MS/MS analysis using ECD or ETD fragmentation

Abstract

A method of mass spectrometry is disclosed comprising: providing supercharged analyte ions; and supplying electrons or reagent ions to said analyte ions so as to transfer charge from said reagent ions or electrons to said analyte ions, said transfer of charge causing at least some of said analyte ions to dissociate. The charge transfer step is performed at a relatively high pressure and preferably substantially at atmospheric pressure.

Claims

1. A method of mass spectrometry comprising: (a) providing supercharged analyte ions, wherein said step of providing supercharged analyte ions comprises adding a reagent to analyte and then ionising the analyte so as to produce said analyte ions with a higher charge state than they would have been produced without having added the reagent to the analyte prior to ionisation; and (b) supplying electrons or reagent ions to said analyte ions so as to transfer charge from said reagent ions or electrons to said analyte ions, said transfer of charge causing at least some of said analyte ions to dissociate; wherein step (b) is performed at a pressure selected from the group of >0.1 mbar; >1 mbar; >5 mbar; >10 mbar; >100 mbar; about 1 bar; or substantially at atmospheric pressure.

2. The method of claim 1, wherein the electrons or reagent ions cause said analyte ions to dissociate via electron capture dissociation (ECD) or via electron transfer dissociation (ETD).

3. The method of claim 1, said transfer of charge causing at least some of said analyte ions to dissociate and others of said analyte ions not to dissociate but to form intermediate ions of altered charge; the method further comprising: (c) isolating at least some of said intermediate ions from other ions; (d) exciting at least some of the isolated intermediate ions so as to cause them to dissociate into daughter ions; and (e) mass analysing at least some of said intermediate ions and/or mass analysing at least some of said daughter ions.

4. The method of claim 3, wherein step (b) comprises supplying said reagent ions to a mixture of different analyte ions so as to cause the analyte ions to dissociate and/or to form the intermediate ions.

5. The method of claim 3, wherein the intermediate ions are precursor analyte ions that have been reduced in charge due to interactions with said reagent ions or electrons.

6. The method of claim 3, wherein the electrons or reagent ions are supplied to the analyte ions in an ion source or reaction cell and wherein the intermediate ions are selectively transmitted downstream of the ion source or reaction cell and subsequently excited and dissociated into said daughter ions.

7. The method of claim 3, comprising: providing said analyte ions; analysing said analyte ions without first exposing them to said electrons or reagent ions so as to generate a first signal; exposing said analyte ions to said electrons or reagent ions so that some of said analyte ions form said intermediate ions, and mass analysing the resulting ions so as to generate a second signal; comparing the first and second signals so as to determine a difference between the signals, the difference having been caused by the generation of said intermediate ions and serving to identify a characteristic of the ions which are the intermediate ions; and performing said step of isolating at least some of said intermediate ions based on said characteristic determined by comparing said signals.

8. The method of claim 7, wherein the first and second signals are generated by mass analysing the ions and the mass or mass to charge ratio of the intermediate ions is the characteristic determined by comparing said signals; and/or comprising mass analysing the analyte ions to generate the first signal and mass analysing said resulting ions to generate the second signal; comparing the first and second signals so as to determine if one or more ion peaks present in both signals has shifted in mass to charge ratio between the signals; and determining that the ions which give rise to the one or more shifted peaks are intermediate ions.

9. The method of claim 3, wherein both the intermediate ions and their daughter ions are analysed in a manner so as to associate the intermediate ions with their daughter ions; and wherein at least some of the intermediate ions that have been dissociated to form daughter ions are identified from their daughter ions.

10. The method of claim 9, wherein the identified intermediate ions are used to identify the analyte molecules or analyte ions from which these intermediate ions derived.

11. The method of claim 1, wherein step (a) comprises providing a mixture of different supercharged analyte ions; and wherein step (b) comprises supplying electrons or reagent ions to said mixture of different analyte ions so as to transfer charge from said reagent ions or electrons to said analyte molecules or ions, said transfer of charge causing at least some of said analyte molecules or analyte ions to dissociate and others of said analyte molecules or analyte ions not to dissociate but to form intermediate ions of altered charge; and the method further comprises: isolating at least some of said intermediate ions from other ions; exciting at least some of the isolated intermediate ions so as to cause them to dissociate into daughter ions; analysing at least some of the intermediate ions and at least some of their daughter ions so as to associate at least some of the intermediate ions with their daughter ions; and identifying intermediate ions from their daughter ions.

