Self-calibration of spectra using known differences of precursor mass / charge ratios and fragment mass / charge ratios

Abstract

A method for checking or adjusting the calibration of a mass spectrometer, comprising: Fragmentation of parent ions and generation of fragment ion mass spectrum data, Detecting first neutral loss ions in the fragment ion mass spectrum data, where neutral loss ions correspond to parent ions that have lost one or more neutral atoms or molecules, Determining a first mass loss difference between the parent ions and the first neutral loss ions and Determining whether the first mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the first mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters, wherein the step of fragmenting parent ions and generating fragment ion mass spectrum data comprises: Scanning a mass-to-charge ratio transmission window of a mass filter and fragmenting parent ions passed through the mass filter.

Description

FIELD OF THE PRESENT INVENTION

[0001] The present invention relates generally to mass spectrometry and, more particularly, to methods for checking or adjusting the calibration of mass spectrometers, methods of mass spectrometry, and mass spectrometers. BACKGROUND

[0002] It is known to perform a mass-to-charge ratio scale calibration of a mass spectrometer by fitting data from known ion peaks, for example, using a reference standard, to the underlying scan protocol used by a mass spectrometer. The underlying scan protocol used by a mass spectrometer is typically a time-of-flight function.

[0003] It is known to perform a mass / charge ratio calibration before, during, or after acquisition of an unknown analyte.

[0004] Internal calibration refers to the addition of a known standard to an analyte sample itself. However, known internal calibration techniques can be particularly problematic because the standard must produce ions with similar intensity to those of the unknown analyte to avoid saturation. Furthermore, the reference ions must have mass / charge ratios sufficiently different from those of the analyte ions to avoid interference.

[0005] An external calibration, or lock mass correction of a calibration, relies on the stability of the system between the time of calibration and the time of analyte acquisition. However, this approach can be problematic, particularly when transient perturbations occur to components within the system, for example, due to effects such as voltage or temperature drifts or spikes.

[0006] External calibration, or lock mass calibration, is also problematic and costly because it typically requires a separate, dedicated ionization source. Furthermore, the mass spectrometer must temporarily switch between the analyte ions and the reference ions, which can result in a loss of analyte data.

[0007] US 2006 / 0136158 A1 discloses a method for recalibrating a mass spectrum of macromolecules or fragments. Information regarding molecules believed to be present within the sample (e.g., information regarding the isotopic envelope of molecules believed to be present in the sample) is used to tentatively assign specific molecular peaks in the spectrum. In the case of peptides, fragment peaks in the mass spectrum are difficult to label as belonging to specific sequences of amino acids due to combinations of amino acids with similar masses. Therefore, instead of assigning the fragment peaks themselves, mass differences between pairs of fragment ion peaks are determined and tentatively assigned to specific amino acids.Calibration parameters are then adjusted to reduce the difference between the measured mass / charge ratio values ​​of the differences between peaks and their “true” values ​​(i.e., the mass values ​​of the corresponding experimentally assigned molecule).

[0008] Relevant prior art is also known from WO 2013 / 081581 A1, US 2013 / 0 214 146 A1, US 000005300771 A and US 020080237458 A1.

[0009] It is therefore desirable to provide an improved method for calibrating or recalibrating a mass spectrometer. SUMMARY

[0010] According to one aspect of the invention, a method having the features of claim 1 is proposed.

[0011] Various embodiments relate to a method for self-calibrating data.

[0012] It should be noted that the various embodiments differ from the methods described in US 2006 / 0136158 A1, wherein pairs of fragment ions are identified in the fragment ion mass spectrum data, the mass difference between the fragment ions in each pair is determined and tentatively assigned to specific molecules, and then the determined mass differences are compared with the expected mass differences of these specific molecules, because the various embodiments do not require pairs of fragment ions to be detected. According to the various embodiments, neutral loss ions (rather than pairs of fragment ions) are detected in the fragment spectrum data, and the mass loss difference between parent or precursor ions and the first neutral loss ions is determined. It is then determined whether this mass loss difference corresponds to an expected or predetermined mass loss difference.

[0013] The step of detecting first neutral loss ions in the fragment or product ion mass spectral data optionally includes the following: Plotting or otherwise analyzing the mass / charge ratio of fragment or product ions as a function of the mass / charge ratio of corresponding parent or precursor ions and

[0014] Identify one or more trend lines in the fragment or product ion mass spectrum data.

