GB2457769A

GB2457769A - Method of ion mobility separation and determining multiple molecular conformations

Info

Publication number: GB2457769A
Application number: GB0816761A
Authority: GB
Inventors: Robert Harold Bateman; Kevin Giles
Original assignee: Micromass UK Ltd
Current assignee: Micromass UK Ltd
Priority date: 2007-09-14
Filing date: 2008-09-12
Publication date: 2009-09-02
Anticipated expiration: 2028-09-12
Also published as: GB2457769B; GB0816761D0

Abstract

A method of ion mobility separation is disclosed wherein ions having multiple molecular conformations may be recognised. Ions are separated temporally by an ion mobility spectrometer. The arrival time distributions of ions which are temporally separated by the ion mobility spectrometer are analysed. Ion peaks having particular ion arrival time profiles may be flagged as possibly relating to two different unresolved ion species having essentially the same mass to charge ratio but different molecular conformations. The experiment is then re-run and ions of interest are again subjected to separation in an ion mobility spectrometer. Ions which emerge from the ion mobility spectrometer are fragmented in a downstream collision cell and the arrival time profiles of the resulting fragment ions are then analysed. If two or more substantially different arrival time distributions are observed for the fragment ions then the parent ions of interest are considered as comprising ions having two different molecular conformations.

Description

METHOD OF ION MOBILITY SEPARATION

The present invention relates to a mass spectrometer and a method of mass spectrometry. The preferred embodiment relates to a method of determining structural variants of ions using ion mobility mass spectrometry.

Tandem mass spectrometric (MS/MS) characterisatiOn of peptides has become a central analytical approach to the analysis of the proteomes of cells and organisms. Both qualitative and quantitative applications provide critical data to enable the modelling of cells and organisms. Accordingly, it is desirable to understand the gas-phase ion chemistry that underpins MS/MS analysis of peptide ions in order to optimize the application of the technique.

The structures of N-terminal a-and b-fragment ions formed from the fragmentation of collisionally activated protonated peptides in the gas phase has been a topic of investigation and discussion for several years. The b-ions have generally been considered to consist of a linear peptide chain terminating in a cyclic oxazolone structure. Subsequent formation of a-ions by loss of CO has been presumed to follow opening of the oxazolone ring. These hypotheses alone, however, do not readily explain all the fragment ions observed in low energy collision-induced dissociation experiments.

It is desired to provide an improved mass spectrometer and method of mass spectrometry.

According to an aspect of the present invention there is provided a method of mass spectrometry comprising: * * 30 separating first ions in an ion mobility spectrometer or separator into one or more ion mobility fractions; *** S estimating, predicting, recognising or determining whether or not first ions in an ion mobility fraction may comprise S...

: multiple structures and/or molecular conformations; temporally separating at least some first ions of interest in an ion mobility spectrometer or separator; fragmenting the first ions of interest which emerge from the ion mobility spectrometer or separator in a collision, * fragmentation or reaction device to produce a plurality of second ions; analysing one or more characteristics of one or more of the second iOnS; and determining or confirming based upon the analysis of the one or more characteristics of the one or more second ions whether or not the first ions of interest have or are likely to have either: (i) a single structure and/or molecular conformation; or (ii) multiple structures and/or molecular conformations.

According to the preferred embodiment the two stages of separating ions in an ion mobility spectrometer or separator are preferably performed in the same ion mobility spectrometer or separator. However, other embodiments are contemplated wherein the two stages of ion mobility separation may be performed in two separate ion mobility spectrometers or separators.

According to an embodiment either: (1) the first ions in the ion mobility fraction comprise ions having a single structure and/or molecular conformation; or (ii) the first ions in the ion mobility fraction comprise ions having multiple structures and/or molecular conformations which are substantially unresolved by the ion mobility spectrometer or separator.

According to an embodiment either: (1) the first ions comprise parent or precursor ions or first or subsequent generation fragment ions; and/or (ii) the second ions comprise first generation fragment ions or second or subsequent generation fragment ions; and/or (iii) the first ions of interest comprise first ions in an ion mobility fraction which are estimated, predicted, recognised or determined to comprise multiple structures and/or molecular conformations; and/or (iv) the first ions of interest comprise first ions in an ion mobility fraction which are estimated, predicted, recognised *...30 or determined to comprise a single structure and/or molecular conformation.

* ** ,-.

