Self-calibration of spectra using precursor mass to charge ratio and fragment mass to charge ratio known differences

Abstract

A method of checking or adjusting the calibration of a mass spectrometer is disclosed. The method comprises fragmenting parent or precursor ions and generating fragment or product ion mass spectral data and recognizing first neutral loss ions in the fragment or product ion mass spectral data. The method further comprises determining a first mass loss difference between the parent or precursor ions and the first neutral loss ions and determining whether the first mass loss difference corresponds with an expected or pre-determined mass loss difference, wherein if it is determined that the first mass loss difference does not correspond with an expected or pre-determined mass loss difference then the method further comprises adjusting one or more calibration parameters.

Claims

1. A method of checking or adjusting the calibration of a mass spectrometer comprising: fragmenting parent or precursor ions and generating fragment or product ion mass spectral data; recognising first neutral loss ions in said fragment or product ion mass spectral data; determining a first mass loss difference between said parent or precursor ions and said first neutral loss ions; determining whether said first mass loss difference corresponds with an expected or pre-determined mass loss difference, wherein if it is determined that said first mass loss difference does not correspond with an expected or pre-determined mass loss difference then said method further comprises adjusting one or more calibration parameters; and wherein said step of fragmenting parent or precursor ions and generating fragment or product ion mass spectral data comprises: scanning a mass to charge ratio transmission window of a mass filter; and fragmenting parent or precursor ions which are transmitted by said mass filter.

2. A method as claimed in claim 1, wherein said step of recognising first neutral loss ions in said fragment or product ion mass spectral data comprises: plotting or otherwise analysing the mass to charge ratio of fragment or product ions as a function of the mass to charge ratio of corresponding parent or precursor ions; and identifying one or more trend lines in said fragment or product ion mass spectral data; and wherein said step of determining a first mass loss difference between said parent or precursor ions and said first neutral loss ions comprises determining a line of best fit between said first neutral loss ions in said fragment or product ion mass spectral data.

3. A method as claimed in claim 1, wherein said first neutral loss ions comprise parent or precursor ions which have lost one or more neutral molecules or atoms; wherein said 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) NH.sub.3; (xxx) CH.sub.2; (xxxi) O.sub.2; (xxxii) CO.sub.2; (xxxiii) PO.sub.2; (xxxiv) PO.sub.3; (xxxv) HPO.sub.3; and (xxxvi) H.sub.3PO.sub.4.

4. A method as claimed in claim 1, wherein the step of adjusting one or more calibration parameters comprises: adjusting the calibration of said mass spectrometer so that when said mass spectrometer has been re-calibrated said first mass loss difference exactly or substantially corresponds with an expected or pre-determined mass loss difference; or adjusting the calibration of said mass spectrometer so that when said mass spectrometer has been re-calibrated the difference between said first mass loss difference and an expected or pre-determined mass loss difference is reduced.

5. A method as claimed in claim 1, further comprising: recognising second neutral loss ions in said fragment or product ion mass spectral data; determining a second mass loss difference between said parent or precursor ions and said second neutral loss ions; and determining whether said second mass loss difference corresponds with an expected or pre-determined mass loss difference, wherein if it is determined that said second mass loss difference does not correspond with an expected or pre-determined mass loss difference then said method further comprises adjusting one or more calibration parameters.

6. A method as claimed in claim 1, further comprising: recognising third neutral loss ions in said fragment or product ion mass spectral data; determining a third mass loss difference between said parent or precursor ions and said third neutral loss ions; and determining whether said third mass loss difference corresponds with an expected or pre-determined mass loss difference, wherein if it is determined that said third mass loss difference does not correspond with an expected or pre-determined mass loss difference then said method further comprises adjusting one or more calibration parameters.

7. A method as claimed in claim 1, further comprising: recognising fourth neutral loss ions in said fragment or product ion mass spectral data; determining a fourth mass loss difference between said parent or precursor ions and said fourth neutral loss ions; and determining whether said fourth mass loss difference corresponds with an expected or pre-determined mass loss difference, wherein if it is determined that said fourth mass loss difference does not correspond with an expected or pre-determined mass loss difference then said method further comprises adjusting one or more calibration parameters.

8. A method as claimed in claim 1, wherein the step of fragmenting said parent or precursor 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 or precursor ions.

9. A method as claimed in claim 1, 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 or precursor ion and fragment or product ion pairs.

10. A method as claimed in claim 1, further comprising: recognising first adduct ions in said fragment or product ion mass spectral data; determining a first mass gain difference between said parent or precursor ions and said first adduct ions; and determining whether said first mass gain difference corresponds with an expected or pre-determined mass gain difference, wherein if it is determined that said first mass gain difference does not correspond with an expected or pre-determined mass gain difference then said method further comprises adjusting one or more calibration parameters.