12. A method of mass spectrometry comprising: (a) providing analyte molecules or analyte ions using a MALDI ion source; (b) supplying electrons or reagent ions to said analyte molecules or analyte ions so as to transfer charge from said reagent ions or electrons to said analyte molecules or ions, said transfer of charge causing at least some of said analyte molecules or analyte ions to dissociate and others of said analyte molecules or analyte ions not to dissociate but to form intermediate ions of altered charge; (c) isolating at least some of said intermediate ions from other ions; (d) exciting at least some of the isolated intermediate ions so as to cause them to dissociate into daughter ions; and (e) mass analysing at least some of said intermediate ions and/or mass analysing at least some of said daughter ions.

13. The method of claim 12, wherein the MALDI ion source is an atmospheric pressure MALDI ion source or a liquid MALDI ion source, preferably producing multiply charged analyte ions.

14. The method of claim 12, wherein step (a) comprises: providing a mixture of different analyte molecules or analyte ions using a MALDI ion source; and wherein step (b) comprises supplying electrons or reagent ions to said mixture of different analyte molecules or analyte ions so as to transfer charge from said reagent ions or electrons to said analyte molecules or ions, said transfer of charge causing at least some of said analyte molecules or analyte ions to dissociate and others of said analyte molecules or analyte ions not to dissociate but to form intermediate ions of altered charge; and wherein step (e) comprises analysing at least some of the intermediate ions and at least some of their daughter ions so as to associate at least some of the intermediate ions with their daughter ions; and the method further comprises: identifying intermediate ions from their daughter ions.

15. A mass spectrometer arranged and configured for performing the method of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

(2) FIG. 1A shows an MS mass spectrum obtained from a sample using a conventional technique, and FIG. 1B shows a mass spectrum obtained when the sample analysed in FIG. 1A is subjected to AP-ECD and then analysed;

(3) FIG. 2A shows a mass spectrum obtained from a sample that has been subjected to conventional ETD in a vacuum, whereas FIG. 2B shows a mass spectrum obtained by a technique according to a preferred embodiment of the present invention;

(4) FIG. 3 shows a mass spectrum obtained by mass analysing a sample comprising glufibrinopeptide in accordance with a preferred embodiment of the present invention;

(5) FIG. 4 shows a mass spectrum obtained by mass analysing a sample comprising bovine insulin in accordance with a preferred embodiment of the present invention;

(6) FIG. 5 shows a first embodiment of an atmospheric pressure MALDI-atmospheric pressure ECD instrument; and

(7) FIG. 6 shows a second embodiment of an atmospheric pressure MALDI-atmospheric pressure ECD instrument.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(8) FIG. 1A shows a mass spectrum obtained by mass analysing a sample (substance-P) using a conventional technique so as to obtain MS data. FIG. 1B shows a mass spectrum obtained by subjecting the same sample to conventional AP-ECD and then mass analysing the resulting ions. The ECD conditions were provided by using a UV lamp to generate photo-electrons and allowing the photo-electrons to interact with the sample ions so as to achieve ECD.

(9) As can be seen by comparing the two spectra of FIGS. 1A and 1B, the AP-ECD process causes parent ions shown in FIG. 1A to fragment into daughter ions shown in FIG. 1B. In this example, the sample being analysed is known (substance-P) and it is possible to identify some of the daughter ions peaks. However, the spectrum of FIG. 1B includes many other peaks of unknown origin and it is not possible to know directly from the experiment which peaks are due to parent ions or fragment ions. It will be appreciated that if the sample being analysed contained mixtures of unknown substances then the data would be even more complex and even more difficult to identify parent and daughter ion peaks.

(10) FIG. 2A shows a mass spectrum obtained by subjecting a sample to conventional ETD fragmentation in a travelling wave ion guide of a quadrupole Time of Flight mass analyser (QTOF) at a pressure of 0.05 mBar and then mass analysing the resulting ions. According to this conventional technique, a precursor ion is selected using the quadrupole rod set of the QTOF. The precursor ion is then subjected to ETD fragmentation under vacuum conditions so as to dissociate the precursor ions. The resulting ions were then mass analysed in the Time of Flight mass analyser so as to obtain the spectrum shown in FIG. 2A. The nature of this conventional technique ensures that the precursor ions and their daughter ions are able to be directly correlated to each other since each precursor ion is selected and then fragmented to produce its daughter ions. However, this technique is not able to associate parent and daughter ions if the parent ions have already been subjected to the ETD or ECD conditions present in the ion source or upstream of the precursor ion selection.