The step of analysing one or more characteristics of one or more of the second ions preferably comprises: :. (i) measuring or determining the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of the second ions; and/or * (ii) comparing the arrival time distributions, mean or * average arrival times and/or arrival time profiles of at least some of the second ions with other second ions and/or with the arrival time distributions, mean or average arrival times and/or arrival time profiles of one or more of the first ions; and/or (iii) determining whether or not the one or more second ions have substantially different arrival time distributions, mean or average arrival times and/or arrival time profiles.

The arrival times preferably correspond with the ion mobility drift times of parent ions or the ion mobility drift times of corresponding precursor ions for observed fragment ions.

Fragmentation of ions which emerge from the ion mobility spectrometer is preferably assumed to occur substantially instantaneously. According to an embodiment the arrival time profiles may correspond with ion mobility elution profiles or the ion mobility elution profiles of precursor ions to observed fragment ions.

According to an embodiment either: (i) if it is determined that two or more of the second ions have substantially different arrival time distributions, mean or average arrival times and/or arrival time profiles then it is determined or confirmed that the first ions of interest have multiple structures and/or molecular conformations; and/or (ii) if it is determined that two or more of the second ions have only one arrival time distribution, mean or average arrival time and/or arrival time profile then it is determined that the first ions of interest have a single structure and/or molecular conformation.

The arriyal time distributions and profiles referred to above correspond with the drift time or ion mobility profiles of ions emerging from the ion mobility spectrometer or separator and optionally fragmented.

The first ions and/or the second ions preferably comprise: (i) one or more biopolymers, proteins, peptides, *30 polypeptides, oligionucleotides, oligionucleosides, amino acids, carbohydrates, sugars, lipids, fatty acids, vitamins, hormones, *..

portions or fragments of DNA, portions or fragments of cDNA, *: portions or fragments of RNA, portions or fragments of mRNA, portions or fragments of tRNA, polyclonal antibodies, monoclonal antibodies, ribonucleases, enzymes, metabolites, polysaccharides, : ,"* phosphorolated peptides, phosphorolated proteins, glycopeptides, " glycoproteins or steroids and/or fragments thereof; and/or S....

* * (ii) biomolecules bound to polyethelene glycol (PEG) and/or fragments thereof; and/or (iii) antibodies and/or fragments thereof; and/or (iv) pegylated proteins andLor fragments thereof; and/or (v) antibodies encapsulated in or protected by polyethelene glycol (PEG) and/or fragments thereof; and/or (vi) polyethelene glycol (PEG) and/or fragments thereof; and/or (vii) multiple conformations of polyethelene glycol (PEG) and/or fragments thereof; and/or (viii) PEG modified proteins, lipsomes or nanoparticles and/or fragments thereof.

According to an embodiment the first ions may correspond with a first sample and either: (i) the first sample is taken from a diseased organism and/or a non-diseased organism; (ii) the first sample is taken from a treated organism and/or a non-treated organism; or (iii) the first sample is taken from a mutant organism and/or from a wild type organism.

According to another aspect of the present invention there is provided a mass spectrometer comprising: an ion mobility spectrometer or separator; a collision, fragmentation or reaction device arranged downstream of the ion mobility spectrometer or separator; and a control system arranged and adapted: (i) to pass first ions to the ion mobility spectrometer or separator so that the first ions are separated into one or more ion mobility fractions; (ii) to estimate, predict, recognise or determine whether or not first ions in an ion mobility fraction may comprise multiple structures and/or molecular conformations; (iii) to cause at least some first ions of interest to be separated temporally in the ion mobility spectrometer or separator; (iv) to cause first ions of interest which emerge, in use, from the ion mobility spectrometer or separator to be fragmented in the collision, fragmentation or reaction device to produce a plurality of second ions; (v) to analyse one or more characteristics of one or more of * 35 the second ions; and : ** (vi) to determine or confirm based upon the analysis of the one or more characteristics of the one or more second ions whether or not the first ions of interest have or are likely to have either: (a) a single structure and/or molecular conformation; or (b) multiple structures and/or molecular conformations. -5-.

According to an embodiment a collision, fragmentation or reaction device may be arranged upstream of said ion mobility spectrometer or separator and/or a collision, fragmentation or reaction device may be arranged downstream of said ion mobility spectrometer. The collision, fragmentation or reaction device preferably comprises an ion guide wherein ions are accelerated into the ion guide such that they fragment upon entering the ion guide.

The control system is preferably arranged and adapted: (1) to measure or determine the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of the second ionS; and/or (ii) to compare the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of the second ions with other second ions and/or with the arrival time distributions, mean or average arrival times and/or arrival time profiles of one or more of the first ions; and/or (iii) to determine whether or not the one or more second ions have two or more different arrival time distributions, mean or average arrival times and/or arrival time profiles.

