Patent Application
20130206974
The present invention relates to a method of mass spectrometry and a mass spectrometer.
It is known to separate ions according to their ion mobility in an ion mobility spectrometer.
A mass filter may be provided upstream of an on mobility spectrometer and may be set to transmit only ions having a certain mass to charge ratio. In some circumstances two ions having slightly different ion mobilities can be partially resolved by the ion mobility spectrometer suggesting that the ions comprise two isomers having, different conformations and with the difference in ion mobility being due to the different conformations. However, conventional ion mobility separation techniques provide limited information about the different conformations and it may be desired to have both a greater degree of understanding concerning the nature of the two different conformations and also a greater degree of confidence that the observed ion peaks in an ion chromatogram do in fact represent ions having different conformations.
In other circumstances the ion mobility spectrometer may be unable to resolve ions having different conformations so that a single ion peak is observed in a resulting ion chromatogram. However, it may be desired to see whether a single ion peak in an ion chromatogram actually comprises two (or more) isomers having different conformations.
Conventional ion mobility spectrometry techniques provide only a limited amount of information and a limited degree of certainty when seeking to analyse isomeric ions.
It is desired to provide an improved method of mass spectrometry and mass spectrometer.
According to an aspect of the present invention there is provided a method of mass spectrometry comprising:
subjecting first and second analyte ions to hydrogen-deuterium exchange within a first device wherein one or more hydrogen atoms of the first and second analyte ions exchange with one or more deuterium atoms to form first and second deuterated ions;
passing the first and second deuterated ions from the first device to an ion mobility spectrometer;
mass analysing deuterated ions which emerge from the ion mobility spectrometer at a first time to produce first mass spectral data;
mass analysing deuterated ions which emerge from the ion mobility spectrometer at a second later time to produce second mass spectral data; and
comparing the first mass spectral data with the second mass spectral data to aid differentiation between either: (i) the first and second acolyte ions; and/or (ii) the first deuterated ions and the second deuterated ions.
The preferred embodiment relates to a method wherein ions are subjected to hydrogen-deuterium exchange (“HDX”) by, for example, passing the ions through a gas cell containing deuterated ammonia gas. One of more hydrogen atoms in the analyte ions are exchanged for deuterium which results in an increase in the resulting mass to charge ratio of the ions. The deuterated ions may then be passed to an ion mobility separator or spectrometer wherein the ions may be partially separated temporally or partially resolved by virtue of having slightly different drift times through the ion mobility separator or spectrometer on account of having different conformations (i.e. structural arrangements). For example, ions having a compact structure may emerge from the ion mobility spectrometer prior to ions having a more elongated structure. An ion may give rise to an isotope pattern in a final mass spectrum. The isotope pattern can be analysed or deconvoluted to determine the relative number of hydrogen atoms which have been exchanged for deuterium. As a result, further information concerning the conformational properties of two ions having similar or identical masses can be determined and a greater degree of confidence can be obtained that two ions which are believed to be isomeric ions having different conformations do in fact have different conformations. Other embodiments are contemplated wherein the ions may first be passed through an ion mobility spectrometer before then passing to a gas cell where the ions are subjected to hydrogen-deuterium exchange.
Hydrogen-deuterium exchange probes the surface of an ion and ion mobility spectrometry differentiates ions on the basis of cross-section. These two parameters are linked in that if the cross-section is different then it is highly likely the surface conformation is also different and vice versa.
The step of passing the first and second deuterated ions from the first device to the ion mobility spectrometer preferably further comprises temporally separating the first and second deuterated ions within the ion mobility spectrometer.
According to an aspect of the present invention there is provided a method of mass spectrometry comprising:
passing first and second analyte ions to an ion mobility spectrometer;
subjecting the first and second analyte ions which emerge from the ion mobility spectrometer to hydrogen-deuterium exchange within a first device wherein one or more hydrogen atoms of the first and second analyte ions exchange with one or more deuterium atoms to form first and second deuterated ions;
mass analysing deuterated ions which emerge from the first device at a first time to produce first mass spectral data;
mass analysing deuterated ions which emerge from the first device at a second later time to produce second mass spectral data; and
comparing the first mass spectral data with the second mass spectral data to aid differentiation between either: (i) the first and second analyte ions; and/or (ii) the first deuterated ions and the second deuterated ions.
