GB2576786A

GB2576786A - Method of characterising ions

Info

Publication number: GB2576786A
Application number: GB1814278.6A
Authority: GB
Inventors: Tomczyk Nick; Giles Kevin; Ujma Jakub
Original assignee: Micromass UK Ltd
Current assignee: Micromass UK Ltd
Priority date: 2018-09-03
Filing date: 2018-09-03
Publication date: 2020-03-04
Also published as: GB201814278D0

Abstract

A method of characterising ions is disclosed. The method comprises comparing an ion mobility value of first product ions to an ion mobility value of second product ions, and identifying and/or determining a location of a difference between the first and second ions based on the comparison between the ion mobility values of the first and second product ions. The first product ions are produced by fragmenting or reacting first ions, the second product ions are produced by fragmenting or reacting second ions, and the first and second ions are isomeric or isobaric.

Description

METHOD OF CHARACTERISING IONS

CROSS-REFERENCE TO RELATED APPLICATION

None.

FIELD OF THE INVENTION

The present invention provides a method of mass and/or ion mobility spectrometry, and in particular a method for characterising modifications to chemical species.

BACKGROUND

The development of chemical species as drugs, pesticides or other species of commercial interest is a time consuming and expensive process. A wide range of analytical tools are used to characterise and understand the production, storage and variation of such species both prior to release and during production. Examples of such tools include, e.g., NMR techniques, production of model compounds, exhaustive chemical synthesis, etc.

Changes to chemical species that involve addition or subtraction to the molecular weight of the species can be ascertained on the basis of mass-to-charge ratio (m/z).

The Applicants believe that there remains scope for improvements to methods of mass spectrometry.

SUMMARY

According to an aspect, there is provided a method of characterising ions, the method comprising:

comparing an ion mobility value of first product ions to an ion mobility value of second product ions, the first product ions being produced by fragmenting or

-2reacting first ions, the second product ions being produced by fragmenting or reacting second ions, the first and second ions being isomeric or isobaric; and identifying and/or determining a location of a difference between the first and second ions based on the comparison between the ion mobility values of the first and second product ions.

According to various embodiments, at least one difference, such as an isomeric or isobaric modification, between first and second isomeric or isobaric ions can be identified and/or the location (site) of the difference(s) can be determined (located). According to various embodiments, this is done by comparing ion mobility values (e.g. drift times) of product (fragment) ions derived from the first and second isomeric or isobaric ions.

As will be described in more detail below, by comparing the ion mobility of product ions in the manner of various embodiments, additional structural information concerning the first and second isomeric or isobaric ions can be obtained that would otherwise be unobtainable.

In particular, various embodiments allow a difference between first and second isomeric or isobaric ions to be identified and/or the location (site) of the difference to be determined where the difference between the first and second ions comprises a difference such that the product (fragment) ions derived from the first ions have substantially the same mass to charge ratios as the product (fragment) ions derived from the second ions. This may be the case, for example, where the difference between the first and second ions is such that one or more particular “first” product (fragment) ions derived from the first ions and one or more particular “second” product (fragment) ions derived from the second ions are isomeric or isobaric (but where other product (fragment) ions derived from the first ions are the same as (have the same structure(s) as) other product (fragment) ions derived from the second ions).

It will be appreciated, therefore, that the various embodiments provide an improved method of mass and/or ion mobility spectrometry.

According to an aspect, there is provided a method of mass spectrometry and/or ion mobility spectrometry, the method comprising:

fragmenting or reacting first ions to produce first product ions and fragmenting or reacting second ions to produce second product ions, wherein the first and second ions are isomeric or isobaric;

-3determining an ion mobility value of the first product ions and an ion mobility value of the second product ions;

comparing the ion mobility value of the first product ions to the ion mobility value of the second product ions; and identifying and/or determining a location of a difference between the first and second ions based on the comparison between the ion mobility values of the first and second product ions.

The first product ions and the second product ions may be isomeric or isobaric.

The difference between the first and second ions may be identified and/or located as comprising a difference between the first and second product ions when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

The method may comprise determining a mass to charge ratio value of the first product ions and a mass to charge ratio value of the second product ions.

The method may comprise comparing a mass to charge ratio value of the first product ions to a mass to charge ratio value of the second product ions; and identifying and/or determining the location of the difference between the first and second ions based on the comparisons between the ion mobility values and the mass to charge ratio values of the first and second product ions.

The difference between the first and second ions may be identified and/or located as comprising a difference between the first and second product ions when the mass to charge ratio value of the first product ions is equal to or sufficiently similar to the mass to charge ratio value of the second product ions, and when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

The method may comprise determining that the first and second product ions are isomeric or isobaric when the mass to charge ratio value of the first product ions is equal to or sufficiently similar to the mass to charge ratio value of the second product ions, and when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

The method may comprise determining a mass to charge ratio value of the first ions and a mass to charge ratio value of the second ions.