The ion mobility spectrometer preferably comprises: (i) a gas phase electrophoresis device; and/or (ii) a drift tube wherein an axial DC voltage gradient is maintained, in use, along at least a portion of the drift tube; and/or (iii) one or more multipole rod sets; and/or (iv) a plurality of electrodes through which ions are transmitted in use; and/or (v) a plurality of plate or mesh electrodes and wherein at *.. 30 least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the plate or mesh electrodes are arranged generally in the plane in which ions travel in use. * S6

.. : According to an embodiment the ion mobility spectrometer or separator may be axially segmented.

According to an embodiment the mass spectrometer may further : .". comprise:

I

* (i) DC voltage means arranged and adapted to maintain a * ** 1.1 * 1 substantially constant DC voltage gradient along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the ion mobility spectrometer or separator in order to urge ions along the axial length of the ion mobility spectrometer or separator; and/or (ii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes forming the ion mobility spectrometer or separator in order to urge at least some ions along at least a portion of the axial length of the ion mobility spectrometer or separator; and/or (iii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the ion mobility spectrometer or separator.

According to an embodiment the mass spectrometer may further comprise means arranged and adapted to maintain at least a portion of the ion mobility spectrometer or separator at a pressure selected from the group consisting of: (i) > 1.0 x i0 mbar; (ii) > 1.0 x 102 inbar; (iii) > 1.0 x 10' mbar; (iv) > 1 mbar; (v) > 10 mbar; (vi) > 100 mbar; (vii) > 5.0 x 10 athar; (viii) > 5.0 x 102 inbar; (ix) 103_102 nthar; and (x) 104_101 mbar.

The fragmentation, collision or reaction device preferably comprises either: (i) a multipole rod set; and/or (ii) plurality of electrodes through which ions are transmitted in use; and/or (iii) a plurality of plate or mesh electrodes and wherein at least 50%, 555, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the plate or mesh electrodes are arranged generally in the plane in which ions travel in use.

The fragmentation, collision or reaction device is *0.

preferably axially segmented.

*:::: According to an embodiment the mass spectrometer may further * comprise: * 35 (i) a DC voltage means arranged and adapted to maintain a : *, substantially constant DC voltage gradient along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the fragmentation, collision or reaction device in order to urge ions along the axial length of the fragmentation, collision or reaction device; and/or (ii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes forming the fragmentation, collision or reaction device in order to urge ions along the axial length of the fragmentation, collision or reaction device; and/or (iii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the fragmentation, collision or reaction device.

According to an embodiment the mass spectrometer may further comprise a device arranged and adapted to maintain at least a portion of the fragmentation, collision or reaction device at a pressure selected from the group consisting of: (i) > 1.0 x mbar; . (ii) > 1.0 x 102 mbar; (iii) > 1.0 x 10 mbar; (iv) > 1 mbar; (v) > 10 mbar; (vi) > 100 mbar; (vii) > 5.0 x i0 mbar; (viii) > 5.0 x 102 mbar; (ix) 103_102 mbary and (x) 10--10' mbar.

The control system is preferably arranged and adapted to switch or repeatedly switch the fragmentation, collision or reaction device between a first mode of operation wherein ions are substantially fragmented and a second mode of operation wherein substantially fewer or no ions are fragmented.

According to an embodiment either: (a) in the first mode of operation ions exiting the ion mobility spectrometer or separator are accelerated through a potential difference selected from the group consisting of: (i) �= * 30 10 V; (ii) �= 20 V; (iii) �= 30 V; (iv) �= 40 V; (v) �= 50 V; (vi) �= V; (vii) �= 70 V; (viii) �= 80 V; (ix) �= 90 V; and (x) �= 100 V; and/or (b) in the second mode of operation ions exiting the ion * mobility spectrometer or separator are accelerated through a *** * 35 potential difference selected from the group consisting of: (i) �= * *, 20 V; (ii) �= 15 V; (iii) �= 10 V; (iv) �= 5V; and (v) �= lv; and/or :.: (C) the control system is arranged and adapted to switch the fragmentation, collision or reaction device between the first mode of operation and the second mode of operation at least once every 1 ms, 5 ins, 10 ins, 15 ms, 20 ins, 25 ins, 30 ins, 35 ms, 40 ins, 45 ins, 50 ms, 55 ins, 60 ins, 65 ins, 70 ins, 75 ins, 80 ms, 85 ins, 90 ins, ms, 100 ms, 200 ins, 300 ms, 400 ins, 500 ms, 600 ms, 700 ms, 800 ms, 900 ms, 1 s, 2 Sf 3 Sf 4 s, 5 s, 6 s, 7 S1 8 s, 9 s or 10 S. The collision, fragmentation or reaction device is preferably selected from the group consisting of: (i) a Collisional Induced Dissociation ("Cm") 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 ion-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 lon-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 reactiori 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 *S.