(11) FIG. 2B shows a mass spectrum obtained by mass analysing a sample comprising substance-P in accordance with a preferred embodiment of the present invention. In this embodiment a mixture of precursor ions was subjected to ECD fragmentation at atmospheric pressure using a UV lamp to generate the reagent electrons. The resulting ions were then mass analysed to obtain spectral data. When precursor ions are subjected to ECD reaction conditions many of the precursor ions dissociate into fragment ions, but some of the precursor ions may not dissociate and may simply change charge state so as to form intermediate ions known as ECnoD ions. In this technique ECnoD intermediate ions were identified and then isolated from the other ions by being mass selectively transmitted through a quadrupole rod set whilst rejecting other ions. These intermediate ions were then subjected to mild CID conditions so as to induce the intermediate ions to dissociate into fragment ions. The fragment ions were then mass analysed. The spectral data obtained from this technique is shown in FIG. 2B.

(12) In the preferred embodiment, identification of the ECnoD ions was performed by searching for precursor ion mass peaks in a mass spectrum that were shifted in mass to charge ratio due to a change in their charge state. In this example, a sample containing substance-P was ionised and then mass analysed to produce first mass spectral data (shown in FIG. 1A). The triply protonated cation of substance-P was observed at a mass to charge ratio of 450 and the doubly protonated cation of substance-P was also observed in the first mass spectral data at a mass to charge ratio of 674. The parent ions were then subjected to ECD conditions at atmospheric pressure and mass spectral data was obtained (FIG. 1B). This was achieved by using a UV lamp to generate reagent electrons and allowing these electrons to interact with the parent ions. Subjecting the parent ions to ECD conditions resulted in the production of intermediate ECnoD ions, i.e. non-dissociated parent ions of reduced charge. The ions resulting from the ECD conditions were then mass analyzed to produce second mass spectral data. It was then possible to identify intermediate ECnoD ions by recognising that the triply and/or doubly protonated cations of substance-P that were observed in the first mass spectral data had been charge reduced by the ECD conditions such that the singly charged species of substance-P (having one or two electron-neutralized protons) were observed at mass to charge ratios of 1348 and 1349 in the second mass spectral data. The intermediate ions were therefore identified as having mass to charge ratios of 1348 and 1349. Once these intermediate ECnoD ions had been identified they were then isolated by transmitting the ions through a quadrupole rod set that was set to selectively transmit only these intermediate ions. Once these intermediate ions had been isolated they were then subjected to Collisionally Induced Dissociation (CID) so as to dissociate the intermediate ions into daughter ions. These daughter ions were then mass analysed so as to produce the mass spectrum shown in FIG. 2B.

(13) A comparison of FIGS. 2A and 2B shows that the daughter ions generated by the preferred embodiment shown in FIG. 2B are of similar nature to those shown in FIG. 2A. In other words, the two techniques generate similar c and/or z ions, showing that the preferred embodiment may be used to reliably identify precursor or parent ions from the daughter ions.

(14) It is to be noted that the collision energy required to promote the supplemental excitation of the intermediate ions so as to dissociate into daughter ions is significantly lower in the preferred embodiment than that which would be normally required for conventional CID fragmentation. In fact the collision energy can be set low enough to reduce the inclusion of conventional CID fragment ions. Despite this, for some samples, y-ions may be generated. It is not known whether the y-ions, which are traditionally associated with CID fragmentation, are in fact derived from the ECD process.

(15) FIG. 3 shows a mass spectrum obtained by mass analysing a sample comprising glufibrinopeptide in accordance with a preferred embodiment of the present invention. A sample containing glufibrinopeptide was ionised and then mass analysed to produce first mass spectral data. A mixture of 2+ and 3+ ions (and other ions) was detected in the first mass spectral data. The parent ions were then subjected to ECD conditions at atmospheric pressure. Subjecting the parent ions to ECD conditions resulted in the production of intermediate ECnoD ions, i.e. non-dissociated parent ions of reduced charge. The ions resulting from the ECD conditions were then mass analyzed to produce second mass spectral data. It was then possible to identify intermediate ECnoD ions by recognising that the triply and doubly protonated cations that were observed in the first mass spectral data had been charge reduced by the ECD conditions such that the signal of the singly charged cation (having one or two electron-neutralized protons) had significantly increased in the second mass spectral data. The intermediate ions were therefore identified as the ions providing the increased signal in the second mass spectral data. Once these intermediate ECnoD ions had been identified they were then isolated by transmitting the ions through a quadrupole rod set that was set to selectively transmit only these intermediate ions. Once these intermediate ions had been isolated they were then subjected to Collisionally Induced Dissociation (CID) so as to dissociate the intermediate ions into daughter ions. These daughter ions were then mass analysed so as to produce the mass spectrum shown in FIG. 3, showing the z ions.