[0015] The step of determining a first mass loss difference between the parent or precursor ions and the first neutral loss ions optionally comprises determining a best fit line between the first neutral loss ions in the fragment or product ion mass spectral data.

[0016] The first neutral loss ions optionally include parent or precursor ions that have lost one or more neutral molecules or atoms.

[0017] According to one embodiment, the one or more neutral molecules or atoms may comprise molecules or atoms selected from the group consisting of: (i) H, (ii) CH3, (iii) OH, (iv) H2O, (v) F, (vi) HF, (vii) C2H3, HCN, (viii) C2H4, CO, (ix) CH2O, (x) CH3O, (xi) CH4O, S, (xii) CH3+ H2O, HS, (xiii) H2S, (xiv) Cl, (xv) HCl, (xvi) C3H6, C2H2O, C2H4N, (xvii) C3H7, CH3CO, (xviii) CO2O, CONH2, (xix) C2H5O, (xx) C4H7, (xxi) C4H9, (xxii) C2H3O2, (xxiii) C2H4O2, (xxiv) SO2, (xxv) Br, (xxvi) HBr, (xxvii) I, (xxviii) HI, (xxix) NH3, (xxx) CH2, (xxxi) O2, (xxxii) CO2, (xxxiii) PO2, (xxxiv) PO3, (xxxv) HPO3 and (xxxvi) H3PO4.

[0018] The step of adjusting one or more calibration parameters optionally includes adjusting the calibration of the mass spectrometer such that when the mass spectrometer has been recalibrated, the first mass loss difference exactly or substantially corresponds to an expected or predetermined mass loss difference.

[0019] The step of adjusting one or more calibration parameters optionally includes adjusting the calibration of the mass spectrometer such that when the mass spectrometer has been recalibrated, the difference between the first mass loss difference and an expected or predetermined mass loss difference is reduced.

[0020] The procedure optionally further includes the following: Detection of second neutral loss ions in the fragment or product ion mass spectrum data, Determining a second mass loss difference between the parent or precursor ions and the second neutral loss ions and Determining whether the second mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the second mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters.

[0021] The procedure optionally further includes the following: Detecting third neutral loss ions in the fragment or product ion mass spectrum data, Determining a third mass loss difference between the parent or precursor ions and the third neutral loss ions and Determining whether the third mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the third mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters.

[0022] The procedure optionally further includes the following: Detecting fourth neutral loss ions in the fragment or product ion mass spectrum data, Determining a fourth mass loss difference between the parent or precursor ions and the fourth neutral loss ions and Determining whether the fourth mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the fourth mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters. The step of fragmenting the parent or precursor ions optionally comprises fragmenting at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 different species of parent or precursor ions. The method optionally further comprises generating at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 different parent or precursor ions and fragment or product ion pairs.

[0023] The procedure optionally further includes the following: Detection of first adductions in the fragment or product ion mass spectrum data, Determining a first mass gain difference between the parent or precursor ions and the first adducts and Determining whether the first mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the first mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters.

[0024] According to another aspect of the invention, a method having the features of claim 13 is proposed.fa

[0025] The step of detecting first adductions in the fragment or product ion mass spectrum data optionally includes the following: Plotting or otherwise analyzing the mass / charge ratio of fragment or product ions as a function of the mass / charge ratio of corresponding parent or precursor ions and Identify one or more trend lines in the fragment or product ion mass spectrum data.

[0026] The step of determining a first mass gain difference between the parent or precursor ions and the first adducts optionally comprises determining a best fit line between the first adducts in the fragment or product ion mass spectral data.

[0027] The step of adjusting one or more calibration parameters optionally includes adjusting the calibration of the mass spectrometer such that when the mass spectrometer has been recalibrated, the first mass gain difference exactly or substantially corresponds to an expected or predetermined mass gain difference.

[0028] The step of adjusting one or more calibration parameters optionally includes adjusting the calibration of the mass spectrometer such that when the mass spectrometer has been recalibrated, the difference between the first mass gain difference and an expected or predetermined mass gain difference is reduced.

[0029] The procedure optionally further includes the following: Detecting second adductions in the fragment or product ion mass spectrum data, Determining a second mass gain difference between the parent or precursor ions and the second adducts and Determining whether the second mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the second mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters.

[0030] The procedure optionally further includes the following: Detecting third adductions in the fragment or product ion mass spectrum data, Determining a third mass gain difference between the parent or precursor ions and the third adducts and Determining whether the third mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the third mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters.

