; and (xiv) a linear acceleration-time-of-flight mass analyzer; and / or (h) one or more energy analyzers or electrostatic energy analyzers; and / or (i) one or more ion detectors; and / or (j) one or more mass filters selected from the group consisting of: (i) a quadrupole mass analyzer; (ii) a 2D or linear quadrupole ion trap; (iii) a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a time-of-flight mass filter; and (viii) a Wien filter; and / or (k) a device or ion gate for pulsing ions; and / or (l) an apparatus for converting a substantially continuous ion beam into a pulsed ion beam.

[0035] The mass spectrometer can further comprise either: (i) a C-trap and a mass analyzer comprising a cylinder-like outer electrode and a coaxial spindle-like inner electrode generating an electrostatic field with a quadrolologarithmic potential distribution, wherein ions are conveyed to the C-trap in a first operating mode and thereafter injected into the mass analyzer, and wherein ions are conveyed to the C-trap in a second operating mode and thereafter to a collision cell or electron transfer dissociation device, wherein at least some of the ions are fragmented into fragment ions, and wherein the fragment ions are then conveyed to the C-trap before being injected into the mass analyzer; and / or (ii) a stacked ring ion guide comprising a plurality of electrodes each having an aperture through which ions are conveyed in use, and wherein the spacing of the electrodes increases along the length of the ion path, and wherein the apertures in the electrodes in an upstream portion of the ion guide have a first diameter, and wherein the apertures in the electrodes in a downstream portion of the ion guide have a second diameter smaller than the first diameter, and wherein opposite phases of an AC or RF voltage are applied in use to successive electrodes.

[0036] According to one embodiment, the mass spectrometer further comprises a device configured and adapted to supply an alternating (AC) or RF voltage to the electrodes. The AC or RF voltage may have an amplitude selected from the group consisting of: (i) <50 V peak to peak; (ii) 50-100 V peak to peak; (iii) 100-150 V peak to peak; (iv) 150-200 V peak to peak; (v) 200-250 V peak to peak; (vi) 250-300 V peak to peak; (vii) 300-350 V peak to peak; (viii) 350-400 V peak to peak; (ix) 400-450 V peak to peak; (x) 450-500 V peak to peak; and (xi) > 500 V peak to peak.

[0037] The AC or RF voltage may have a frequency selected from the group consisting of: (i) < 100 kHz; (ii) 100-200 kHz; (iii) 200-300 kHz; (iv) 300-400 kHz; (v) 400-500 kHz; (vi) 0.5-1.0 MHz; (vii) 1.0-1.5 MHz; (viii) 1.5-2.0 MHz; (ix) 2.0-2.5 MHz; (x) 2.5-3.0 MHz; (xi) 3.0-3.5 MHz; (xii) 3.5-4.0 MHz; (xiii) 4.0-4.5 MHz; (xiv) 4.5-5.0 MHz; (xv) 5.0-5.5 MHz; (xvi) 5.5-6.0MHz; (xvii) 6.0-6.5MHz; (xviii) 6.5-7.0 MHz; (xix) 7.0-7.5MHz; (xx) 7.5-8.0MHz; (xxi) 8.0-8.5MHz; (xxii) 8.5-9.0MHz; (xxiii) 9.0-9.5MHz; (xxiv) 9.5-10.0MHz; and (xxv) > 10.0 MHz.

[0038] The mass spectrometer may further comprise a chromatography or other separation device upstream of an ion source. According to one embodiment, the chromatography separation device comprises a liquid chromatography or gas chromatography device. According to another embodiment, the separation device may comprise: (i) a capillary electrophoresis (CE) separation device; (ii) a capillary electrochromatography (CEC) separation device; (iii) a multilayer microfluidic substrate separation device based on substantially rigid ceramic (ceramic tiles); or (vi) a supercritical fluid chromatography separation device.