(16) FIG. 4 shows a mass spectrum obtained by mass analysing a sample comprising bovine insulin (molecular weight 5730) in accordance with a preferred embodiment of the present invention. The sample was analysed in substantially the same manner as described above with respect to FIGS. 2B and 3. The precursor ions were subjected to ECD conditions at atmospheric pressure, resulting in precursor ions being charge reduced to 2+ so as to form intermediate ECnoD ions. The 2+ intermediate ECnoD ions were then selected by a quadrupole rod set for excitation and fragmentation by CID fragmentation. This technique resulted in high sequence coverage including N and C terminal fragmentation of the beta chain of the bovine insulin. The resulting daughter ion spectrum is shown in FIG. 4. It is important to note that the alpha and beta chains are doubly linked by disulfide bonds that are conventionally very difficult to fragment, even by conventional vacuum ECD or ETD. The preferred embodiment therefore provides an improved method for fragmenting these types of bonds.

(17) FIG. 5 shows an embodiment of an atmospheric pressure MALDI-ECD mass spectrometer. The instrument comprises a laser 2, a lens 4, a MALDI sample plate 6, and a reaction region 8. An analyte conduit 10 is provided connecting the sample plate 6 to the reaction region 10. An auxiliary gas conduit 12 feeds into the analyte conduit 10. A heater 14 is provided for heating the analyte conduit 10. A photo-ionisation lamp 16 is arranged for emitting photons into the reaction region 8. A wire mesh 18 is provided between the reaction region 8 and the analyte conduit 10.

(18) In operation, the laser 2 fires a laser beam at a first side of the MALDI sample plate 6 and ionises analyte on a second side thereof. The laser beam 2 is focussed onto the sample plate 6 by the lens 4. The analyte on the sample plate 6 is ionised by the laser beam 2 to form multiply protonated analyte ions 20 that pass into the analyte conduit 10 on the second side of the sample plate 6. The sample plate 6 may be moved in directions extending in the plane of the sample plate 6 so as to expose analyte on different areas of the sample plate 6 to the laser beam 2 and to generate ions therefrom.

(19) An auxiliary gas is flowed into the analyte conduit 10 through the auxiliary gas conduit 12. The auxiliary gas contains dopant molecules and flows from the auxiliary gas conduit 12, through the analyte conduit 10, passed the wire mesh 18 and into the reaction region 8. The gas flow carries the dopant molecules and analyte ions into the reaction region 8. The photo-ionisation lamp 16 emits photons into the reaction region 8, which cause electrons to be released from the dopant molecules. The free electrons are then captured by the analyte ions and the analyte ions are fragmented by electron capture dissociation (ECD). The gas flow carries the resulting ions downstream towards a mass analyser (not shown). The fragment ions are then ionised in the mass analyser.

(20) FIG. 6 shows another embodiment of an atmospheric pressure MALDI-ECD mass spectrometer. This embodiment is substantially the same as that of FIG. 5, except for the way in which the laser beam 2 is directed onto the sample plate 6. According to the embodiment of FIG. 6, a compound lens 30 having a hole therethrough and a mirror 32 having a hole therethrough are arranged between the sample plate 6 and the analyte conduit 10. The holes in the mirror 32 and the compound lens 30 are arranged along an axis extending from the sample plate 6 to the analyte conduit 10. The mirror 32 is substantially planar and is arranged with its reflective surface at 45 degrees to the axis.

(21) In operation, expanded laser light 2 is directed towards the mirror 32 and is reflected from the mirror 32 onto the compound lens 30. The lens 30 focuses the light onto the sample plate 6 and causes analyte thereon to be ionised. The sample plate 6 may be moved as described above in relation to FIG. 5. Analyte ions generated at the sample plate 6 travel through the holes in the lens 30 and the mirror 32 and into the analyte conduit 10. The analyte ions are then subjected to ECD reactions and the resulting fragments ions are mass analysed, as described above in relation to FIG. 5.

(22) It is also envisages that the apparatus may be used for AP-MALDI-ECD ion imaging from a sample surface on an X-Y sample stage.

(23) It is also contemplated that IR-MALDI-ECD may be performed using water as a matrix.

(24) It is also contemplated that the apparatus may be used for charge stripping (CS). atmospheric pressure MALDI-atmospheric pressure charge stripping may be used for charge stripping where a particular charge is required within the mass spectrometer, e.g. CCS studies, CID or ETD. Charge stripping may also be used to remove singly charged background ions from the MALDI matrix, thereby differentially enhancing the signal to noise of the remaining charge states having a charge greater than +1.

(25) Although the present invention has been described with reference to preferred 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.