The method may comprise determining an ion mobility value of the first ions and an ion mobility value of the second ions.

-4The method may comprise comparing a mass to charge ratio value of the first ions to a mass to charge ratio value of the second ions, and comparing an ion mobility value of the first ions to an ion mobility value of the second ions; and determining that the first and second ions are isomeric or isobaric based on the comparisons between the mass to charge ratio values and the ion mobility values of the first and second ions.

The first and second ions may be determined as being isomeric or isobaric when the mass to charge ratio value of the first ions is equal to or sufficiently similar to the mass to charge ratio value of the second ions, and when the ion mobility value of the first ions is different to or sufficiently different to the ion mobility value of the second ions.

The method may comprise determining a mass to charge ratio value of third ions, wherein the first, second and third ions are isomeric or isobaric.

The method may comprise determining an ion mobility value of the third ions.

The method may comprise comparing a mass to charge ratio value of the third ions to the mass to charge ratio value of the first and/or second ions, and comparing an ion mobility value of the third ions to the ion mobility value of the first and/or second ions; and determining that the first, second and third ions are isomeric or isobaric based on the comparisons between the mass to charge ratio values and the ion mobility values of the third and first and/or second ions.

The first, second and third ions may be determined as being isomeric or isobaric when the mass to charge ratio value of the third ions is equal to or sufficiently similar to the mass to charge ratio value of the first and/or second ions, and when the ion mobility value of the third ions is different to or sufficiently different to the ion mobility value of the first and/or second ions.

The method may comprise fragmenting or reacting the first ions to produce the first product ions and fourth product ions and fragmenting or reacting the second ions to produce the second product ions and fifth product ions.

The method may comprise determining an ion mobility value of fourth product ions and an ion mobility value of fifth product ions, the first and fourth product ions being produced by fragmenting or reacting the first ions, the second and fifth product ions being produced by fragmenting or reacting the second ions;

-5comparing the ion mobility value of the fourth product ions to the ion mobility value of the fifth product ions; and identifying and/or determining the location of the difference between the first and second ions based on the comparison between the ion mobility values of the fourth and fifth product ions.

The fourth product ions and the fifth product ions may be the same.

The difference between the first and second ions may be identified and/or located as not comprising a difference between the fourth and fifth product ions when the ion mobility value of the fourth product ions is the same as or sufficiently similar to the ion mobility value of the fifth product ions.

The method may comprise determining a mass to charge ratio value the fourth product ions and a mass to charge ratio value of the fifth product ions.

The method may comprise comparing a mass to charge ratio value of the fourth product ions to a mass to charge ratio value of the fifth product ions; and identifying and/or determining the location of the difference between the first and second ions based on the comparisons between the ion mobility values and the mass to charge ratio values of the fourth and fifth product ions.

The difference between the first and second ions may be identified and/or located as not comprising a difference between the fourth and fifth product ions when the mass to charge ratio value of the fourth product ions is equal to or sufficiently similar to the mass to charge ratio value of the fifth product ions, and when the ion mobility value of the fourth product ions is equal to or sufficiently similar to the ion mobility value of the fifth product ions.

The method may comprise determining that the fourth and fifth product ions are the same or indistinguishable when the mass to charge ratio value of the fourth product ions is equal to or sufficiently similar to the mass to charge ratio value of the fifth product ions, and when the ion mobility value of the fourth product ions is equal to or sufficiently similar to the ion mobility value of the fifth product ions.

The method may comprise fragmenting or reacting third ions to produce sixth product ions, wherein the first, second and third ions may be isomeric or isobaric.

The method may comprise determining a mass to charge ratio value of the sixth product ions.

The method may comprise comparing a mass to charge ratio value of sixth product ions to the mass to charge ratio value of the first and/or second product

-δίοπε, the sixth product ions being produced by fragmenting or reacting third ions, the first, second and third ions being isomeric or isobaric; and identifying and/or determining a location of a difference between the third ions and the first and/or second ions based on the comparison between the mass to charge ratio values of the sixth and first and/or second product ions.

The first ions may be known ions having a known structure, and the location of the difference between the first and second ions may be identified and/or located using the known structure of the first ions.

According to an aspect there is provided a computer readable medium storing software code, which when executing on a processor, causes the processor to perform the method described above.

According to an aspect, there is provided an analytical instrument such as a mass and/or ion mobility spectrometer, comprising:

one or more collision, fragmentation or reaction devices;

one or more mass and/or ion mobility analysers; and a control system, wherein the control system is configured:

to cause the one or more collision, fragmentation or reaction devices to fragment or react first ions to produce first product ions and to fragment or react second ions to produce second product ions, wherein the first and second ions are isomeric or isobaric;

to cause the one or more mass and/or ion mobility analysers to determine an ion mobility value of the first product ions and an ion mobility value of the second product ions;

to compare the ion mobility value of the first product ions to the ion mobility value of the second product ions; and to identify and/or determine a location of a difference between the first and second ions based on the comparison between the ion mobility values of the first and second product ions.