[0039] The ion guide may be maintained at a pressure selected from the group consisting of: (i) < 0.0001 mbar; (ii) 0.0001-0.001 mbar; (iii) 0.001-0.01 mbar; (iv) 0.01-0.1 mbar; (v) 0.1-1 mbar; (vi) 1-10 mbar; (vii) 10-100 mbar; (viii) 100-1000 mbar; and (ix) > 1000 mbar.

[0040] According to one embodiment, analyte ions can be subjected to electron transfer dissociation (ETD) fragmentation in an electron transfer dissociation fragmentation device. Analyte ions can be caused to interact with ETD reagents within an ion guide or fragmentation device.

[0041] According to one embodiment, to effect electron transfer dissociation, either: (a) analyte ions are fragmented or induced to dissociate and form product or fragment ions after reacting with reagent ions; and / or (b) electrons are transferred from one or more reagent anions or negatively charged ions to one or more multiply charged analyte cations or positively charged ions, thereafter inducing at least some of the multiply charged analyte ions or positively charged ions to dissociate and form product or fragment ions; and / or (c) analyte ions are fragmented or induced to dissociate and form product or fragment ions after reacting with neutral reagent gas molecules or atoms or a non-ionic reagent gas;and / or (d) transporting electrons from one or more neutral, non-ionic, or uncharged basic gases or vapors to one or more multiply charged analyte cations or positively charged ions, thereafter inducing at least some of the multiply charged analyte cations or positively charged ions to dissociate and form product or fragment ions; and / or (e) transporting electrons from one or more neutral, non-ionic, or uncharged superbase reagent gases or vapors to one or more multiply charged analyte cations or positively charged ions, thereafter inducing at least some of the multiply charged analyte cations or positively charged ions to dissociate and form product or fragment ions;and / or (f) electrons are transported from one or more neutral, non-ionic or uncharged alkali metal gases or vapors to one or more multiply charged analyte cations or positively charged ions, thereafter inducing at least some of the multiply charged analyte cations or positively charged ions to dissociate and form product or fragment ions;and / or (g) electrons are transferred from one or more neutral, non-ionic or uncharged gases, vapors or atoms to one or more multiply charged analyte cations or positively charged ions, thereafter inducing at least some of the multiply charged analyte cations or positively charged ions to dissociate and form product or fragment ions, wherein the one or more neutral, non-ionic or uncharged gases, vapors or atoms are selected from the group consisting of: (i) sodium vapor or atoms; (ii) lithium vapor or atoms; (iii) potassium vapor or atoms; (iv) rubidium vapor or atoms; (v) cesium vapor or atoms; (vi) frankium vapor or atoms; (vii) C; 60 vapor or atoms; and (viii) magnesium vapor or atoms.

[0042] The multiply charged analyte cations or positively charged ions can include peptides, polypeptides, proteins or biomolecules.

[0043] According to one embodiment, to effect electron transfer dissociation: (a) the reagent anions or negatively charged ions are derived from a polyaromatic hydrocarbon or a substituted polyaromatic hydrocarbon; and / or (b) the reagent anions or negatively charged ions are derived from the group consisting of: (i) anthracene; (ii) 9,10-diphenylanthracene; (iii) naphthalene; (iv) fluorine; (v) phenanthrene; (vi) pyrene; (vii) fluoranthene; (viii) chrysene; (ix) triphenylene; (x) perylene; (xi) acridine; (xii) 2,2'-dipyridyl; (xiii) 2,2'-biquinoline; (xiv) 9-anthracenecarbonitrile; (xv) dibenzothiophene; (xvi) 1,10'-phenanthroline; (xvii) 9'-anthracenecarbonitrile; and (xviii) anthraquinone; and / or (c) the reagent ions or negatively charged ions comprise azobenzene anions or azobenzene radical anions.

[0044] According to one embodiment, the electron transfer dissociation fragmentation process comprises interacting analyte ions with reagent ions, wherein the reagent ions comprise dicyanobenzene, 4-nitrotoluene, or azulene. DETAILED DESCRIPTION

[0045] An embodiment will now be described.

[0046] The molecular weight of an analyte molecule calculated from two different charge state analyte ion peaks, where the analyte ions were charged by the addition of known adduct species (e.g., a proton or a sodium or potassium atom), should be the same for a perfectly calibrated mass-to-charge ratio scale.