[0031] The procedure optionally further includes the following: Detecting fourth adductions in the fragment or product ion mass spectrum data, Determining a fourth mass gain difference between the parent or precursor ions and the fourth adducts and Determining whether the fourth mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the fourth mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters.

[0032] Also disclosed is a method for mass spectrometry comprising a method as described above.

[0033] According to another aspect of the invention, a mass spectrometer having the features of claim 21 is proposed.

[0034] According to another aspect of the invention, a mass spectrometer having the features of claim 22 is proposed.

[0035] Also disclosed is a method for ionizing a known class of compounds for analysis by MS / MS, comprising: (i) Identifying a plurality of characteristic constant neutral loss peaks and measuring the neutral loss differences in the MS / MS data and (ii) Using values ​​from a variety of peaks to improve the mass / charge ratio calibration.

[0036] According to the embodiment, the preferred method further comprises: (i) scanning a precursor ion with a first mass filter, sequentially fragmenting a plurality of precursor ions to generate mass / charge ratio data which are recorded by a second mass analyzer, (ii) Plotting the fragment mass / charge ratio against the precursor mass / charge ratio, (iii) applying an automated algorithm to determine the best-fit lines corresponding to known neutral losses of precursors of the compound class and subtracting the best-fit lines to obtain statistically valid measurements of the apparent neutral losses, and (iv) Comparing the apparent neutral loss mass / charge ratio with the known expected mass / charge ratio value and correcting and / or recalibrating the entire mass / charge ratio scale based on the measured error function.

[0037] 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 (“El”) 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 glow discharge ionization (“ASGDI”) ion source, (xx) a glow discharge (“GD”) ion source, (xxi) an impactor ion source, (xxii) a direct analysis in real-time (“DART”) ion source, (xxii) a laser spray ionization (“LSI”) ion source, (xxiv) a sonic spray ionization (“SSl”) ion source, (xxv) a matrix-assisted Inlet ionization (“MAII”) ion source, (xxvi) a solvent-assisted inlet ionization (“SAII”) ion source, (xxvii) a desorption electrospray ionization (“DESI”) ion source, and (xxviii) a laser ablation electrospray ionization (“LAESI”) 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 confinement areas 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 impact or impact dissociation fragmentation device, (vi) a photo-induced dissociation (“PID”) fragmentation device, (vii) a laser-induced dissociation fragmentation device, (viii) an infrared radiation-induced dissociation device, (ix) a Ultraviolet radiation-induced dissociation device, (x) a nozzle-skimmer interface fragmentation device, (xi) an in-source fragmentation device,(xii) an in-source collision-induced dissociation fragmentation device, (xiii) a thermal or temperature source fragmentation device, (xiv) an electric field-induced fragmentation device, (xv) a magnetic field-induced 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 adducts or product ions, (xxvi) an ion-metastable ion reaction device for reacting ions to form adducts or product ions, (xxvii) an ion-metastable molecule reaction device for reacting ions to form adducts or product ions, (xxviii) an ion-metastable atom reaction device for reacting ions to form adducts 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 two-dimensional or linear quadrupole mass analyzer, (iii) a Paul or three-dimensional 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 arranged to generate an electrostatic field having a quadrolologarithmic potential distribution, (x) an electrostatic Fourier transform 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 filter, (ii) a two-dimensional or linear quadrupole ion trap, (iii) a Paul or three-dimensional 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.

[0038] The mass spectrometer may further comprise one of the following: (i) a C-trap and a mass analyzer having an outer tubular electrode and a coaxial inner spindle-like electrode forming an electrostatic field with a quadrolologarithmic potential distribution, wherein in a first mode of operation, ions are transferred to the C-trap and then injected into the mass analyzer, and wherein in a second mode of operation, ions are transferred to the C-trap and then transferred to a collision cell or electron transfer dissociation device where at least some ions are fragmented into fragment ions, and wherein the fragment ions are then transferred to the C-trap before being injected into the mass analyzer, and / or (ii) a ring stack ion guide comprising a plurality of electrodes each having an aperture through which ions are passed in use, and wherein the distance between the electrodes increases along 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.