The first product ions and the second product ions may be isomeric or isobaric.

The control system may be configured to identify and/or determine the location of a difference between the first and second ions may as comprising a difference between the first and second product ions when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

-7The control system may be configured to cause one or more mass and/or ion mobility analysers to determine a mass to charge ratio value of the first product ions and a mass to charge ratio value of the second product ions.

The control system may be configured to compare a mass to charge ratio value of the first product ions to a mass to charge ratio value of the second product ions.

The control system may be configured to identify and/or determine the location of the difference between the first and second ions based on the comparisons between the ion mobility values and the mass to charge ratio values of the first and second product ions.

The control system may be configured to identify and/or determine the difference between the first and second ions as comprising a difference between the first and second product ions when the mass to charge ratio value of the first product ions is equal to or sufficiently similar to the mass to charge ratio value of the second product ions, and when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

The control system may be configured to determine that the first and second product ions are isomeric or isobaric when the mass to charge ratio value of the first product ions is equal to or sufficiently similar to the mass to charge ratio value of the second product ions, and when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine a mass to charge ratio value of the first ions and a mass to charge ratio value of the second ions.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine an ion mobility value of the first ions and an ion mobility value of the second ions.

The control system may be configured to compare a mass to charge ratio value of the first ions to a mass to charge ratio value of the second ions, and compare an ion mobility value of the first ions to an ion mobility value of the second ions.

-8The control system may be configured to determine that the first and second ions are isomeric or isobaric based on the comparisons between the mass to charge ratio values and the ion mobility values of the first and second ions.

The control system may be configured to determine the first and second ions as being isomeric or isobaric when the mass to charge ratio value of the first ions is equal to or sufficiently similar to the mass to charge ratio value of the second ions, and when the ion mobility value of the first ions is different to or sufficiently different to the ion mobility value of the second ions.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine a mass to charge ratio value of third ions, wherein the first, second and third ions are isomeric or isobaric.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine an ion mobility value of the third ions.

The control system may be configured to compare a mass to charge ratio value of the third ions to the mass to charge ratio value of the first and/or second ions, and to compare an ion mobility value of the third ions to the ion mobility value of the first and/or second ions.

The control system may be configured to determine that the first, second and third ions are isomeric or isobaric based on the comparisons between the mass to charge ratio values and the ion mobility values of the third and first and/or second ions.

The control system may be configured to determine that the first, second and third ions are isomeric or isobaric when the mass to charge ratio value of the third ions is equal to or sufficiently similar to the mass to charge ratio value of the first and/or second ions, and when the ion mobility value of the third ions is different to or sufficiently different to the ion mobility value of the first and/or second ions.

The control system may be configured to cause the one or more collision, fragmentation or reaction devices to fragment or react the first ions to produce the first product ions and fourth product ions and to fragment or react the second ions to produce the second product ions and fifth product ions.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine an ion mobility value of fourth product ions and an ion mobility value of fifth product ions.

The control system may be configured to compare the ion mobility value of the fourth product ions to the ion mobility value of the fifth product ions.

-9The control system may be configured to identify and/or determine the location of the difference between the first and second ions based on the comparison between the ion mobility values of the fourth and fifth product ions.

The fourth product ions and the fifth product ions may have the same structure.

The control system may be configured to identify and/or determine the difference between the first and second ions as not comprising a difference between the fourth and fifth product ions when the ion mobility value of the fourth product ions is the same as or sufficiently similar to the ion mobility value of the fifth product ions.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine a mass to charge ratio value the fourth product ions and a mass to charge ratio value of the fifth product ions.

The control system may be configured to compare a mass to charge ratio value of the fourth product ions to a mass to charge ratio value of the fifth product ions.

The control system may be configured to identify and/or determine the location of the difference between the first and second ions based on the comparisons between the ion mobility values and the mass to charge ratio values of the fourth and fifth product ions.

The control system may be configured to identify and/or determine the difference between the first and second ions as not comprising a difference between the fourth and fifth product ions when the mass to charge ratio value of the fourth product ions is equal to or sufficiently similar to the mass to charge ratio value of the fifth product ions, and when the ion mobility value of the fourth product ions is equal to or sufficiently similar to the ion mobility value of the fifth product ions.

The control system may be configured to determine that the fourth and fifth product ions are the same or indistinguishable when the mass to charge ratio value of the fourth product ions is equal to or sufficiently similar to the mass to charge ratio value of the fifth product ions, and when the ion mobility value of the fourth product ions is equal to or sufficiently similar to the ion mobility value of the fifth product ions.