[0047] In practice, however, the determined molecular weights differ slightly because the mass-to-charge ratio scale has probably drifted and is therefore not perfectly calibrated.

[0048] According to the embodiment, if the mass-to-charge ratio scale has drifted by an instrument drift factor, it is possible to calculate (and thus correct) the instrument drift based on the difference in the calculated molecular weight of an analyte based on corresponding analyte ions with different charge states.

[0049] For a mass spectrum containing a singly charged ion and a corresponding doubly charged ion of the same unknown analyte species, the mass-to-charge ratio M1 of the singly charged (protonated) analyte ion can be expressed as follows: M1=mw+H where mw is the molecular weight of the analyte molecule and H is the mass of a proton.

[0050] Similarly, the mass-to-charge ratio M2 of the doubly charged species can be expressed as: M2=(mw+2H) / 2

[0051] This results in the relationship: 2M2=M1+H H=2M2−M1

[0052] A linear drift α in the calibration would lead to measured mass-to-charge values: M1'=αM1 M2'=αM2

[0053] Accordingly: H'=αH α=H' / H H'=2M2'−M1'

[0054] For this reason, the linear drift α in the calibration with respect to singly charged and doubly charged ions can be expressed as follows: α=2M2'−M1'H

[0055] The above expression, which refers to the linear drift α expressed in terms of singly and doubly charged ions, can be generalized to cover any two charge states (a and b), yielding the following expression: α=aMa'−bMb'(a−b)H

[0056] If the charge state difference is due to the inclusion of adducts such as sodium or potassium (instead of H), the larger mass of the adduct then improves the accuracy of the correction factor.

[0057] In the embodiment described above, the same adduct is used to achieve the two different charge states of analyte ions. However, embodiments are contemplated in which one type of analyte ion is used to achieve a first charge state and a second, different type of analyte ion is used to achieve a second charge state.

[0058] In one embodiment, the standard deviation of the determined linear drift α can be calculated using a probabilistic approach, as shown below.

[0059] The two observed species are related by the unknown common mass mw, differing by known adduct masses Δ1 and Δ2, measured at known charge states a and b, mw+Δ1 mw+Δ2 measured at known charge states a and b. Note that the adduct masses Δ1 and Δ2 include any change in mass due to ionization (e.g., addition of protons or loss of electrons). In these expressions, the subscripts 1 and 2 refer to the adduct index, not the charge state. In fact, the two charge states can be equal, in which case a = b.

[0060] The two species result in two measured m / z values ​​M'1 and M'2, with corresponding uncertainties σ1 and σ2, M'1±σ1 M'2±σ2

[0061] The measured mass axis is perturbed by an unknown gain or linear drift α, so that (cf. equations (5) and (6)): M'1∼αmw+Δ1a M'2∼αmw+Δ2b

[0062] Given that the measurements of M'1 and M'2 are not perfect and carry uncertainties, the linear drift α obtained using these measurements would also carry uncertainties that can be characterized by its standard deviation.

[0063] Assuming that the measurement of M'1 is likely to take the form of a Gaussian distribution, then the probability that the value M'1 is observed is: P(M'1∨ mw,α)=12πσexp (−(M'1−αmw+Δ1a)2σ122)

[0064] Similarly for M'2: P(M'2∨ mw,α)=12πσ2exp (−(M'2−αmw+Δ2b)2σ222)

[0065] Other distribution choices can be made instead, but given reasonable mass spectrometric peak shapes, the Gaussian shape is usually appropriate.

[0066] Since the value of mw is unknown and not of primary interest, it can be treated as a “noise variable” and mathematically removed from equations (18) and (19) (this is known as “marginalization of the mw variable”).