The step of passing the first and second analyte ions to the ion mobility spectrometer preferably further comprises temporally separating the first and second analyte ions within the ion mobility spectrometer.
The preferred embodiment relates to methods of improved differentiation and determination of ionic conformations by combining hydrogen-deuterium exchange reactions with ion mobility separation techniques.
The step of comparing the first mass spectral data with the second mass spectral data to aid differentiation preferably comprises determining differences in structural or conformational properties or reactiveness with a gas in the first device between either: (i) the first and second analyte ions; and/or (ii) the first deuterated ions and the second deuterated ions.
The step of comparing the first mass spectral data with the second mass spectral data preferably comprises:
(i) determining or approximating the degree to which hydrogen atoms in the first and/or second analyte ions are exchanged for deuterium atoms; and/or
(ii) determining or approximating structural or conformational properties of the first and/or second analyte ions; and/or
(iii) determining or approximating structural or conformational properties of the first and/or second deuterated ions; and/or
(iv) determining or approximating the relative compactness or elongation of the first and/or second analyte ions; and/or
(v) determining or approximating the relative compactness or elongation of the first and/or second deuterated ions; and/or
(vi) determining or approximating the degree to which the first and/or second analyte ions react with the gas in the first device to form adduct ions; and/or
(vii) determining or approximating the number of surface sites on the first and/or second analyte ions at which hydrogen atoms may exchange for deuterium atoms; and/or
(viii) comparing one or more isotope patterns and/or one or more isotope distributions and/or one or more isotope ratios related to the first deuterated ions with one or more isotope patterns and/or one or more isotope distributions and/or one or more isotope ratios related to the second deuterated ions.
The method preferably further comprises providing a mass filter upstream of the ion mobility spectrometer and/or the first device, and operating the mass filter to selectively transmit the first and second analyte ions having substantially the same mass to charge ratio and to filter out or attenuate other ions having different mass to charge ratios.
According to an aspect of the present invention there is provided a mass spectrometer comprising:
a first device arranged and adapted to subject first and second analyte ions to hydrogen-deuterium exchange wherein one or more hydrogen atoms of the first and second analyte ions exchange with one or more deuterium atoms to form first and second deuterated ions;
an ion mobility spectrometer, wherein the first and second deuterated ions are passed, in use, from the first device to the ion mobility spectrometer;
a mass analyser arranged and adapted to mass analyse deuterated ions which emerge from the ion mobility spectrometer at a first time to produce first mass spectral data and to mass analyse deuterated ions which emerge from the on mobility spectrometer at a second later time to produce second mass spectral data; and
a control system arranged and adapted to compare the first mass spectral data with the second mass spectral data to aid differentiation between either: (i) the first and second analyte ions; and/or (ii) the first deuterated ions and the second deuterated ions.
According to an aspect of the present invention there is provided a mass spectrometer comprising:
an ion mobility spectrometer, wherein first and second analyte ions are passed to the ion mobility spectrometer;
a first device arranged and adapted to subject the first and second analyte ions which emerge from the ion mobility spectrometer to hydrogen-deuterium exchange within the first device wherein one or more hydrogen atoms of the first and second analyte ions exchange with one or more deuterium atoms to form first and second deuterated ions;
a mass analyser arranged and adapted to mass analyse deuterated ions which emerge from the first device at a first time to produce first mass spectral data and to mass analyse deuterated ions which emerge from the first device at a second later time to produce second mass spectral data; and
a control system arranged and adapted to compare the first mass spectral data with the second mass spectral data to aid differentiation between either: (i) the first and second analyte ions; and/or (ii) the first deuterated ions and the second deuterated ions.