- 10The control system may be configured to cause the one or more collision, fragmentation or reaction devices to fragment or react third ions to produce sixth product ions, wherein the first, second and third ions may be isomeric or isobaric.

The control system may be configured to cause one or more mass and/or ion mobility analysers to determine a mass to charge ratio value of the sixth product ions.

The control system may be configured to compare a mass to charge ratio value of sixth product ions to the mass to charge ratio value of the first and/or second product ions, the sixth product ions being produced by fragmenting or reacting third ions, the first, second and third ions being isomeric or isobaric.

The control system may be configured to identify and/or determine a location of a difference between the third ions and the first and/or second ions based on the comparison between the mass to charge ratio values of the sixth and first and/or second product ions.

The first ions may be known ions having a known structure, and the control system may be configured to identify and/or determine the location of the difference between the first and second ions using the known structure of the first ions.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig. 1 shows schematically an analytical instrument according to various embodiments;

Fig. 2 shows schematically an analytical instrument according to various embodiments;

Fig. 3A shows schematically the structure of three theoretical isomeric or isobaric molecules, and Fig. 3A shows their expected mass spectra;

Fig. 4A and 4B show theoretical product ion mass spectra for each of the three theoretical isomeric or isobaric molecules;

Fig. 5 shows expected ion mobility drift time-mass to charge ratio data for two of the three theoretical isomeric or isobaric molecules, where the centre plots show drift time versus mass to charge ratio data, the upper plot shows a mass to charge ratio contraction, and the left-most plot shows a drift-time contraction;

Figs. 6 and 7 show expected ion mobility drift time-mass to charge ratio data for product ions derived from two of the three theoretical isomeric or isobaric molecules, where the centre plots show drift time versus mass to charge ratio data,

- 11 the upper plots show a mass to charge ratio contraction, and the left-most plots show a drift-time contraction;

Fig. 8A shows a comparison of expected product ion data obtained for two of the three theoretical isomeric or isobaric molecules, and Fig. 8B shows schematically a method of locating a region of a difference between two of the three theoretical isomeric or isobaric molecules according to various embodiments; and

Fig. 9 shows schematically a method for locating isobaric or isomeric differences between ions according to various embodiments.

DETAILED DESCRIPTION

Figs. 1 and 2 show schematically an analytical instrument such as a mass spectrometer according to various embodiments. The analytical instrument comprises a collision, fragmentation or reaction device 1 upstream of (and coupled to) an ion mobility separator 2, which is upstream of (and coupled to) a mass analyser 4.

It should be noted that Figs. 1 and 2 are merely schematic, and that the analytical instrument may (and in various embodiments does) include other components, devices and functional elements to those shown in Figs. 1 and 2.

For example, the analytical instrument may comprise an ion source (not shown) upstream of (and coupled to) the a collision, fragmentation or reaction device 1. The ion source may comprise any suitable ion source such as 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 (El) ion source; (ix) a Chemical Ionisation (Cl) ion source; (x) a Field Ionisation (Fl) 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 Ionisation (DESI) ion source; (xvi) a Nickel63 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; (xx) a

- 12 Glow Discharge (“GD”) ion source; (xxi) an Impactor ion source; (xxii) a Direct Analysis in Real Time (DART) ion source; (xxiii) a Laserspray Ionisation (LSI) ion source; (xxiv) a Sonicspray Ionisation (SSI) ion source; (xxv) a Matrix Assisted Inlet Ionisation (MAH) ion source; (xxvi) a Solvent Assisted Inlet Ionisation (SAII) ion source; (xxvii) a Desorption Electrospray Ionisation (“DESI”) ion source; (xxviii) a Laser Ablation Electrospray Ionisation (“LAESI”) ion source; (xxix) a Surface Assisted Laser Desorption Ionisation (“SALDI”) ion source; (xxx) a Low Temperature Plasma (“LTP”) ion source; and (xxxi) a Helium Plasma Ionisation (“HePI”) ion source.

The analytical instrument may comprise a chromatography or other separation device upstream of (and coupled to) the ion source. The chromatography separation device may comprise a liquid chromatography or gas chromatography device. Alternatively, the separation device may comprise: (i) a Capillary Electrophoresis (“CE”) separation device; (ii) a Capillary Electrochromatography (“CEC”) separation device; (iii) a substantially rigid ceramicbased multilayer microfluidic substrate (“ceramic tile”) separation device; or (iv) a supercritical fluid chromatography separation device.

The collision, fragmentation or reaction device 1 may comprise any suitable such device. The analytical instrument may comprise 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 ion reaction fragmentation device; (xxi) an ion-metastable molecule reaction fragmentation

- 13device; (xxii) an ion-metastable atom reaction fragmentation device; (xxiii) an ionion reaction device for reacting ions to form adduct or product ions; (xxiv) an ionmolecule 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.

The ion mobility separator 2 may comprise any suitable ion mobility separator that is configured to separate ions according to their ion mobility.