[0067] Using Bayes' theorem and (for simplicity) assuming uniform prior probabilities with respect to mw and α, an expression for the probability distribution (inference) for α is obtained: P(α∨M'1,M'2)=Kαexp(−(α−α0)22∑2) where K is a normalization constant, α0=aM'1−bM'2Δ1−Δ2

[0068] In summary, the linear drift or correction factor α to be used for calibration is the difference in the measured masses in different charge states (i.e., the product of the mass-to-charge ratio and the charge) divided by the difference in the adduct masses used to obtain the different charge states. Note that when Δ1 = aH and Δ2 = bH, M1' = M a ' and M2' = M b ', this expression is identical to equation (11). ∑2=∑12+∑22(Δ1−Δ2)2 ∑1=aσ1 ∑2=bσ2

[0069] For data of sufficient quality, the probability distribution of α is approximately a Gaussian probability distribution centered on α0 with a standard deviation of Σ. For example, if M'1 and M'2 are individually loaded and charged with 1 ppm (mw x 10 -6 Da) are measured, then the uncertainty in the linear displacement α is: Δ1−Δ2 V∑=2mw ppm

[0070] The achievable accuracy, which is inversely proportional to the standard deviation Σ, is therefore directly related to the difference in adduct mass divided by the molecular weight |Δ1 - Δ2| / mw. In other words, the accuracy can be improved by increasing the mass difference between the two types of adducts used to achieve the two different charge states.

[0071] For this reason, it is desirable to minimize the factor mw / (Δ1 - Δ2). This can be achieved, for example, by using larger charge-bearing adducts and / or by generating larger charge differences using, for example, charge removal or charging techniques. (Note that, although charge differences do not appear explicitly in this factor, it is usually the case that the adduct difference also increases with increasing charge.)

[0072] The same logic can be applied to the scenario where three or more known adducts are present. In the case of three adducts: α0=aM'1(Δ1−Δ3∑12∑32+Δ1−Δ2∑12∑22)+bM'2(Δ2−Δ3∑22∑32+Δ2−Δ1∑12∑22)+cM'3( Δ3−Δ2∑22∑32+Δ3−Δ1∑12∑32)(Δ2−Δ3)2∑22∑32+(Δ1−Δ3)2∑12∑32+(Δ1−Δ2)2∑12∑22 ∑2=1∑12+1∑22+1∑32(Δ2−Δ3)2∑22∑32+(Δ1−Δ3)2∑12∑32+(Δ1−Δ2)2∑12∑22

[0073] Equation (27) reduces to the 2-adduct equation (25) when one of the measurements becomes uninformative (e.g., the limit Σ3 tends to infinity).

[0074] In addition to fluctuations from effects such as temperature and voltage stability, which introduce systematic error into the mass accuracy, other sources of error should also be considered. For example, an absolute measurement error, which depends on systematic calibration errors, also introduces error. Center of gravity design errors also introduce errors, and chemical and electronic interference equally influence the measurements.

[0075] According to embodiments, a linear drift or a mass correction may be determined using measurements from a single experiment with analyte ions in two different charge states and / or derived from two different adducts. There may be cases where multiple distinct sets of analyte ion species are present, each set of species comprising a base compound of unknown molecular weight and a known set of adducts, e.g., in the analysis of complex mixtures. In these cases, the linear drifts or mass corrections determined from each set of species may be combined as needed, and the corresponding uncertainties may be determined and accounted for to provide a linear drift / mass correction with improved accuracy / reduced uncertainty.

[0076] There may be cases where the uncertainty in the applied linear drift is determined to be comparable to or higher than the drift that may have occurred. According to further embodiments, the calculated uncertainty may then be used to determine whether the correction should be applied. Additionally, or alternatively, the uncertainty may be used to determine whether more sets of analyte species should be located in the data and used to calculate a linear drift to reduce the uncertainty to a predefined threshold or below.

[0077] Embodiments are contemplated wherein instrument recalibration may be induced if the magnitude of the calibration correction or linear drift exceeds a predefined threshold. The instrument calibration may be a calibration update using, for example, Lockmass, or a calibration of the entire instrument. Example 1

[0078] According to a first example, an analyte with an approximate molecular weight of 1000 can be considered.