The control system is preferably arranged and adapted to determine differences in structural or conformational properties or reactiveness with a gas in the first device between either: (i) the first and second analyte ions; and/or (ii) the first deuterated ions and the second deuterated ions.
The control system is preferably arranged and adapted to:
(i) determine or approximate the degree to which hydrogen atoms in the first and/or second analyte ions are exchanged for deuterium atoms; and/or
(ii) determine or approximate structural or conformational properties of the first and/or second analyte ions; and/or
(iii) determine or approximate structural or conformational properties of the first and/or second deuterated ions; and/or
(iv) determine or approximate the relative compactness or elongation of the first and/or second analyte ions; and/or
(v) determine or approximate the relative compactness or elongation of the first and/or second deuterated ions; and/or
(vi) determine or approximate the degree to which the first and/or second analyte ions react with the gas in the first device to form adduct ions; and/or
(vii) determine or approximate the number of surface sites on the first and/or second analyte ions at which hydrogen atoms may exchange for deuterium atoms; and/or
(viii) compare one or more isotope patterns and/or one or more isotope distributions and/or one or more isotope ratios related to the first deuterated ions with one or more isotope patterns and/or one or more isotope distributions and/or one or more isotope ratios related to the second deuterated ions.
The mass analyser preferably comprises a Time of Flight mass analyser.
The mass spectrometer preferably further comprises a mass filter arranged upstream of the ion mobility spectrometer and/or the first device, wherein in a mode of operation the mass filter is arranged and adapted to selectively transmit the first and second analyte ions having substantially the same mass to charge ratio and to filter out or attenuate other ions having different mass to charge ratios.
According to an aspect of the present invention there is provided a method of mass spectrometry comprising:
subjecting analyte ions to hydrogen-deuterium exchange within a first device wherein one or more hydrogen atoms of the analyte ions exchange with one or more deuterium atoms to form deuterated ions;
passing the deuterated ions from the first device to an ion mobility spectrometer;
mass analysing first deuterated ions which emerge from the ion mobility spectrometer at a first time to produce first mass spectral data;
deconvoluting one or more isotope ratio patterns in the first mass spectral data to determine or approximate the number of hydrogen atoms which have been exchanged for deuterium atoms in the first deuterated ions;
mass analysing second deuterated ions which emerge from the ion mobility spectrometer at a second later time to produce second mass spectral data; and
deconvoluting one or more isotope ratio patterns in the second mass spectral data to determine or approximate the number of hydrogen atoms which have been exchanged for deuterium atoms in the second deuterated ions.
The method may further comprise comparing the number of hydrogen atoms which have been determined to have been exchanged for deuterium atoms on the first deuterated ions with the number of hydrogen atoms which have been determined to have been exchanged for deuterium atoms on the second deuterated ions.
According to an aspect of the present invention there is provided a mass spectrometer comprising:
a first device arranged and adapted to subject analyte ions to hydrogen-deuterium exchange wherein one or more hydrogen atoms of the analyte ions exchange with one or more deuterium atoms to form deuterated ions;
an ion mobility spectrometer, wherein the deuterated ions are passed, in use, from the first device to the ion mobility spectrometer;
a control system and mass analyser arranged and adapted:
(i) to mass analyse first deuterated ions which emerge from the ion mobility spectrometer at a first time to produce first mass spectral data;
(ii) to deconvolute one or more isotope ratio patterns in the first mass spectral data to determine or approximate the number of hydrogen atoms which have been exchanged for deuterium atoms in the first deuterated ions;
(iii) to mass analyse second deuterated ions which emerge from the on mobility spectrometer at a second later time to produce second mass spectral data; and
(iv) to deconvolute one or more isotope ratio patterns in the second mass spectral data to determine or approximate the number of hydrogen atoms which have been exchanged for deuterium atoms in the second deuterated ions.
The control system may be arranged and adapted to compare the number of hydrogen atoms which have been determined to have been exchanged for deuterium atoms on the first deuterated ions with the number of hydrogen atoms which have been determined to have been exchanged for deuterium, atoms on the second deuterated ions.