* to form adduct or product ions; and (xxix) an Electron lonisation * ** 35 Dissociation ("EID") fragmentation device. * S *

* According to an embodiment the collision, fragmentation or reaction device may comprise one or more ion guides arranged upstream and/or downstream of the ion mobility spectrometer or separator. According to an embodiment ions may be accelerated into the one or more ion guides in order to fragment ions by Collision Induced Dissociation ("CID").

According to an embodiment the mass spectrometer may further comprise: (a) an ion source selected from the group consisting of: (i) an Electrospray ionisation ("ESI") ion source; (ii) an Atmospheric Pressure Photo lonisation ("APPI") ion source; (iii) an Atmospheric Pressure Chemical lonisation ("APCI") ion source; (iv) a Matrix Assisted Laser Desorption lonisation ("MALDI") ion source; (v) a Laser Desorption lonisation ("LDI") ion source; (vi) an Atmospheric Pressure lonisation ("API") ion source; (vii) a Desorption lonisation on Silicon ("DIOS") ion Source; (viii) an Electron Impact ("El') ion source; (ix) a Chemical lonisation ("CI") ion source; (x) a Field lonisation ("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 lonisation ("DESI") ion source; (xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption lonisation ion source; and (xviii) a Thermospray ion source; and/or (b) a continuous or pulsed ion source; and/or (C) one or more ion guides arranged upstream and/or downstream of the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device; and/or (d) one or more ion traps or one or more ion trapping regions arranged upstream and/or downstream of the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device; and/or ** (e) a mass analyser arranged upstream and/or downstream of the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device, the mass analyser being selected :. from the group consisting of: (i) a quadrupole mass analyser; (ii) S. a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D *Ss * quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) * .. 35 an ion trap mass analyser; (vi) a magnetic sector mass analyser; * . S * (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; -10 -and (xiv) a linear acceleration Time of Flight mass analyser; and/or (t) one or more energy analysers or electrostatic energy analysers arranged upstream and/or downstream of the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device; and/or (g) one or more ion detectors arranged upstream and/or downstream of the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device; and/or (h) one or more mass filters arranged upstream and/or downstream of the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device, wherein the one or more mass filters are 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 (1) a device or ion gate for pulsing ions into the ion mobility spectrometer or separator and/or the collision, fragmentation or reaction device; and/or (j) a device for converting a substantially continuous ion beam into a pulsed ion beam.

According to an embodiment the mass spectrometer may further comprise: a C-trap; and an orbitrap mass analyser; wherein in a first mode of operation ions are transmitted to the C-trap and are then injected into the orbitrap mass analyser; * ** and * * * * ** 30 wherein in a second mode of operation ions are transmitted * to the C-trap and then to a collision cell wherein at least some ions are fragmented into fragment ions, and wherein the fragment * I I * ions are then transmitted to the C-trap before being injected into the orbitrap mass analyser.

The collision cell downstream of the C-trap referred to above preferably comprises or corresponds with the collision, *:*** fragmentation or reaction device which forms part of the present invention.

According to another aspect of the present invention there is provided a computer program executable by the control system of a mass spectrometer comprising an ion mobility spectrometer or -11 -separator and a collision, fragmentation or reaction device, the computer program being arranged to cause the control system: (i) to pass first ions to the ion mobility spectrometer or separator so that the first ions are separated into one or more ion mobility fractions; (ii) to estimate, predict, recognise or determine whether or not first ions in an ion mobility fraction may comprise multiple structures and/or molecular conformations; (iii) to cause at least some first ions of interest to be separated temporally in the ion mobility spectrometer or separator; (iv) to cause first ions of interest which emerge, in use, from the ion mobility spectrometer or separator to be fragmented in the collision, fragmentation or reaction device to produce a plurality of second ions; (v) to analyse one or more characteristics of one or more of the second ions; and (vi) to determine or confirm based upon the analysis of the one or more characteristics of the one or more second ions whether or not the first ions of interest have or are likely to have either: (a) a single structure and/or molecular conformation; or (b) multiple structures and/or molecular conformations.