11. A method of mass spectrometry comprising a method as claimed in claim 1.

12. A method of checking or adjusting the calibration of a mass spectrometer comprising: reacting parent or precursor ions and generating fragment or product ion mass spectral data; recognising first adduct ions in said fragment or product ion mass spectral data; determining a first mass gain difference between said parent or precursor ions and said first adduct ions; determining whether said first mass gain difference corresponds with an expected or pre-determined mass gain difference, wherein if it is determined that said first mass gain difference does not correspond with an expected or pre-determined mass gain difference then said method further comprises adjusting one or more calibration parameters; and wherein said step of reacting parent or precursor ions and generating fragment or product ion mass spectral data comprises: scanning a mass to charge ratio transmission window of a mass filter; and reacting parent or precursor ions which are transmitted by said mass filter.

13. A method as claimed in claim 12, wherein said step of recognising first adduct ions in said fragment or product ion mass spectral data comprises: plotting or otherwise analysing the mass to charge ratio of fragment or product ions as a function of the mass to charge ratio of corresponding parent or precursor ions; and identifying one or more trend lines in said fragment or product ion mass spectral data; and wherein said step of determining a first mass gain difference between said parent or precursor ions and said first adduct ions comprises determining a line of best fit between said first adduct ions in said fragment or product ion mass spectral data.

14. A method as claimed in claim 12, wherein the step of adjusting one or more calibration parameters comprises: adjusting the calibration of said mass spectrometer so that when said mass spectrometer has been re-calibrated said first mass gain difference exactly or substantially corresponds with an expected or pre-determined mass gain difference; or adjusting the calibration of said mass spectrometer so that when said mass spectrometer has been re-calibrated the difference between said first mass gain difference and an expected or pre-determined mass gain difference is reduced.

15. A method as claimed in any of claim 12, further comprising: recognising second adduct ions in said fragment or product ion mass spectral data; determining a second mass gain difference between said parent or precursor ions and said second adduct ions; and determining whether said second mass gain difference corresponds with an expected or pre-determined mass gain difference, wherein if it is determined that said second mass gain difference does not correspond with an expected or pre-determined mass gain difference then said method further comprises adjusting one or more calibration parameters.

16. A method as claimed in any of claim 12, further comprising: recognising third adduct ions in said fragment or product ion mass spectral data; determining a third mass gain difference between said parent or precursor ions and said third adduct ions; and determining whether said third mass gain difference corresponds with an expected or pre-determined mass gain difference, wherein if it is determined that said third mass gain difference does not correspond with an expected or pre-determined mass gain difference then said method further comprises adjusting one or more calibration parameters.

17. A method as claimed in claim 12, further comprising: recognising fourth adduct ions in said fragment or product ion mass spectral data; determining a fourth mass gain difference between said parent or precursor ions and said fourth adduct ions; and determining whether said fourth mass gain difference corresponds with an expected or pre-determined mass gain difference, wherein if it is determined that said fourth mass gain difference does not correspond with an expected or pre-determined mass gain difference then said method further comprises adjusting one or more calibration parameters.

18. A mass spectrometer comprising: a fragmentation device for fragmenting ions; and a control system arranged and adapted: (i) to fragment parent or precursor ions and to generate fragment or product ion mass spectral data by scanning a mass to charge ratio transmission window of a mass filter and fragmenting parent or precursor ions which are transmitted by said mass filter; (ii) to recognise first neutral loss ions or first adduct ions in said fragment or product ion mass spectral data; (iii) to determine a first mass loss difference between said parent or precursor ions and said first neutral loss ions or determine a first mass gain difference between said parent or precursor ions and said first adduct ions; and (iv) to determine whether said first mass loss difference or said first mass gain difference corresponds with an expected or pre-determined mass loss difference or with an expected or predetermined mass gain difference, wherein if said control system determines said first mass loss difference or said first mass gain difference does not correspond with said expected or pre-determined mass loss difference or said expected or predetermined mass gain difference then said control system is further arranged and adapted to adjust one or more calibration parameters.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Various embodiments will now be described, by way of example only, and with reference to the accompanying drawing in which:

(2) FIG. 1 shows a heat map showing a parent or precursor scan line and trend lines relating to constant neutral loss ions.

DETAILED DESCRIPTION

(3) An embodiment will now be described.

(4) An embodiment utilises the fact that ions of certain classes of compounds (e.g. peptides) when subjected to fragmentation will result in fragment or product ions wherein some of the fragment or product ions are neutral loss ions wherein the ions 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 recognises neutral loss ions in fragmentation mass spectral data and uses the mass difference between the neutral loss ions and the parent ions to self calibrate the mass to charge ratio scale of a mass spectrometer.