According to an aspect of the present invention there is provided a method of mass spectrometry comprising:
subjecting analyte ions to hydrogen-deuterium exchange within a first device wherein one or more hydrogen atoms of the analyte ions exchange with one or more deuterium atoms to form deuterated ions;
mass analysing the deuterated ions to produce mass spectral data; and
deconvoluting one or more isotope ratio patterns in the mass spectral data to determine or approximate the number of hydrogen atoms in the analyte ions which have been exchanged for deuterium atoms.
According to an aspect of the present invention there is provided a mass spectrometer comprising:
a first device arranged and adapted to subject analyte ions to hydrogen-deuterium exchange wherein one or more hydrogen atoms of the analyte ions exchange with one or more deuterium atoms to form deuterated ions;
a mass analyser arranged and adapted to mass analyse the deuterated ions to produce mass spectral data; and
a control system arranged and adapted to deconvolute one or more isotope ratio patterns in the mass spectral data to determine or approximate the number of hydrogen atoms in the analyte ions which have been exchanged for deuterium atoms.
According to the preferred embodiment a method of significantly enhancing the differentiation of ionic conformations within a mass spectrometer equipped with an ion mobility drift cell device is provided. The preferred embodiment relates to improvements in the determination of ion mobility derived cross sectional areas by invoking hydrogen-deuterium exchange reactions. By measuring the differences in mass spectral modifications associated with hydrogen-deuterium exchange labelling either before or after an ion mobility device, ions with very similar cross-sections and identical mass to charge ratios can be differentiated and hence more accurately measured. Complementary methods are disclosed involving pattern matching of deuterated isotope clusters.
The preferred embodiment represents a significant improvement in the art.
According to an embodiment the mass spectrometer preferably further comprises:
(a) an ion source selected from the group consisting of: (i) an Electrospray ionisation (“ESI”) ion source; (ii) an Atmospheric Pressure Photo Ionisation (“APPI”) ion source; (iii) an Atmospheric Pressure Chemical Ionisation (“APCI”) ion source; (iv) a Matrix Assisted Laser Desorption Ionisation (“MALDI”) ion source; (v) a Laser Desorption Ionisation (“LDI”) ion source; (vi) an Atmospheric Pressure Ionisation (“API”) ion source; (vii) a Desorption Ionisation on Silicon (“DIOS”) ion source; (viii) an Electron Impact (“EI”) ion source; (ix) a Chemical Ionisation (“CI”) ion source; (x) a Field Ionisation (“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”) on source; (xv) a Desorption Electrospray Ionisation (“DESI”) ion source; (xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption Ionisation ion source; (xviii) a Thermospray ion source: (xix) an Atmospheric Sampling Glow Discharge Ionisation (“ASGDI”) ion source; and (xx) a Glow Discharge (“GD”) 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 trapping regions; and/or
(f) one or more collision, fragmentation or reaction cells selected from the group consisting of: (i) a Collisional Induced Dissociation (“CID”) fragmentation device; (ii) a Surface Induced Dissociation (“SID”) fragmentation device; (iii) an Electron Transfer Dissociation (“ETD”) fragmentation device; (iv) an Electron Capture Dissociation (“ECD”) fragmentation device; (v) an Electron Collision or 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) an 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 on 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 adduct or product ions; (xxiv) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxv) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxvi) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxvii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; (xxviii) an ion-metastable atom reaction device for reacting ions to form adduct or product ions; and (xxix) an Electron Ionisation Dissociation (“EID”) fragmentation device; and/or