As illustrated in Fig. 1, the ion mobility separator 2 may comprise a drift tube, e.g. that is pressurised with gas. An electric field, e.g. comprising a DC voltage gradient and/or a travelling DC voltage wave, may be arranged to urge ions along the length of the ion mobility separator 2, i.e. through the gas, so that ions separate according to their ion mobility. The ions may optionally be urged against a counter flow of gas. Alternatively, a gas flow may be arranged to urge ions along the length of the ion mobility separator 2, while an electric field, e.g. comprising a DC voltage gradient and/or a travelling DC voltage wave, may be arranged to oppose the gas flow so that ions separate according to their ion mobility.

As illustrated by Fig. 2, it would also be possible for the ion mobility separator 2 to comprise a cyclic (closed-loop) ion mobility separator. In these embodiments, ions may then be caused to separate according to their ion mobility over e.g. a fixed integer number of cycles around the ion mobility separator 2. An ion gate or gate region 5 may be provided which may be closed to allow multi-pass operation. The ion gate 5 may be opened, e.g. after a predetermined time period, to allow ions to exit the ion mobility separator 2 after ions have made one or more circuits of the ion mobility separator 2. Using a cyclic ion mobility separator can allow a higher degree of separation, and so higher ion mobility resolution.

In the illustrated embodiments, the mass analyser 4 comprises an orthogonal acceleration Time of Flight mass analyser. However, more generally the mass analyser may comprise any suitable mass analyser such as 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

- 14mass analyser; (vii) Ion Cyclotron Resonance (ICR) mass analyser; (viii) a Fourier Transform Ion Cyclotron Resonance (FTICR) mass analyser; (ix) an electrostatic mass analyser arranged to generate an electrostatic field having a quadrologarithmic potential distribution; (x) a Fourier Transform electrostatic 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.

The spectrometer may comprise any one or more further devices, as desired. For example, in various embodiments, the analytical instrument may comprise one or more ion guides, one or more ion traps, and/or one or more mass filters, e.g. which may be 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 Wien filter.

As shown in Figs. 1 and 2, the analytical instrument may comprise a control system 6, e.g. that is configured to control the operation of the analytical instrument, e.g. in the manner of the various embodiments described herein. The control system may comprise suitable control circuitry that is configured to cause the instrument to operate in the manner of the various embodiments described herein. The control system may comprise suitable processing circuitry configured to perform any one or more or all of the necessary processing and/or post-processing operations in respect of the various embodiments described herein. In various embodiments, the control system may comprise a suitable computing device, a microprocessor system, a programmable FPGA (field programmable gate array), and the like.

The analytical instrument may be operated in various modes of operation including a mass spectrometry (MS) mode of operation; a tandem mass spectrometry (MS/MS) mode of operation; a mode of operation in which parent or precursor ions are alternatively fragmented or reacted so as to produce fragment or product ions, and not fragmented or reacted or fragmented or reacted to a lesser degree; a Multiple Reaction Monitoring (MRM) mode of operation; a Data Dependent Analysis (DDA) mode of operation; a Data Independent Analysis (DIA) mode of operation a Quantification mode of operation or an Ion Mobility Spectrometry (IMS) mode of operation.

- 15In operation, (parent or precursor) ions from the ion source may be introduced into the collision, fragmentation or reaction device 1. The collision, fragmentation or reaction device 1 may be operated in a fragmentation or reaction mode whereby ions are fragmented or reacted, or may be operated in a nonfragmenting or reacting mode whereby ions from the ion source are not fragmented or reacted.

The ions may then be introduced into the ion mobility separator 2, whereupon the ions may be caused to separate according to their ion mobility, e.g. as they pass through the ion mobility separator 2.

Ions exiting the ion mobility separator 2 may then pass, e.g. via an optional transfer cell 3 or otherwise, to the mass analyser 4 so that ions are then separated according to their mass to charge ratio (time of flight).

In this manner, the mass to charge ratio and ion mobility of ions from the ion source may be determined and/or the mass to charge ratio and ion mobility of product (daughter or fragment) ions derived from the ions from the ion source may be determined.

Other arrangements would be possible.

Various embodiments are directed to a method of site specific location of isobaric or isomeric differences between chemical entities using ion mobility and mass spectrometry. According to various embodiments, IMS-MS/MS data is acquired and processed so as to enable location of isobaric or isomeric changes to chemical species by comparing the mobility of the precursor and fragment ions to an unmodified species.

Using the method(s) described herein, the location(s) of isobaric or isomeric changes may be discovered from a single analysis. The method(s) described here provide sensitive and rapid detection of isobaric or isomeric changes to a chemical species that may complement or reinforce other methods such as NMR, production of model compounds, etc.

Various embodiments may be used, e.g., to distinguish and/or characterise isomeric or isobaric molecules, such as for example peptides, isobaric amino acids or modifications with identical masses, etc.