[0039] According to one embodiment, the mass spectrometer further comprises a device configured and designed to supply an AC or RF voltage to the electrodes. The AC or RF voltage preferably has an amplitude selected from the group consisting of: (i) about < 50 V peak-to-peak, (ii) about 50 - 100 V peak-to-peak, (iii) about 100 - 150 V peak-to-peak, (iv) about 150 - 200 V peak-to-peak, (v) about 200 - 250 V peak-to-peak, (vi) about 250 - 300 V peak-to-peak, (vii) about 300 - 350 V peak-to-peak, (viii) about 350 - 400 V peak-to-peak, (ix) about 400 - 450 V peak-to-peak, (x) about 450 - 500 V peak-to-peak and (xi) about > 500 V peak-to-peak.

[0040] The AC or RF voltage may have a frequency selected from the group consisting of: (i) < about 100 kHz, (ii) about 100 - 200 kHz, (iii) about 200 - 300 kHz, (iv) about 300 - 400 kHz, (v) about 400 - 500 kHz, (vi) about 0.5 - 1.0 MHz, (vii) about 1.0 - 1.5 MHz, (viii) about 1.5 - 2.0 MHz, (ix) about 2.0 - 2.5 MHz, (x) about 2.5 - 3.0 MHz, (xi) about 3.0 - 3.5 MHz, (xii) about 3.5 - 4.0 MHz, (xiii) about 4.0 - 4.5 MHz, (xiv) about 4.5 - 5.0 MHz, (xv) about 5.0 - 5.5 MHz, (xvi) about 5.5 - 6.0 MHz, (xvii) about 6.0 - 6.5 MHz, (xviii) about 6.5 - 7.0 MHz, (xix) about 7.0 - 7.5 MHz, (xx) about 7.5 - 8.0 MHz, (xxi) about 8.0 - 8.5 MHz, (xxii) about 8.5 - 9.0 MHz, (xxiii) about 9.0 - 9.5 MHz, (xxiv) about 9.5 - 10.0 MHz and (xxv) > about 10.0 MHz.

[0041] The mass spectrometer may also 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 separation device comprising a substantially rigid ceramic-based multilayer microfluidic substrate ("ceramic tile"), or (iv) a supercritical fluid chromatography separation device.

[0042] The ion guide may be maintained at a pressure selected from the group consisting of: (i) < about 0.0001 mbar, (ii) about 0.0001 - 0.001 mbar, (iii) about 0.001 - 0.01 mbar, (iv) about 0.01 - 0.1 mbar, (v) about 0.1 - 1 mbar, (vi) about 1 - 10 mbar, (vii) about 10 - 100 mbar, (viii) about 100 - 1000 mbar and (ix) > about 1000 mbar.

[0043] 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.

[0044] According to one embodiment, to effect electron transfer dissociation, either: (a) analyte ions are fragmented or caused to dissociate and form product or fragment ions after interacting 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, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, and / or (c) analyte ions are fragmented or caused to dissociate and form product or fragment ions after interacting with neutral reagent gas molecules or atoms or a non-ionic reagent gas,and / or (d) electrons are transferred from one or more neutral non-ionic or uncharged source gases or vapors to one or more multiply charged analyte cations or positively charged ions, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, and / or (e) electrons are transferred 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, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused to dissociate and form product or fragment ions, and / or (f) electrons 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, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused 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, whereupon at least some of the multiply charged analyte cations or positively charged ions are caused 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) caesium vapor or atoms, (vi) francium vapor or atoms, (vii) C, 60 vapor or atoms and (viii) magnesium vapor or atoms.

[0045] The multiply charged analyte cations or positively charged ions can include peptides, polypeptides, proteins or biomolecules.

[0046] 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.

[0047] According to one embodiment, the electron transfer dissociation fragmentation process includes interacting analyte ions with reagent ions, wherein the reagent ions comprise dicyanobenzene, 4-nitrotoluene, or azulene. BRIEF DESCRIPTION OF THE DRAWING

[0048] Various embodiments will now be described by way of example only and with reference to the accompanying drawings. They show: Fig. 1 a heatmap showing an initial or precursor scan line and trend lines related to constant neutral loss ions. DETAILED DESCRIPTION

[0049] An embodiment will now be described.

[0050] One embodiment utilizes the fact that ions of certain classes of compounds (e.g., peptides) undergo fragmentation, resulting in fragment or product ions, where some of the fragment or product ions are neutral loss ions that have lost one or more neutral molecules or atoms (e.g., water). The neutral loss ions should have a precise mass difference from that of the parent ions. The embodiment detects neutral loss ions in fragmentation mass spectrum data and uses the mass difference between the neutral loss ions and the parent ions to self-calibrate the mass / charge ratio scale of a mass spectrometer.