(g) a mass analyser selected from the group consisting of (i) a quadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron Resonance (“ICR”) mass analyser; (viii) a Fourier Transform Ion Cyclotron Resonance (“FTICR”) mass analyser; (ix) an electrostatic or orbitrap mass analyser; (x) a Fourier Transform electrostatic or orbitrap mass analyser; (xi) a Fourier Transform mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonal acceleration Time of Flight mass analyser; and (xiv) a linear acceleration Time of Flight mass analyser; and/or
(h) one or more energy analysers or electrostatic energy analysers; 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 2D or linear quadrupole ion trap, (iii) a Paul or 3D quadrupole ion trap, (iv) a Penning ion trap; (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a Time of Flight mass filter, and (viii) a Wein filter; and/or
(k) a device or ion gate for pulsing ions; and/or
(l) a device for converting a substantially continuous ion beam into a pulsed ion beam
The mass spectrometer preferably further comprises either:
(i) a C-trap and an Orbitrap® mass analyser comprising an outer barrel-like electrode and a coaxial inner spindle-like electrode, wherein in a first mode of operation ions are transmitted to the C-trap and are then injected into the Orbitrap® mass analyser and wherein in a second mode of operation ions are transmitted to the C-trap and then to a collision cell or Electron Transfer Dissociation device wherein at least some ions are fragmented into fragment ions, and wherein the fragment ions are then transmitted to the C-trap before being injected into the Orbitrap® mass analyser; and/or
(ii) a stacked ring ion guide comprising a plurality of electrodes each having an aperture through which ions are transmitted in use and wherein the spacing of the electrodes increases along the length of the ion path, and wherein the apertures in the electrodes in an upstream section of the ion guide have a first diameter and wherein the apertures in the electrodes in a downstream section of the ion guide have a second diameter which is smaller than the first diameter, and wherein opposite phases of an AC or RF voltage are applied, in use, to successive electrodes.
The ion mobility spectrometer according to the preferred embodiment may comprise a plurality of electrodes each having an aperture through which ions are transmitted in use. One or more transient DC voltages or potentials or one or more DC voltage or potential waveforms are preferably applied to the electrodes comprising the ion mobility spectrometer in order to urge ions along the length of the ion mobility spectrometer.
According to the preferred embodiment the one or more transient DC voltages or potentials or the one or more DC voltage or potential waveforms create: (i) a potential hill or barrier; (ii) a potential well; (iii) multiple potential hills or barriers; (iv) multiple potential wells; (v) a combination of a potential hill or barrier and a potential well; or (vi) a combination of multiple potential hills or barriers and multiple potential wells.
The one or more transient DC voltage or potential waveforms preferably comprise a repeating waveform or square wave.
An RF voltage is preferably applied to the electrodes of the ion mobility spectrometer and preferably has an amplitude selected from the group consisting of: (i)<50 V peak to peak; (ii) 50-100 V peak to peak; (iii) 100-150 V peak to peak; (iv) 150-200 V peak to peak, (v) 200-250 V peak to peak; (vi) 250-300 V peak to peak; (vii) 300-350 V peak to peak; (viii) 350-400 V peak to peak; (ix) 400-450 V peak to peak; (x) 450-500 V peak to peak; (xi) 500-550 V peak to peak; (xxii) 550-600 V peak to peak; (xxiii) 600-650 V peak to peak; (xxiv) 650-700 V peak to peak; (xxv) 700-750 V peak to peak; (xxvi) 750-800 V peak to peak; (xxvii) 800-850 V peak to peak; (xxviii) 850-900 V peak to peak; (xxix) 900-950 V peak to peak; (xxx) 950-1000 V peak to peak; and (xxxi)>1000 V peak to peak.
The RF voltage preferably have a frequency selected from the group consisting of (i)<100 kHz; (ii) 100-200 kHz; (iii) 200-300 kHz; (iv) 300-400 kHz; (v) 400-500 kHz; (vi) 0.5-1.0 MHz; (vii) 1.0-1.5 MHz; (viii) 1.5-2.0 MHz; (ix) 2.0-2.5 MHz; (x) 2.5-3.0 MHz; (xi) 3.0-3.5 MHz; (xii) 3.5-4.0 MHz; (xiii) 4.0-4.5 MHz; (xiv) 4.5-5.0 MHz; (xv) 5.0-5.5 MHz; (xvi) 5.5-6.0 MHz; (xvii) 6.0-6.5 MHz; (xviii) 6.5-7.0 MHz; (xix) 7.0-7.5 MHz, (xx) 7.5-8.0 MHz; (xxi) 8.0-8.5 MHz; (xxii) 8.5-9.0 MHz; (xxiii) 9.0-9.5 MHz; (xxiv) 9.5-10.0 MHz; and (xxv) >10.0 MHz.