According to another aspect of the present invention there is provided a computer readable medium comprising computer executable instructions stored on the computer readable medium, the instructions being arranged to be executable by a control system of a mass spectrometer comprising an ion mobility spectrometer or separator and a collision, fragmentation or * ** *** reaction device, to cause the control system: (i) to pass first ions to the ion mobility spectrometer or *S*.

separator so that the first ions are separated into one or more ion mobility fractions; as * * (ii) to estimate, predict, recognise or determine whether or *S.

* not first ions in an ion mobility fraction may comprise multiple * ** 35 structures and/or molecular conformations; * S * * (iii) to cause at least some first ions of interest to be separated temporally in the ion mobility spectrometer or separator; (iv) to cause first ions of interest which emerge, in use, from the ion mobility spectrometer or separator to be fragmented -12 -in the collision, fragmentation or reaction device to produce a plurality of second ions; (v) to analyse one or more characteristics of one or more of the second ions; and (vi) to determine or confirm based upon the analysis of the one or more characteristics of the one or more second ions whether or not the first ions of interest have or are likely to have either: (a) a single structure and/or molecular conformation; or (b) multiple structures and/or molecular conformations.

The computer readable medium is preferably selected from the group consisting of: (i) a ROM; (ii) an EAROM; (iii) an EPROM; (iv) an EEPROM; (v) a flash memory; and (vi) an optical disk.

The preferred embodiment relates to a method of determining whether or not a precursor ion in a sample of ions is present in two or more different molecular conformations.

According to an embodiment a method of investigating the structures of a-or b-fragment ions is preferably provided.

According to another embodiment a method of analysing ions which may form macrocyclic or other structures is provided.

According to an embodiment the overall size of ion species may be determined in order to complement characterization by mass to charge ratio.

According to an embodiment a method of ion mobility separation is provided wherein the structural variants of ions may be determined. According to an embodiment ions may be separated temporally by an ion mobility spectrometer. The arrival time or ion mobility distributions of ions which are temporally separated * *, by the ion mobility spectrometer are then analysed. Ion peaks * * . * * having a certain arrival time' or ion mobility profile may be **S.

flagged as possibly relating to two different (unresolved) ions species having essentially the same mass to charge ratio but different molecular conformations. The experiment is then preferably re-run and in a subsequent mode of operation ions of interest are preferably (again) passed through the ion mobility * S. :.: * 35 spectrometer or separator. Ions which emerge from the ion mobility spectrometer or separator are then preferably fragmented in a collision cell. The arrival time or ion mobility elution profiles of the resulting fragment ions are preferably analysed.

If two or more substantially different arrival time distributions or ion mobility elution profiles are observed within the fragment -13 -ion population then the ions of interest are considered as comprising two different species of parent ions having different molecular conformations.

According to the preferred embodiment an ion mobility spectrometer or separator is provided. Ion species are preferably driven or urged through a background neutral gas under the influence of an electric field within the ion mobility spectrometer or separator. The ions are preferably caused to separate temporally according to their differing ion mobilities.

The mobility of a particular ion species will be dependent upon a number factors including the charge state, mass and the interaction cross section of the ion with background gas molecules. Ion species having the same or substantially similar mass to charge ratios but different molecular conformations may be separated or resolved due to the ions having different interaction cross-sections with the neutral gas.

According to an embodiment isobaric a-ions or b-ions having different structures or molecular conformations may be separated or distinguished by ion mobility separation followed by ion fragmentation.

According to an embodiment a mass spectrometer may be operated either in an IMS/MS mode or an IMS/MS/MS mode. In an IMS/MS mode of operation ions are first separated temporally according to their ion mobility in an ion mobility separator or spectrometer. Ions emerging from the ion mobility separator or spectrometer are then mass analysed and the arrival time or ion mobility profile of the parent ions may be determined. In an * ** ***. IMS/MS/MS mode of operation ions are first separated temporally according to their ion mobility in an ion mobility separator or **..

spectrometer. Ions emerging from the ion mobility separator or spectrometer are then subjected to fragmentation in a collision or * fragmentation cell. The resulting fragment ions are then mass analysed and the arrival time or effective ion mobility or elution : *. profile of the fragment ions may be determined. A strong * * 35 confirmation of the structure of parent and fragment ions can * * preferably be provided according to the preferred embodiment.

This in turn enables the structural variety of various species of ions to be determined.

-14 -The preferred embodiment enables a significant enhancement in the understanding of the fragmentation properties of gas-phase peptide ions to be achieved and increases the power of MS/MS mass spectrometry techniques in applications such as proteome characterisation.