[0051] According to one embodiment, ions are transferred from an ion source, such as an electrospray ionization ("ESI") ion source, to a quadrupole mass filter. The quadrupole mass filter is optionally set to pass a 1 Da mass range of parent or precursor ions at a given time. The mass-to-charge ratio pass window of the quadrupole mass filter is optionally scanned. For example, according to one embodiment, the mass-to-charge ratio pass window can be progressively scanned from a mass-to-charge ratio of 400 to a mass-to-charge ratio of 900 in 1 Da increments.

[0052] Once the quadrupole mass filter has passed ions with a mass-to-charge ratio of 900, the quadrupole mass filter is then optionally reset to return to passing ions with a mass-to-charge ratio of 400, and the scanning process is then optionally repeated one or more times.

[0053] Parent or precursor ions passed through the quadrupole mass filter are optionally fragmented in a fragmentation cell or device. According to one embodiment, the fragmentation cell or device may comprise a collision-induced dissociation ("CID") fragmentation cell or device.

[0054] However, according to other embodiments, the fragmentation cell or device may comprise an electron transfer dissociation ("ETD") device or another form of fragmentation cell or device.

[0055] The parent or precursor ions that are fragmented or otherwise dissociated in the fragmentation cell or device are optionally fragmented to result in multiple fragments or product ions. The resulting fragments or product ions are then mass-analyzed, for example, by a time-of-flight mass analyzer. Some of the resulting fragments or product ions optionally include neutral loss ions, i.e., parent or precursor ions that have lost one or more neutral molecules or atoms. For example, peptide ions may lose a water molecule, and the resulting dehydrated neutral loss ions then have a mass-to-charge ratio 18 Da smaller than that of the parent peptide ion.

[0056] Dehydrogenation of peptides is frequently observed, with a corresponding peak observed at 18 mass units below the mass / charge ratio of the parent or precursor ion. Fig. Figure 1 shows results following electrospray ionization ("ESI") of the neuropeptide substance P. The peptide ions ionized by the electrospray ionization ion source were passed through a quadrupole mass filter. The quadrupole mass filter was progressively scanned in 1 Da steps, and the parent or precursor ions passed through at each setting of the quadrupole mass filter were fragmented in a collision-induced dissociation ("CID") fragmentation device. The resulting fragments or product ions were then mass analyzed.

[0057] Fig. Figure 1 shows the mass-to-charge ratio of the fragment or product ions plotted as a function of the scan time of the quadrupole mass filter. Note that the scan time of the quadrupole mass filter corresponds to the mass-to-charge ratio of the parent or precursor ions passed through the quadrupole mass filter. Accordingly, Fig. 1 can be considered as showing along the x-axis the mass / charge ratio of parent or precursor ions passed by the quadrupole mass filter at a given time, with the y-axis showing the mass / charge ratio of the resulting fragment or product ions.

[0058] It is known that singly charged substance P ions have a mass / charge ratio of 1347.7, doubly charged substance P ions have a mass / charge ratio of 674.4, and triply charged substance P ions have a mass / charge ratio of 449.9.

[0059] Fig. Figure 1 shows a vertical line around the mass / charge ratio 450, which corresponds to fragment or product ions resulting from the fragmentation of triply charged substance P ions.

[0060] A particularly important feature of the embodiment is that, as Fig. 1, various trend lines can be observed in the fragmentation mass spectrum data.

[0061] When in Fig. In the specific example shown in Figure 1, a parent or precursor ion scan line is indicated, and three additional trend lines are indicated below the parent or precursor ion scan line. The parent or precursor ion scan line shows that when the parent or precursor ions were scanned from 400 to 900 Da, unfragmented ions with the same mass-to-charge ratio were observed in the fragmentation ion mass spectral data.

[0062] The three other Fig. The trend lines indicated in Figure 1 (which appear below the parent or precursor ion scan line) are particularly important.

[0063] One of the highlighted trend lines corresponds to substance P ions that have been dehydrated (i.e., have lost a water molecule). The dehydrated peptide ions are neutral loss ions, and the mass-to-charge ratio of the neutral loss ions should be 18 Da smaller than the mass-to-charge ratio of the corresponding hydrogenated parent or precursor peptide ions.

[0064] The various trend lines that appear in Fig. 1 correspond to joint neutral losses of parent or precursor peptide ions.