[0079] According to this example, the mass of the adducts (potassium adducts) is M1 = 156 and M2 = 39 and according to this example, the first charge state is a = 4 and the second charge state is b = 1.

[0080] If the mass spectrometer has an instrument drift of 10 ppm, the difference in measured molecular weight between the fourth and first charge states according to the embodiment is 1.2 ppm (± 0.2 ppm standard deviation for each peak), and this results in a required correction of 10 ppm ± 1.7 ppm.

[0081] The embodiment therefore leads to a significant improvement after the correction (approximately a factor of x 5).

[0082] It may be desirable to use the factor (mw / (M a × (ab))), and this can be done by using larger adducts and by creating larger charge differences using charge removal techniques. Example 2

[0083] A second example will now be explained. According to the second example, an axial matrix-assisted laser desorption ionization mass spectrometer can be used, which leads to the production of ions with a relatively high charge number. The ions are generated by laser spray ionization.

[0084] An analyte with a molecular weight of 5700 can be considered. The mass-to-charge ratio of MALDI matrix ions can be 224, and according to this example, the first charge state a = 8 and the second charge state b = 1.

[0085] If the mass spectrometer had an instrument drift of 100 ppm, the difference in molecular weights calculated according to the embodiment would be 27 ppm (± 5 ppm standard deviation for each peak). This would result in a correction of 100 ppm ± 18 ppm, which corresponds to an approximately 5-fold improvement.

[0086] Although the technology described herein has been described with reference to the embodiments, those skilled in the art will understand that various changes in form and details may be made without departing from the scope of the following claims.