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. lA shows an ion mobility distribution for the (M+H) parent ion of YAGFL-N1-12 having a mass to charge ratio of 569.2 together with various (labelled) first generation a-fragment ions and first generation b-fragment ions derived from the parent ion and Fig. lB shows arrival time distributions of two parent ions having two different molecular conformations together with certain first generation fragment ions; Fig. 2 shows an arrival time distribution for first generation a4 fragment ions together with arrival time distributions for two second generation fragment ions; and Fig. 3 shows a mass spectrum of fragment ions derived from fragmenting linear YAGFL-NH2 parent ions (having a mass to charge ratio of 569.2).

A preferred embodiment of the present invention will now be described in more detail. In order to illustrate various aspects of the preferred embodiment, the ion mobility and fragmentation characteristics of [d-ala(2)]-leucine enkephalin ("YAGFL") peptide ions were studied.

The studies detailed below confirmed that YAGFL parent ions existed both as linear protonated YAGFL-NH2 ions and as cyclo- (YAGFL) parent peptide ions. Fragment ions resulting from * S..

fragmenting both molecular conformations of YAGFL parent ions were :. investigated and compared using a Waters Synapt HDMS (RTM) mass S. * :. spectrometer (Waters Corp., Manchester, UK). The mass spectrometer comprised a first ion guide (which may be operated as : ** a collision cell in a mode of operation), a quadrupole mass S.. * filter, an ion mobility spectrometer or separator, a second ion * * S guide (which may be operated as a collision cell in a mode of operation) and an orthogonal acceleration Time of Flight mass analyser. Samples were introduced into the mass spectrometer by infusion from an Electrospray ionization source.

-15 -The ion mobility spectrometer or separator comprised a plurality of electrodes having apertures through which ions were transmitted in use. One or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms were applied to the electrodes of the ion mobility spectrometer or separator so that ions were urged along the length of the ion mobility spectrometer or separator. The ion mobility spectrometer or separator was maintained at a pressure of 0.5 mbar of nitrogen.

The wave parameters of the one or more transient DC voltages or potentials which were applied to the electrodes of the ion mobility spectrometer or separator were optimized so as to ensure that the ion arrival time distributions of mobility-separated ion species were less than the 13 ms acquisition time per mobility experiment.

First generation fragment ions were obtained by fragmehting parent or precursor ions in the first ion guide (which was operated as a collision cell) arranged upstream both of the quadrupole mass filter and the ion mobility spectrometer or separator.

Fig. lA shows ion mobility distributions for (M+}fl* parent ions of YAGFL-NH2. The parent YAGFL-NH2 ions have a mass to charge ratio of 569.2 and although not labelled in Fig. lA can be seen above the ions identified as b5 fragment ions (which have a mass to charge ratio of 552.2).

Fig. 1A also shows the ion mobility distributions of various first generation a-fragment ions and first generation b-fragment ions resulting from the fragmentation of (M+H)' parent ions of * *.

s.. YAGFL-NH2. * S..

Fig. lB shows arrival time distributions of linear YAGFL-NH2 *:. parent ions together with corresponding first generation a4, b4 S. * and b5 fragment ions. It is apparent from Fig. lB that the * arrival time distribution of first generation b5 fragment ions : *** having a mass to charge ratio of 552.2 is displaced to a shorter 5.5 arrival time when compared with the arrival time distribution of I.....

* linear YAGFL-NH2 parent or precursor ions which have a substantially similar mass to charge ratio of 569.2. The small mass to charge ratio difference between the linear YAGFL-NH2 parent ions (569.2) and the first generation b5 fragment ions -16 - (552.2) is predicted as having a negligible effect on ion mobility and hence the resulting arrival time. Therefore, since both species are singly charged and have substantially similar mass to charge ratios then the observed difference in arrival time and hence their ion mobility is, according to the preferred embodiment, attributed to structural or conformational differences between the linear parent ions and the first generation b5 fragment ions. As an initial assumption it is assumed that the b fragment ions have a non-linear (e.g. cyclic or other) structure.

In order to test this assumption, Fig. lB also shows the arrival time distribution for cyclo-(YAGFL) parent ions having a mass to charge ratio of 552.2. The ion mobility data for cyclo- (YAGFL) parent ions was derived from a separate experiment and the arrival time distribution for cyclo-(YAGFL) parent ions has been superimposed upon the other arrival time distributions shown in Fig. lB.

The difference in the arrival time distribution between cyclo-(YAGFL) parent ions and linear YAGFL-NH2 parent ions is consistent with the cyclo-(YAGFL) parent ions having a smaller collision cross-section and hence a more compact structure relative to that of the linear YAGFL-NH2 parent ions.