The ion mobility spectrometer is preferably maintained at a pressure selected from the group comprising: (i)>0.001 mbar; (ii)>0.01 mbar; (iii)>0.1 mbar; (iv)>1 mbar; (v)>10 mbar; (vi)>100 mbar; (vii) 0.001-0.01 mbar; (viii) 0.01-0.1 mbar; (ix) 0.1-1 mbar; (x) 1-10 mbar; and (xi) 10-100 mbar.
Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:
A preferred embodiment of the present invention will now be described. According to the preferred embodiment a mass spectrometer comprising a modified Waters Synapt® hybrid quadrupole Time of Flight mass spectrometer is provided.
The mass spectrometer comprises a first (inlet) vacuum chamber which is followed by a second vacuum chamber housing a travelling wave ion guide. Ions pass from the first vacuum chamber into the second vacuum chamber. The axis of the second vacuum chamber is preferably inclined at 90° to the axis of the first vacuum chamber.
The travelling wave ion guide arranged in the second vacuum chamber preferably comprises a plurality of ring electrodes each having an aperture through which ions are transmitted. Ions are confined radially within the travelling wave ion guide by applying opposite phases of an RF voltage to alternate electrodes thereby generating a radial pseudo-potential barrier which acts to confine ions radially within the ion guide. Ions which are transmitted through the ion guide in the second vacuum chamber are then passed to a third vacuum chamber which is located downstream of the second vacuum chamber.
The third vacuum chamber preferably houses a quadrupole rod set mass filter (MS1). In a mode of operation parent ions of interest may be selected by the quadrupole rod set mass filter MS1 and are passed onwards to a fourth vacuum chamber located downstream of the third vacuum chamber. Ions having mass to charge ratios which are not of interest are preferably filtered out or are otherwise attenuated by the mass filter MS1.
The fourth vacuum chamber downstream of the third vacuum chamber preferably comprises an ion trap gas cell, an ion mobility spectrometer (“IMS”) and a transfer section.
According to the preferred embodiment analyte ions of interest are preferably selectively transmitted by the quadrupole rod set mass filter MS1 and are then preferably subjected to hydrogen-deuterium exchange CHDX″) reactions within the ion trap gas cell in the fourth vacuum chamber.
Deuterated ammonia is preferably admitted into the ion trap gas cell located in the fourth vacuum chamber. As a result, analyte ions which are transmitted into the ion trap gas cell are preferably caused to be subjected to hydrogen-deuterium exchange reactions. The hydrogen-deuterium exchange reactions preferably occur on or at the surface of the analyte ions as the analyte ions pass through the ion trap gas cell.
Analyte ions which are modified by hydrogen-deuterium exchange reactions are shifted to higher masses or mass to charge ratios depending upon the surface conformation of the analyte ions and the number of sites on the surface of the analyte ion which are available for hydrogen-deuterium exchange.
Deuterated analyte ions which emerge from the ion trap gas cell are then preferably passed to the ion mobility spectrometer (“IMS”) which is arranged to separate the deuterated ions on the basis of their ion mobility. The deuterated ions which emerge from the ion mobility spectrometer then preferably pass through the transfer section and are onwardly transmitted to a Time of Flight mass analyser (MS2) located in a fifth vacuum chamber arranged downstream of the fourth vacuum chamber.
According to a less preferred embodiment the ion mobility spectrometer may be arranged upstream of the ion trap gas cell and analyte ions may first be separated temporally according to their ion mobility before being subjected to hydrogen-deuterium exchange in the ion trap gas cell.