(5) According to an embodiment ions from an ion source such as an Electrospray Ionisation (ESI) ion source are passed to a quadrupole mass filter. The quadrupole mass filter is optionally set to transmit a 1 Da mass range of parent or precursor ions at any particular point in time. The mass to charge ratio transmission window of the quadrupole mass filter is optionally scanned. For example, according to an embodiment the mass to charge ratio transmission window may be progressively scanned from a mass to charge ratio of 400 to a mass to charge ratio of 900 in steps of 1 Da.

(6) Once the quadrupole mass filter has transmitted ions having a mass to charge ratio of 900 the quadrupole mass filter is then optionally reset so as to return to transmitting ions having a mass to charge ratio of 400 and the scan process is then optionally repeated one or more times.

(7) Parent or precursor ions which are transmitted by the quadrupole mass filter are optionally fragmented in a fragmentation cell or device. According to an embodiment the fragmentation cell or device may comprise a Collision Induced Dissociation (CID) fragmentation cell or device. 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.

(8) The parent or precursor ions which are fragmented or otherwise dissociated in the fragmentation cell or device are optionally fragmented so as to result in a plurality of fragment or product ions. The resulting fragment or product ions are then mass analysed by, for example, a Time of Flight mass analyser. Some of the resulting fragment or product ions optionally comprise neutral loss ions i.e. parent or precursor ions which 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 will have a mass to charge ratio which is 18 Da less than that of the parent peptide ion.

(9) Dehydration of peptides is often observed with a corresponding peak observed at 18 mass units lower than the mass to charge ratio of the parent or precursor ion. FIG. 1 shows results following Electrospray Ionisation (ESI) of the neuropeptide Substance P. The peptide ions ionised by the Electrospray Ionisation ion source were transmitted to a quadrupole mass filter. The quadrupole mass filter was progressively scanned in 1 Da steps and the transmitted parent or precursor ions at each setting of the quadrupole mass filter were fragmented in a Collision Induced Dissociation (CID) fragmentation device. The resulting fragment or product ions were then mass analysed.

(10) FIG. 1 shows the mass to charge ratio of the fragment or product ions plotted as function of the scan time of the quadrupole mass filter. It will be understood that the scan time of the quadrupole mass filter corresponds with the mass to charge ratio of the parent or precursor ions which were transmitted by the quadrupole mass filter. Accordingly, FIG. 1 may be considered as showing along the x-axis the mass to charge ratio of parent or precursor ions which were transmitted at any instance in time by the quadrupole mass filter and wherein the y-axis shows the mass to charge ratio of the resulting fragment or product ions.

(11) It is known that singly charged Substance P ions have a mass to charge ratio of 1347.7, doubly charged Substance P ions have a mass to charge ratio of 674.4 and triply charged Substance P ions have a mass to charge ratio of 449.9.

(12) FIG. 1 shows a vertical line around mass to charge ratio 450 which corresponds with fragment or product ions resulting from the fragmentation of triply charged Substance P ions.

(13) A particularly important feature of the embodiment is that it is apparent from FIG. 1 that various trend lines may be observed in the fragmentation mass spectral data.

(14) In the particular example shown in FIG. 1 a parent or precursor ion scan line is indicated and three further trend lines are indicated below the parent or precursor ion scan line. The parent or precursor ion scan line shows that as the parent or precursor ions were scanned from 400 to 900 Da, unfragmented ions having the same mass to charge ratio were observed in the fragmentation ion mass spectral data.

(15) The three further trend lines indicated in FIG. 1 (which appear below the parent or precursor ion scan line) are particularly important.

(16) One of the highlighted trend lines corresponds with Substance P ions which have become 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 less than the mass to charge ratio of the corresponding hydrated parent or precursor peptide ions.

(17) The various trend lines which are observed in FIG. 1 correspond to common neutral losses from parent or precursor peptide ions.

(18) It is apparent from FIG. 1 that ion peaks are observed effectively at every parent or precursor ion mass to charge ratio value. The ion peaks are mostly of unknown structure but will be related to the class of compound (e.g. peptides) being analysed. For example, the ions may comprise non-specific peptides, clusters, adducts, modifications, fragments, or ions resulting from partial digestion etc.

(19) It is possible to extract accurate values for the neutral losses observed by applying best fit lines to the mass spectral data. According to the embodiment if the neutral loss value is, for example, 5 ppm too high compared with a pre-determined or expected mass loss then the mass spectral data set may be corrected by 5 ppm in order to obtain more accurate values for the unknown ions. According to the embodiment one or more calibration parameters may be adjusted so that when the mass spectrometer has been re-calibrated the neutral loss ions have a mass difference which optionally exactly matches a pre-determined or expected mass difference.