Fig. 3A shows three theoretical isomeric molecules which are desired to be characterised in accordance with various embodiments: species A, species B and species C. As shown in Fig 3A, species A, B and C share a common core structure, schematically represented by circles, and differ by various isomeric

- 16modifications (functional groups) attached to the core structure, which are schematically represented by triangles and rectangles.

Species A may be a known species, and species B and C may be unknown isomeric species sharing the same core structure. The core structure may be known before the analysis, or may be determined as part of the same analysis. It may be desired to characterise the structural differences between known species A and unknown isomeric species B and C.

As illustrated by Fig. 3B, even though species A, B and C differ in their structures, all three species share the same molecular weight, namely 274 Da, and the same mass to charge ratio, namely 274, and so are indistinguishable on the basis of mass to charge ratio alone. As illustrated by Fig. 3A, this is because each of the “circles” has the same elemental composition and structure, and so has the same molecular weight (e.g. 50 Da), while although the “triangles” and “rectangles” may have different elemental compositions and/or different structures, they have the same (e.g. total) molecular weight (e.g. 12 Da).

More information to help distinguish between the different ion species may be obtained by fragmenting the precursor ions of each of the species A, B and C, and then mass analysing the resulting fragment or product ions, for example in a tandem mass spectrometry (MS/MS) experiment or otherwise (e.g. in the manner described above with respect to Figs. 1 and 2).

Fig. 4A shows theoretical product ion spectra from the isomeric molecular species A, B, and C shown in Fig. 3. Each peak is labelled with its fragment composition. Fig. 4B shows the same data where diagnostic ions that may be used to locate differences between species B and A are highlighted. A mass difference of 12 Da is shown by

As shown in Fig. 4A, fragmenting species A results in various product ions 41A-45A, as well as un-fragmented precursor ions 40A. Similarly, fragmenting species B and C results in respective different sets of product ions being produced.

As can be seen from Fig. 4B, some of the product ions produced from fragmenting species A (40A, 41A, 44A and 45A) are indistinguishable on the basis of mass to charge ratio from corresponding product ions produced from fragmenting species B. Other product ions (42A, 43A), however, differ in mass to charge ratio (e.g. by 12 Da) from corresponding product ions (42B, 43B) produced from fragmenting species B. Accordingly, these differing, or diagnostic, product ions can be used to distinguish between species A and B.

- 17Furthermore, where the structure and fragmentation pattern of species A is known, this difference information can be used to infer the location of the difference (e.g. modification) between species A and B.

However, in the case of species A and C, even though fragmenting these species results in different product ions being produced (as illustrated in Fig. 4A), for each product ion resulting from fragmenting species A, there is a corresponding product ion or ions resulting from fragmenting species C which has the same mass to charge ratio, i.e. which is the same or which is isomeric or isobaric.

For example, as illustrated in Fig. 4A, the fragmentation of species A results in fragment ions 45A, represented schematically by a triangle, which has a mass to charge ratio of 12 Da. The fragmentation of species C, on the other hand, results in “triangle” fragments 45C as well as isomeric fragment ions 46C, represented schematically by rectangles, which also has a mass to charge ratio of 12Da. Accordingly, “triangle” and “rectangle” fragment ions are indistinguishable from each other on the basis of mass to charge ratio.

Accordingly, species A and C are indistinguishable from each other on the basis of the mass to charge ratios of their respective product ions (as well as being indistinguishable from each other on the basis of their (un-fragmented) mass to charge ratios). The differences between A and C could be due to, amongst other things, differences in atom connectivity, three dimensional orientations, etc.

The Applicants have recognised that structural differences of this nature or otherwise may be more readily identified using ion mobility, e.g. of the product (fragment) ions. Ion mobility is orthogonal to mass to charge ratio and can separate ions based on their size and/or shape.

Fig. 5 shows a two-dimensional plot of ion mobility (drift time) versus mass to charge ratio (m/z) data for species A and species C. In Fig. 5, and the following figures, the centre of the circle indicates the mass to charge ratio and the ion mobility value of the chemical species being analysed, while the radius indicates its intensity.

As shown in Fig. 5, species A and C have different ion mobilities (but equal mass to charge ratios), and so can be distinguished from each other on the basis of ion mobility. Since the mass to charge ratio values are the same, and the ion mobilities are different, it may be assumed that the differences between A and C are structural in nature (i.e. that species A and C are isomeric or isobaric).

- 18Fig. 6 shows a two-dimensional plot of ion mobility (drift time) versus mass to charge ratio (m/z) data for product ions 40A-45A produced from fragmenting species A, and for product ions 40C-46C produced from fragmenting species C. It can be seen that although for each product ion 40A-45A produced from fragmenting species A, there is a corresponding product ion or ions 40C-46C produced from fragmenting species C having the same mass to charge ratio, some of the corresponding product ions differ in ion mobility.