Replicate experiments (data not shown) indicated high analysis precision with the difference between the arrival time distributions recorded for t.he same ion in separate experiments being much less than the difference between the arrival time distributions of the linear and cyclic peptide ions.

With regard to Fig. lB. it is noticeable that the arrival * time distribution of the first generation b5 fragment ions (having a mass to charge ratio of 552.2) is almost indistinguishable from that of the cyclo-(YAGFL) parent ions (which also have a mass to *: charge ratio of 552.2). This strongly suggests that first generation b5 fragment ions possess a compact and most likely cyclic structure which may be substantially similar to that of the : *. cyclo-(YAGFL) parent ions. This observation also suggests that *** * first generation b5 fragment ions can exist in cyclic form and can * serve as a precursor for second generation fragment ions which The mobility data obtained suggests that cyclic forms of ions are stable at least on a millisecond time scale. In further -17 -experiments first generation b5 fragment ions (which were believed to have a cyclic,structure) and cyclo-(YAGFL) parent ions were mass selectively selected and compared by collision induced fragmentation and mobility separation of the resulting product ions. No differences were observed (data not shown) between the arrival time distributions or relative signal intensities of equivalent fragment ions from the two precursor species.

It is also apparent from the data shown in Fig. lB that there is a difference in the mobilities of two other first generation fragment ions which have otherwise substantially similar mass to charge ratios. The arrival time distributions for first generation a4 fragment ions and first generation b4 fragment ions are shown in Fig. lB. It can be seen that the first generation a4 fragment ions have a wider arrival time distribution than corresponding first generation b4 fragment ions. An aspect of the preferred embodiment is that by analysing and/or comparing the arrival time or ion mobility or elution distributions of first generation fragment ions (or other ions), certain first generation fragment ions (or other ions) may be flagged for further investigation. In particular, flagged ions may be investigated to see or confirm whether or not they have one or more particular molecular conformations.

According to the preferred embodiment the increased width of the first generation a4 fragment ions relative to the width of the arrival time distribution for first generation b4 fragment ions suggests that first generation a4 fragment ions may exist or be present in multiple conformations. However, the ion mobility * ** s. resolution of the ion mobility spectrometer or separator is insufficient to be able to separate or resolve discrete components * *s of the first generation a4 ion peak into two or more resolved ion peaks (assuming of course that the ion peak does in fact relate to I. * ions having two different molecular conformations).

According to the preferred embodiment if an ion peak : .. observed in an ion mobility spectrum is suspected of relating to *S. * * 35 two or more (unresolved) species of ions having substantially the **.*.

same or similar mass to charge ratios but possibly having two or more different conformations, then the ion species is preferably further investigated in a subsequent experimental run. According to the preferred embodiment the ions (e.g. first generation -18 -fragment ions) which were unresolved by the ion mobility spectrometer or separator are transmitted again to the ion mobility spectrometer or separator and the ions which emerge from the ion mobility spectrometer are fragmented in a collision cell which is preferably arranged downstream of the ion mobility spectrometer or separator.

According to an embodiment as ions of interest exit the ion mobility spectrometer or separator the ions are preferably subjected to an accelerating voltage. The ions of interest preferably undergo collisional activation in an ensuing lower pressure region of the mass spectrometer such as an ion guide or collision cell arranged downstream of the ion mobility spectrometer or separator. Precursor and corresponding fragment ions are preferably assigned through correlation of their respective arrival time distributions.

In order to illustrate certain aspects of the preferred embodiment first generation a4 fragment ions which were believed, from an analysis of the arrival time distribution shown in Fig. lB and discussed above, to co-exist in two different conformations, were selected by a quadrupole mass filter arranged upstream of an ion mobility spectrometer or separator for further investigation.

According to this embodiment parent ions were first fragmented in a first ion guide or collision cell and then desired first generation fragment ions of interest were onwardly transmitted by the mass filter to the ion mobility spectrometer or separator whilst undesired fragment ions were attenuated.

First generation a4 fragment ions of interest having a mass to charge ratio of 411.2 were selectively transmitted by the mass * *,,* filter. The first generation a4 fragment ions of interest were * 30 then passed to an ion mobility spectrometer or separator.