Claims

[1] A method for calibrating a mass spectrometer, comprising: Mass analyzing first ions derived from an analyte molecule, wherein the first ions have a first charge state, Determining a first mass-to-charge ratio of the first ions, Mass analyzing second ions derived from the analyte molecule, wherein the second ions have a second charge state and wherein the second ions comprise protonated or adduct variants of the first ions, Determining a second mass-to-charge ratio of the second ions, Calculating a calibration correction based on the first mass-to-charge ratio and the second mass-to-charge ratio, wherein the step of calculating the calibration correction comprises calculating a linear drift α of the mass-to-charge ratio, Determining an uncertainty value for the calibration correction, wherein the step of determining the uncertainty value comprises determining a standard deviation Σ of the calibration correction, and Determine, based on the determined uncertainty value, whether the calibration correction should be applied. [2] The method of claim 1, wherein the calibration correction is determined based on the difference in charge between the first charge state and the second charge state, wherein the accuracy of the calibration correction is improved by increasing the difference in charge between the first charge state and the second charge state. [3] A method for calibrating a mass spectrometer, comprising: Mass analyzing first ions derived from an analyte molecule and first adducts, Determining a first mass-to-charge ratio of the first ions, Mass analyzing second ions derived from the analyte molecule and second adducts, wherein the second adducts have a second mass different from the first adducts, Determining a second mass-to-charge ratio of the second ions, Calculating a calibration correction based on the first mass-to-charge ratio and the second mass-to-charge ratio, wherein the step of calculating the calibration correction comprises calculating a linear drift α of the mass-to-charge ratio, Determining an uncertainty value for the calibration correction, wherein the step of determining the uncertainty value comprises determining a standard deviation Σ of the calibration correction, and Determine, based on the determined uncertainty value, whether the calibration correction should be applied. [4] The method of claim 3, wherein the calibration correction is determined based on the difference in mass between the first adductions and the second adductions, wherein the accuracy of the calibration correction is improved by increasing the difference in mass between the first adductions and the second adductions. [5] Method according to claim 1 or 2, wherein the linear drift α is determined using the relationship α=aMa'−bMb'(a−b)H where a is the charge state of the first ions, M' a is the mass-to-charge ratio of the first ions, b is the charge state of the second ions, M' b is the mass-to-charge ratio of the second ions and H is the mass of a proton. [6] Method according to one of claims 1 to 4, wherein the linear drift α is determined using the relationship α=aMa'−bMb'Δa−Δb where a is the charge state of the first ions, M a' is the mass-to-charge ratio of the first ions, Δ a is the mass of adducts from which the first ions are derived, b is the charge state of the second ions, M' b is the mass-to-charge ratio of the second ions and Δ b is the mass of adducts from which the second ions are derived. [7] Method according to one of the preceding claims, wherein the standard deviation Σ is determined using the relationship ∑2=∑a2+∑b2(Δa−Δb)2 is determined, where Σ a is the standard deviation associated with the measurement of the first ions, Δ a is the mass of adducts from which the first ions are derived, Σ b is the standard deviation associated with the measurement of the second ions, and Δ b is the mass of adducts from which the second ions are derived. [8] A method according to any one of the preceding claims, comprising: Mass analyzing third ions derived from the analyte molecule, wherein the third ions have a third charge state and wherein the third ions comprise protonated or adduct variants of the first and second ions, Determining a third mass-to-charge ratio of the third ions, and Calculate the calibration correction based on the first mass-to-charge ratio, the second mass-to-charge ratio, and the third mass-to-charge ratio. [9] Method according to claim 8, wherein the standard deviation Σ is determined using the relationship ∑2=1∑a2+1∑b2+1∑c2(Δb−Δc)2∑b2∑c2+(Δa−Δc)2∑a2∑c2+(Δa−Δb)2∑a2∑b2 is determined, where Δ a is the mass of adducts from which the first ions are derived, Σ a is the standard deviation associated with the measurement of the first ions, Δ bis the mass of adducts from which the second ions are derived, Σ b is the standard deviation associated with the measurement of the second ions, Δ c is the mass of adducts from which the third ions are derived, Σ c is the standard deviation associated with the measurement of the third ions. [10] A method according to any one of the preceding claims, wherein the calibration correction is applied when the determined uncertainty value is below a predefined threshold. [11] A method according to any preceding claim, wherein the calibration correction is calculated for each of a plurality of different analyte molecules and the calculated calibration corrections are combined to obtain a combined calibration correction. [12] A method according to any preceding claim, wherein the mass spectrometer comprises a time-of-flight mass spectrometer. [13] Mass spectrometry method which includes: a method for calibrating a mass spectrometer according to any one of the preceding claims. [14] The mass spectrometry method of claim 13, further comprising performing a recalibration of the instrument when the calibration correction exceeds a predefined threshold. [15] Mass spectrometer comprising a control system designed and arranged to: (i) mass-analyzing first ions derived from an analyte molecule, the first ions having a first charge state, (ii) determining a first mass-to-charge ratio of the first ions, (iii) mass-analyzing second ions derived from the analyte molecule, wherein the second ions have a second charge state and wherein the second ions comprise protonated or adduct variants of the first ions, (iv) determining a second mass-to-charge ratio of the second ions, (v) calculating a calibration correction based on the first mass-to-charge ratio and the second mass-to-charge ratio, wherein the step of calculating the calibration correction comprises calculating a linear drift α of the mass-to-charge ratio, (vi) determining an uncertainty value for the calibration correction, wherein the step of determining the uncertainty value comprises determining a standard deviation Σ of the calibration correction, and (vii) Determine, based on the uncertainty value obtained, whether the calibration correction should be applied. [16] Mass spectrometer comprising a control system designed and arranged to: (i) mass analyzing first ions derived from an analyte molecule and first adducts, (ii) determining a first mass-to-charge ratio of the first ions, (iii) mass-analyzing second ions derived from the analyte molecule and second adducts, wherein the second adducts have a second mass different from the first adducts, (iv) determining a second mass-to-charge ratio of the second ions, (v) calculating a calibration correction based on the first mass-to-charge ratio and the second mass-to-charge ratio, wherein the step of calculating the calibration correction comprises calculating a linear drift α of the mass-to-charge ratio, (vi) determining an uncertainty value for the calibration correction, wherein the step of determining the uncertainty value comprises determining a standard deviation Σ of the calibration correction, and (vii) Determine, based on the uncertainty value obtained, whether the calibration correction should be applied.