These differences are highlighted in Fig. 7. As shown in Fig. 7, “triangle” fragment ions 45A differ in ion mobility to “rectangle” fragment ions 46C, even though they share the same mass to charge ratio. Similarly, products ions 40A, 41A and 43A each differ in ion mobility from corresponding product ions 40C, 41C and 43C due to the presence of a “rectangle” instead of a “triangle” in species C as compared to species A. Accordingly, it is possible to distinguish between species A and C on the basis of the ion mobilities of their respective product ions.

The Applicants have furthermore recognised that this information can be further leveraged to derive additional information on the nature of the structural differences between species A and C. This process is illustrated by Fig. 8.

Fig. 8A shows mass to charge ratio and ion mobility (drift time) data determined for each product ion 40A-45A produced from fragmenting species A, together with mass to charge ratio and ion mobility (drift time) data determined for each corresponding product ion 40C-46C produced from fragmenting species C. This information may be combined with knowledge of the structure of species A to determine the site of (to locate) a chemical modification. The knowledge of the structure may be prior knowledge, or may be determined as part of the method before hand.

In various embodiments, a “process of elimination” is used to determine the site of a structural modification by “mapping” the data onto the known structure of species of interest A.

As illustrated in Fig. 8B, at a first step 81, it is determined that for fragments 2 and 4 (42A and 44A) produced from fragmenting species A, there are corresponding fragments 2 and 4 (42C and 44C) produced from fragmenting species C having respectively the same mass to charge ratios and ion mobilities. Since these corresponding fragments share the same mass to charge ratio and ion mobility values, the structure corresponding to these fragments may be ruled out as a location for the isomeric modification causing the overall difference in ion mobility.

- 19Thus, as shown in Fig. 8B, after step 81, the structure corresponding to these fragments, which is schematically represented by circles, is eliminated.

At a second step, 82 it may then be confirmed from a comparison of mass to charge ratio and ion mobility data for fragments 3, 5 and 6, that the modification is as a result of a difference within the highlighted region 84 of species A.

Then, at step 83, from a comparison of corresponding fragments 1 it may be determined that the difference lies in the circled triangle 85.

Other processes could be used to arrive at the same conclusion.

Fig. 9 shows a process in accordance with various embodiment.

At step 91A, mass to charge ratio and ion mobility (drift time) data is measured for a known precursor species A. At step 91C, mass to charge ratio and ion mobility (drift time) data is measured for an unknown precursor species C, being a modified version of known precursor species A. Steps 91A and 91C may be performed separately, e.g. as different experiments runs, but in various embodiments are performed at the same time, i.e. in the same experiment. This may be done in the manner discussed above with reference to Figs. 1 and 2.

Next, precursor species A and C may be fragmented, and then mass to charge ratio and ion mobility (drift time) data may be obtained for product ions produced from species A, at step 92A, and for produced from species C at step 92C. Again, steps 92A and 92C may be performed separately, e.g. as different experiments runs, but in various embodiments are performed at the same time, i.e. in the same experiment. This may be done in the manner discussed above with reference to Figs. 1 and 2.

Once the precursor and product ion data has been obtained, the data may then be processed.

In steps 93A and 93C, product ions may be assigned to corresponding structure of their respective precursor ions.

Then, at step 94, the ion mobility (drift time) data determined for species A may be compared with the ion mobility (drift time) data determined for species C. If no ion mobility differences are detected, then the process may end, and it may be determined that the species A and C are the same or indistinguishable.

Otherwise, if ion mobility differences are detected at step 95, then at step 96 a process of elimination may be used to map the determined ion mobility differences onto the known structure of the known species A. Then, at step 97, the difference may be assigned to a site of the structure.

-20It will be appreciated from the above that various embodiments provide an improved method of mass and/or ion mobility spectrometry.

Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various 5 changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

Claims

1. A method of characterising ions, the method comprising:

comparing an ion mobility value of first product ions to an ion mobility value of second product ions, the first product ions being produced by fragmenting or reacting first ions, the second product ions being produced by fragmenting or reacting second ions, the first and second ions being isomeric or isobaric; and identifying and/or determining a location of a difference between the first and second ions based on the comparison between the ion mobility values of the first and second product ions.

2. A method of mass spectrometry and/or ion mobility spectrometry, the method comprising:

determining an ion mobility value of the first product ions and an ion mobility value of the second product ions;

3. The method of claim 1 or 2, wherein the first product ions and the second product ions are isomeric or isobaric.

4. The method of claim 1, 2 or 3, wherein the difference between the first and second ions is identified and/or located as comprising a difference between the first and second product ions when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

5. The method of any one of the preceding claims, wherein the method comprises:

comparing a mass to charge ratio value of the first product ions to a mass to charge ratio value of the second product ions; and identifying and/or determining the location of the difference between the first and second ions based on the comparisons between the ion mobility values and the mass to charge ratio values of the first and second product ions.