An advantageous aspect of the preferred embodiment is that if two ions have substantially similar ion mobilities then the ions can be differentiated more effectively, if they have different surface conformations, by determining the relative degree to which the ions are susceptible to hydrogen-deuterium exchange reactions.
In order to illustrate aspects of the preferred embodiment experimental data was generated using a modified Waters Synapt® hybrid quadrupole Time of Flight mass spectrometer as described above. The experimental data will be discussed in more detail below.
Substance-P was ionised by a nanospray ion source and triply charged precursor or parent ions having a mass to charge ratio of 450 were selected by the quadrupole rod set mass filter. MS1 arranged in the third vacuum chamber.
The 3+ parent ions of substance P having a mass to charge ratio of 450 which were selected by the quadrupole rod set mass filter MS1 were then passed to the ion trap gas cell located in the fourth vacuum chamber. The 3+ parent ions were then subjected to hydrogen-deuterium exchange reactions with deuterated ammonia reagent gas within the ion trap gas cell. The deuterated ammonia gas was supplied to the ion trap gas cell. The ion trap gas cell was pressurised to around 3×10−2 mbar with helium. An additional gas inlet needle valve was connected to the ion trap gas cell and was used to introduce deuterated ammonia into the ion trap gas cell causing the pressure in the ion trap gas cell to increase from 3×10−2 mbar to 3.5×10−2 mbar.
The parent ions which had been subjected to hydrogen-deuterium exchange within the ion trap gas cell were then passed to the ion mobility spectrometer where the ions were caused to separate temporally according to their ion mobility. Ions which eluted from the ion mobility spectrometer were then mass analysed by the Time of Flight mass analyser MS2.
It was evident from measuring the ion mobility or drift times of the parent ions which had been subjected to hydrogen-deuterium exchange and which were separated according to their ion mobility by the ion mobility spectrometer that the 3+ parent ions of substance P comprise a mixture of ions having two different cross sections or conformations. Two distinct arrival time distributions are observed as eluting from the ion mobility spectrometer.
Admitting deuterated ammonia (ND3) into the ion trap gas cell of the mass spectrometer enabled hydrogen atoms in or on the exposed surface of the 3+ parent ions of substance P to be exchanged for deuterium.
It can be seen from comparing
As will be discussed in more detail below,
When deuterated ammonia was added to the ion trap gas cell, the mass to charge ratio of the analyte ions which were subjected to hydrogen-deuterium exchange were observed to increase. The deuterated ions were separated according to their ion mobility in the ion mobility spectrometer and mass spectra of the deuterated ions eluting from the ion mobility spectrometer were obtained and analysed. In particular, the isotope pattern of the deuterated ions in a resulting mass spectrum were deconvoluted to determine the relative exchange for the different sites of deuteration exposed on the surface of the analyte ions. According to the preferred embodiment deconvolution of the isotope pattern can be used to determine the number of surface sites for hydrogen-deuterium exchange which the ions possess.
It is evident from comparing
The data shown in
By plotting ion mobility drift time chromatograms of isotopes associated with specific hydrogen-deuterium exchange shifts the differing signal profiles enable the differentiation of isomeric species with very similar cross sections.
Furthermore, plotting an ion mobility chromatogram for deuterated substance-P having a mass to charge ratio of 452 and overlapping an ion mobility chromatogram relating to deuterated adduct ions (
Although the experimental data presented above relates to substance-P where it is possible to observe differing ion mobility separator peaks without requiring hydrogen-deuterium exchange, isomeric ions having different conformations and almost identical ion mobility drift times can be deconvoluted and more accurately determined according to the preferred embodiment of the present invention.
Although the present invention has been described with reference to the preferred embodiment, 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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1002445.3 | Feb 2010 | GB | national |
This application claims priority from and the benefit of U.S. Provisional Patent Application Ser. No. 61/307,882 filed on 25 Feb. 2010 and United Kingdom Patent Application No. 1002445.3 filed on 12 Feb. 2010. The entire contents of these applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB11/50273 | 2/14/2011 | WO | 00 | 12/14/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61307882 | Feb 2010 | US |