[0065] It is over Fig. 1 shows that ion peaks are effectively observed at any starting or precursor ion mass-to-charge ratio value. The ion peaks primarily exhibit unknown structures, but they are related to the compound class being analyzed (e.g., peptides). For example, the ions can include non-specific peptides, clusters, adducts, modifications, fragments, or ions resulting from partial digestion, etc.

[0066] Accurate values ​​of the observed neutral losses can be determined by applying the best-fit lines to the mass spectrum data. According to the embodiment, if the neutral loss value is, for example, 5 ppm too high compared to a predetermined or expected mass loss, the mass spectrum data set can be corrected by 5 ppm to obtain more accurate values ​​for the unknown ions. According to the embodiment, one or more calibration parameters can be adjusted so that, when the mass spectrometer has been recalibrated, the neutral loss ions have a mass difference that optionally exactly matches a predetermined or expected mass difference.

[0067] It can then be seen that the approach according to the embodiment is not possible with only a few data points due to the small error values ​​in the mass / charge ratio differences. However, using a complete mass spectrum data set comprising tens or hundreds of parent or precursor ions and fragment ion pairs in a manner described above, the statistical accuracy of the measurement process is significantly improved. Accordingly, after sufficient mass spectrum data has been acquired, the mass spectrometer's control system can then accurately self-calibrate the mass spectrometer or otherwise perform a precise process for calibrating or recalibrating the mass spectrometer using essentially an internal calibration procedure as described above.

[0068] Various further embodiments are contemplated wherein the mass spectrometer may be operated in a mode of operation for obtaining hi-lo detections. For example, in this mode of operation, a collision cell or fragmentation device may be repeatedly switched between a first mode of operation, wherein parent or precursor ions are passed without being fragmented in the collision cell or fragmentation device, and a second mode of operation, wherein parent or precursor ions are fragmented within the collision cell or fragmentation device. In the first mode of operation, parent or precursor ions may be passed by the collision cell or fragmentation device, but the collision cell or fragmentation device may be substantially turned off, such that the collision cell or fragmentation device acts as an ion guide to allow ions to continue to pass without the ions becoming significantly fragmented.Alternatively, in the first mode of operation, parent or precursor ions can be directed to essentially bypass the collision cell or fragmentation device.

[0069] According to a similar operating mode, the mass spectrometer can be used in an MS e -mode. For data-independent analysis (“DIA” or MS e ), the collision energy is switched between a low energy and a high energy to generate precursor and product ion spectra. However, if a complex sample is analyzed, co-elution of parent ions may occur, for which retention time alignment alone is insufficient to determine the MS e-spectra. An ion mobility separation stage can be inserted before the fragmentation device so that both the retention time and the ion mobility elution time can be used to assign parent or precursor ion mass spectral data to corresponding product ion mass spectral data. This approach is known as HDMS. e The self-calibration approach described above can be used to calibrate or recalibrate a mass spectrometer installed in an MS e - or HDMS e -operating mode.

[0070] Parent or precursor ions can lose neutral molecules or atoms and therefore suffer neutral loss. The following table shows several common ways in which parent or precursor ions can suffer neutral loss. MonoisotopischerMassenverlust (amu) Zusammensetzung 1,007825 H 14,01565008 CH2 15,023475 CH3 15,99491463 O 17,00273967 OH 17,02654912 NH3 18,01056471 H2O ~19 F ~20 HF 21,98194 N / a + replaced by H + 27,01089904 HCN 27,02347512 C2H3 27,99491463 CO 38,03130016 C2H4 30,01056471 CH2O 31,01838975 CH3O 32,02621479 CH4O 31,97207 S 31,98983 O2 32,97989573 HS ~33 CH3+H2O 33,98772077 H2S 34,968853(37) CI 35,97667804(38) HCl 42,04695024 C3H6 42,01056471 C2H2O 42,03437416 C2H4N 43,05477528 C3H7 ~43 CH3CO 43,98982926 CO2 44,01363871 CONH2 ~45 C2H5O ~55 C4H7 ~57 C4H9 ~59 C2H3O2 ~60 C2H4O2 62,96359077 PO2 63,96189995 SO2 78,9585054 PO3 79,96633044 HPO3 ~79(81) Br ~80(82) HBr 97,97689515 H3PO4 ~127 I ~128 HI

[0071] Embodiments are contemplated in which one or more mass losses (as exemplified in the table above) may be used to perform self-calibration of the mass spectrometer. However, the present embodiments are not limited to the specific mass losses detailed above, and further embodiments are contemplated in which different mass losses may be used to perform self-calibration of the mass spectrometer.