6. The method of claim 5, wherein the difference between the first and second ions is identified and/or located as comprising a difference between the first and second product ions when the mass to charge ratio value of the first product ions is equal to or sufficiently similar to the mass to charge ratio value of the second product ions, and when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

7. The method of claim 5 or 6, wherein the method comprises:

determining that the first and second product ions are isomeric or isobaric when the mass to charge ratio value of the first product ions is equal to or sufficiently similar to the mass to charge ratio value of the second product ions, and when the ion mobility value of the first product ions is different to or sufficiently different to the ion mobility value of the second product ions.

8. The method of any one of the preceding claims, wherein the method comprises:

comparing a mass to charge ratio value of the first ions to a mass to charge ratio value of the second ions, and comparing an ion mobility value of the first ions to an ion mobility value of the second ions; and determining that the first and second ions are isomeric or isobaric based on the comparisons between the mass to charge ratio values and the ion mobility values of the first and second ions.

9. The method of claim 8, wherein the first and second ions are determined to be isomeric or isobaric when the mass to charge ratio value of the first ions is equal to or sufficiently similar to the mass to charge ratio value of the second ions, and

-23when the ion mobility value of the first ions is different to or sufficiently different to the ion mobility value of the second ions.

10. The method of any one of the preceding claims, wherein the method comprises:

comparing a mass to charge ratio value of third ions to the mass to charge ratio value of the first and/or second ions, and comparing an ion mobility value of the third ions to the ion mobility value of the first and/or second ions; and determining that the first, second and third ions are isomeric or isobaric based on the comparisons between the mass to charge ratio values and the ion mobility values of the third and first and/or second ions.

11. The method of claim 10, wherein the first, second and third ions are determined to be isomeric or isobaric when the mass to charge ratio value of the third ions is equal to or sufficiently similar to the mass to charge ratio value of the first and/or second ions, and when the ion mobility value of the third ions is different to or sufficiently different to the ion mobility value of the first and/or second ions.

12. The method of any one of the preceding claims, wherein the method comprises:

determining an ion mobility value of fourth product ions and an ion mobility value of fifth product ions, the first and fourth product ions being produced by fragmenting or reacting the first ions, the second and fifth product ions being produced by fragmenting or reacting the second ions;

comparing the ion mobility value of the fourth product ions to the ion mobility value of the fifth product ions; and identifying and/or determining the location of the difference between the first and second ions based on the comparison between the ion mobility values of the fourth and fifth product ions.

13. The method of claim 12, wherein the difference between the first and second ions is identified and/or located as other than comprising a difference between the fourth and fifth product ions when the ion mobility value of the fourth product ions is the same as or sufficiently similar to the ion mobility value of the fifth product ions.

14. The method of claim 12 or 13, wherein the method comprises: comparing a mass to charge ratio value of the fourth product ions to a mass to charge ratio value of the fifth product ions; and identifying and/or determining the location of the difference between the first and second ions based on the comparisons between the ion mobility values and the mass to charge ratio values of the fourth and fifth product ions.

15. The method of claim 14, wherein the difference between the first and second ions is identified and/or located as other than comprising a difference between the fourth and fifth product ions when the mass to charge ratio value of the fourth product ions is equal to or sufficiently similar to the mass to charge ratio value of the fifth product ions, and when the ion mobility value of the fourth product ions is equal to or sufficiently similar to the ion mobility value of the fifth product ions.

16. The method of claim 14 or 15, wherein the method comprises: determining that the fourth and fifth product ions are the same or indistinguishable when the mass to charge ratio value of the fourth product ions is equal to or sufficiently similar to the mass to charge ratio value of the fifth product ions, and when the ion mobility value of the fourth product ions is equal to or sufficiently similar to the ion mobility value of the fifth product ions.

17. The method of any one of the preceding claims, wherein the method comprises:

comparing a mass to charge ratio value of sixth product ions to the mass to charge ratio value of the first and/or second product ions, the sixth product ions being produced by fragmenting or reacting third ions, the first, second and third ions being isomeric or isobaric; and identifying and/or determining a location of a difference between the third ions and the first and/or second ions based on the comparison between the mass to charge ratio values of the sixth and first and/or second product ions.

18. The method of any one of the preceding claims, wherein the first ions are known ions having a known structure, and wherein the method comprises

-25identifying and/or determining the location of the difference between the first and second ions using the known structure of the first ions.

19. A computer readable medium storing software code, which when executing on a processor, causes the processor to perform the method any one of the preceding claims.

20. An analytical instrument comprising:

one or more collision, fragmentation or reaction devices;

to compare the ion mobility value of the first product ions to the ion mobility value of the second product ions; and to identify and/or determine a location of a difference between the first and second ions based on the comparison between the ion mobility values ofthe first and second product ions.