(20) It is apparent then the approach according to the embodiment is not possible with just a few data points due to the small error values in mass to charge ratio differences. However, utilising a full mass spectral data set which may comprise tens or hundreds of parent or precursor ion and fragment ion pairs in a manner as described above significantly improves the statistical accuracy of the measurement process. Accordingly, having acquired sufficient mass spectral data, the control system of the mass spectrometer may then accurately self-calibrate the mass spectrometer or otherwise perform an accurate process of calibrating or re-calibrating the mass spectrometer using essentially an internal calibration method as described above.

(21) Various further embodiments are contemplated wherein the mass spectrometer may be operated in a mode of operation so as to obtain Hi-Lo acquisitions. 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 transmitted without being fragmented within 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 transmitted through the collision cell or fragmentation device but the collision cell or fragmentation device may be essentially switched OFF so that the collision cell or fragmentation device acts as an ion guide so as to onwardly transmit ions without substantially fragmenting the ions. Alternatively, in the first mode of operation parent or precursor ions may be directed so as to substantially by-pass the collision cell or fragmentation device.

(22) According to a similar mode of operation the mass spectrometer may be operated in a MS.sup.e mode of operation. Data Independent Analysis (DIA or MS.sup.e) involves switching the collision energy between low energy and high energy in order to produce precursor and product ion spectra. However, if a complex sample is analysed then there may be co-eluting parent ions for which retention time alignment by itself is inadequate to deconvolve the MS.sup.e spectra. An ion mobility separation stage may be introduced prior to the fragmentation device so that both retention time and ion mobility elution time may be used to assign parent or precursor ion mass spectral data to corresponding product ion mass spectral data. This approach is known as HDMS.sup.e. The self-calibration approach as described above may be used to calibrate or re-calibrate a mass spectrometer which is operated in a MS.sup.e or HDMS.sup.e mode of operation.

(23) Parent or precursor ions may lose neutral molecules or neutral atoms and hence may suffer from neutral loss. The following table details a number of common ways in which parent or precursor ions may suffer from neutral loss.

(24) TABLE-US-00001 Monoisotopic mass loss (amu) Composition 1.007825 H 14.01565008 CH.sub.2 15.023475 CH.sub.3 15.99491463 O 17.00273967 OH 17.02654912 NH.sub.3 18.01056471 H.sub.2O ~19 F ~20 HF 21.98194 Na.sup.+ replaced by H.sup.+ 27.01089904 HCN 27.02347512 C.sub.2H.sub.3 27.99491463 CO 38.03130016 C.sub.2H.sub.4 30.01056471 CH.sub.2O 31.01838975 CH.sub.3O 32.02621479 CH.sub.4O 31.97207 S 31.98983 O.sub.2 32.97989573 HS ~33 CH.sub.3 + H.sub.2O, 33.98772077 H.sub.2S 34.968853(37) Cl 35.97667804(38) HCl 42.04695024 C.sub.3H.sub.6 42.01056471 C.sub.2H.sub.2O 42.03437416 C.sub.2H.sub.4N 43.05477528 C.sub.3H.sub.7 ~43 CH.sub.3CO 43.98982926 CO.sub.2 44.01363871 CONH.sub.2 ~45 C.sub.2H.sub.5O ~55 C.sub.4H.sub.7 ~57 C.sub.4H.sub.9 ~59 C.sub.2H.sub.3O.sub.2 ~60 C.sub.2H.sub.4O.sub.2 62.96359077 PO.sub.2 63.96189995 SO.sub.2 78.9585054 PO.sub.3 79.96633044 HPO.sub.3 ~79(81) Br ~80(82) HBr 97.97689515 H.sub.3PO.sub.4 ~127 I ~128 HI

(25) Embodiments are contemplated wherein one or more mass losses (as exemplified in the table above) may be utilised in order to self-calibrate the mass spectrometer. However, the present embodiments are not restricted to the specific mass loses as detailed above and further embodiments are contemplated wherein different mass loses may be used to self-calibrate the mass spectrometer.

(26) Further embodiments are contemplated wherein adduct ions are utilised along with, or instead of, neutral loss ions in order to self-calibrate the mass spectrometer. These various embodiments utilise the fact that when ions of certain classes of compounds are reacted they may result in product ions wherein some of the product ions are adduct ions wherein the ions have gained one or more atoms or molecules. Like neutral loss ions, the adduct ions should have a precise mass difference from that of the precursor ions. In these various embodiments, one or more calibration parameters may be adjusted so that when the mass spectrometer has been re-calibrated the adduct ions have a mass difference which optionally exactly matches the pre-determined or expected mass difference.

(27) 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, 3 Oct. 2008, Pages 71-81, details a number of common ways in which parent or precursor ions may undergo adducts, losses or replacements, and the corresponding precise (expected) mass differences for these reactions. Each or any of these expected mass differences may, as will be understood by those skilled in the art, be utilised in accordance with the methods described herein in order to self-calibrate a mass spectrometer.

(28) Although the present invention has been described with reference to embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.