[0072] Further embodiments are contemplated in which adductants are used together with, or in place of, neutral loss ions to perform self-calibration of the mass spectrometer. These various embodiments utilize the fact that when ions of certain classes of compounds are reacted, they can result in product ions, some of which are adduct ions, where the ions have gained one or more atoms or molecules. Like neutral loss ions, the adduct ions should have a precise mass difference relative to the precursor ions. According to these various embodiments, one or more calibration parameters can be adjusted so that when the mass spectrometer has been recalibrated, the adduct ions have a mass difference that optionally exactly matches the predetermined or expected mass difference.

[0073] "Interferences and contaminants encountered in modern mass spectrometry," Bernd O. Keller, Jie Sui, Alex B. Young, and Randy M. Whittal, Analytica Chimica Acta 627, Issue 1, October 3, 2008, pages 71-81, details several common ways in which parent or precursor ions can undergo adducts, losses, or replacements, and the corresponding precise (expected) mass differences for these reactions. Any of these expected mass differences, as will be understood by those skilled in the art, can be used, according to the procedures described herein, to perform self-calibration of a mass spectrometer.

Claims

[1] A method for checking or adjusting the calibration of a mass spectrometer, comprising: Fragmentation of parent ions and generation of fragment ion mass spectrum data, Detecting first neutral loss ions in the fragment ion mass spectrum data, where neutral loss ions correspond to parent ions that have lost one or more neutral atoms or molecules, Determining a first mass loss difference between the parent ions and the first neutral loss ions and Determining whether the first mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the first mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters, wherein the step of fragmenting parent ions and generating fragment ion mass spectrum data comprises: Scanning a mass-to-charge ratio transmission window of a mass filter and fragmenting parent ions passed through the mass filter. [2] The method of claim 1, wherein the step of detecting first neutral loss ions in the fragment ion mass spectrum data comprises: Analyzing the mass / charge ratio of fragment ions as a function of the mass / charge ratio of corresponding parent ions and Identify one or more trend lines in the fragment ion mass spectrum data. [3] The method of claim 2, wherein the step of determining a first mass loss difference between the parent ions and the first neutral loss ions comprises determining one or more best-fit lines for the trend line or the plurality of trend lines. [4] A process according to any one of the preceding claims, wherein the one or more neutral molecules or atoms are selected from the group consisting of: (i) H, (ii) CH3, (iii) OH, (iv) H2O, (v) F, (vi) HF, (vii) C2H3, HCN, (viii) C2H4, CO, (ix) CH2O, (x) CH3O, (xi) CH4O, S, (xii) CH3+ H2O, HS, (xiii) H2S, (xiv) Cl, (xv) HCl, (xvi) C3H6, C2H2O, C2H4N, (xvii) C3H7, CH3CO, (xviii) CO2O, CONH2, (xix) C2H5O, (xx) C4H7, (xxi) C4H9, (xxii) C2H3O2, (xxiii) C2H4O2, (xxiv) SO2, (xxv) Br, (xxvi) HBr, (xxvii) I, (xxviii) HI, (xxix) NH3, (xxx) CH2, (xxxi) O2, (xxxii) CO2, (xxxiii) PO2, (xxxiv) PO3, (xxxv) HPO3 and (xxxvi) H3PO4. [5] A method according to any one of the preceding claims, wherein the step of adjusting one or more calibration parameters comprises adjusting the calibration of the mass spectrometer such that when the mass spectrometer has been recalibrated, the first mass loss difference corresponds to an expected or predetermined mass loss difference. [6] A method according to any one of claims 1 to 4, wherein the step of adjusting one or more calibration parameters comprises adjusting the calibration of the mass spectrometer such that when the mass spectrometer has been recalibrated, the difference between the first mass loss difference and an expected or predetermined mass loss difference is reduced. [7] A method according to any one of the preceding claims, further comprising: Detection of second neutral loss ions in the fragment ion mass spectrum data, Determining a second mass loss difference between the parent ions and the second neutral loss ions and Determining whether the second mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the second mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters. [8] The method of claim 7, further comprising: Detection of third neutral loss ions in the fragment ion mass spectrum data, Determining a third mass loss difference between the parent ions and the third neutral loss ions and Determining whether the third mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the third mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters. [9] The method of claim 8, further comprising: Detecting fourth neutral loss ions in the fragment ion mass spectrum data, Determining a fourth mass loss difference between the parent ions and the fourth neutral loss ions and Determining whether the fourth mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if it is determined that the fourth mass loss difference does not correspond to an expected or predetermined mass loss difference, the method further comprises adjusting one or more calibration parameters. [10] A method according to any one of the preceding claims, wherein the step of fragmenting the parent ions comprises fragmenting at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 different species of parent ions. [11] A method according to any one of the preceding claims, further comprising generating at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 different parent ion and fragment ion pairs. [12] A method according to any one of the preceding claims, further comprising: Detection of first adductions in the fragment ion mass spectrum data, Determining a first mass gain difference between the parent ions and the first adducts and Determining whether the first mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the first mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters. [13] A method for checking or adjusting the calibration of a mass spectrometer, comprising: Reaction of parent ions and generation of product ions mass spectrum data, Detection of first adductions in the product ion mass spectrum data, Determining a first mass gain difference between the parent ions and the first adducts and Determining whether the first mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the first mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters, wherein the step of reacting parent ions and generating product ion mass spectrum data comprises: Scanning a mass-to-charge ratio transmission window of a mass filter and reacting parent ions passed through the mass filter. [14] The method of claim 13, wherein the step of detecting first adducts in the product ion mass spectrum data comprises: Analyzing the mass / charge ratio of product ions as a function of the mass / charge ratio of corresponding parent ions and Identify one or more trend lines in the product ion mass spectrum data. [15] The method of claim 14, wherein the step of determining a first mass gain difference between the parent ions and the first adducts comprises determining one or more best-fit lines for the one or more trend lines. [16] A method according to any one of claims 13 to 15, wherein the step of adjusting one or more calibration parameters comprises adjusting the calibration of the mass spectrometer such that, when the mass spectrometer has been recalibrated, the first mass gain difference corresponds to an expected or predetermined mass gain difference. [17] A method according to any one of claims 13 to 16, wherein the step of adjusting one or more calibration parameters comprises adjusting the calibration of the mass spectrometer such that, when the mass spectrometer has been recalibrated, the difference between the first mass gain difference and an expected or predetermined mass gain difference is reduced. [18] A method according to any one of claims 13 to 17, further comprising: Detection of second adductions in the product ion mass spectrum data, Determining a second mass gain difference between the parent ions and the second adducts and Determining whether the second mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the second mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters. [19] The method of claim 18, further comprising: Detecting third adductions in the product ion mass spectrum data, Determining a third mass gain difference between the parent ions and the third adducts and Determining whether the third mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the third mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters. [20] The method of claim 19, further comprising: Detecting fourth adducts in the product ion mass spectrum data, determining a fourth mass gain difference between the parent ions and the fourth adducts and Determining whether the fourth mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if it is determined that the fourth mass gain difference does not correspond to an expected or predetermined mass gain difference, the method further comprises adjusting one or more calibration parameters. [21] Mass spectrometer, which includes: a fragmentation device for fragmenting ions and a control system that is equipped and designed to carry out the following: (i) fragmenting output and generating fragment ion mass spectrum data, (ii) Detecting first neutral loss ions in the fragment ion mass spectral data, where neutral loss ions correspond to parent ions that have lost one or more neutral atoms or molecules (iii) determining a first mass loss difference between the parent ions and the first neutral loss ions and (iv) determining whether the first mass loss difference corresponds to an expected or predetermined mass loss difference, wherein, if the control system determines that the first mass loss difference does not correspond to an expected or predetermined mass loss difference, the control system is further arranged and designed to adjust one or more calibration parameters, wherein the step of fragmenting parent ions and generating fragment ion mass spectrum data comprises: Scanning a mass-to-charge ratio transmission window of a mass filter and fragmenting parent ions passed through the mass filter. [22] Mass spectrometer, which includes: a fragmentation device for fragmenting ions and a control system that is equipped and designed to carry out the following: (i) reacting parent ions and generating product ion mass spectrum data, (ii) Detection of first adductions in the product ion mass spectrum data, (iii) determining a first mass gain difference between the parent ions and the first adducts and (iv) determining whether the first mass gain difference corresponds to an expected or predetermined mass gain difference, wherein, if the control system determines that the first mass gain difference does not correspond to an expected or predetermined mass gain difference, the control system is further arranged and designed to adjust one or more calibration parameters, wherein the step of fragmenting parent ions and generating product ion mass spectrum data comprises: Scanning a mass-to-charge ratio transmission window of a mass filter and reacting parent ions passed through the mass filter.

Images