:, However, as expected, the ion mobility spectrometer or separator

S

was unable to resolve the first generation a4 fragment ions into * groups of ions having different conformations. However, in a * ,* subsequent experimental run the first generation a4 fragment ions * * * 35 were again passed to the ion mobility spectrometer and the ions *S*..* * a which emerged from the ion mobility spectrometer or Separator were then arranged to be fragmented in an ion guide or collision cell which was arranged downstream of the ion mobility spectrometer or -19 -separator. The resulting second generation fragment ions were then mass analysed and their arrival time profiles analysed.

Fig. 2 shows an arrival time distribution for the first generation a4 fragment ions together with corresponding arrival time distributions for two particular species of second generation fragment ions. One of the second generation fragment ions [YA- 28+H] resulting from the fragmentation of the first generation a4 fragment ions [YAGF-28+HJ has a mass to charge ratio of 207.1.

The other second generation fragment ion shown [YAF-28-17-28+H] was determined to have mass to charge ratios of 309.2.

It is apparent from Fig. 2 that the arrival time distribution of the second generation fragment ions having a mass to charge ratio of 207.1 is similar to the arrival time distribution of the first generation a4 fragment ions. However, the arrival time distribution of the second generation fragment ions having a mass to charge ratio of 309.2 is observed as being shifted towards the leading edge of the first generation a4 peak.

The arrival time peak for the second generation fragment ions having a mass to charge ratio of 309.2 is also notably narrower.

These results strongly suggest (or effectively confirm) that the first generation a4 fragment ions co-exist in two different conformations.

Further experiments were performed to investigate this further and to provide further confirmation. Subsequent experiments showed that the second generation fragment ions [YAF- 28-17-28-i-H]4 having a mass to charge ratio of 309.2 were only formed when cyclic first generation a4 fragment ions were fragmented. However, second generation fragment ions [YA-28+H]' having a mass to charge ratio of 207.1 were observed either when . cyclic first generation a4 fragment ions were fragmented or when * linear first generation a4 fragment ions were fragmented. 00 *

:. Other second generation fragment ions were observed when fragmenting either linear or cyclic precursor ions. These second : ., generation fragment ions include [YAG+HJ ions having a mass to charge ratio of 292.1, [YA+H) ions having a mass to charge ratio *.S...

* of 235.1, and [GF-28+HY ions having a mass to charge ratio of 177.1. These second generation fragment ions yield a similar arrival time distribution profile as the second generation -20 -fragment ions having a mass to charge ratio of 207.1 as shown in Fig. 2.

However, some other second generation fragment ions were only observed when fragmenting a cyclic precursor ion. These second generation fragment ions include [YAF-28-l7+H] ions having a mass to charge ratio of 337.2. The second generation fragment ions yield similar arrival time distribution profiles as the second generation fragment ions [YAF-28-17-28+H]having a mass to charge ratio of 309.2 as shown in Fig. 2.

These results indicate that the first generation a4 fragment ions which were analysed comprised a heterogeneous population of both linear a4 fragment ions and also cyclic a4 fragment ions.

An important aspect of the preferred embodiment therefore is the step of fragmenting unresolved precursor ions (e.g. first generation fragment ions) after ion mobility separation. This provides the general capability of interrogating an ion population through a mobility distribution and enables an increase in the effective resolution of the ion mobility separation to be achieved.

Further experiments involving the fragmentation of other first generation fragment ions such as first generation b4, b5 and a5 fragment ions after ion mobility separation also suggest a mixture of structures for each ion (data not shown).

The experimental evidence presented above provides strong evidence for the formation of stable macrocyclic structures for b5 fragment ions generated by fragmentation of protonated linear YAGFL-NH2 precursor ions. Additionally, it is apparent that first generation a4 fragment ions resulting from fragmentation of protonated YAGFL-NH2 ions have at least two different structures.

One of the species of ions has a relatively high ion mobility * which is attributed to a macrocyclic structure and is shown to S. * give rise to second generation fragment ions having arrival time distributions which are also consistent with having a cyclic : .*. precursor. S.. *

The preferred embodiment confirms the value of combined ion S.....

* 1 mobility separation and subsequent fragmentation of ion species to investigate further the detailed fragmentation mechanisms of peptide ions. In particular, the ability to generate and analyse fragment ions after precursor ions have been separated according -21 -to their ion mobility enables an effective increase in ion mobility resolution to be achieved.

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. * S. ** . * *5S* * S *

*S. -, * ** I,

S **.

S * S. * J. * : S *

Claims

-22 -Claims 1. A method of mass spectrometry comprising: separating first ions in an ion mobility spectrometer or separator into one or more ion mobility fractions; estimating, predicting, recognising or determining whether or not first ions in an ion mobility fraction may comprise multiple structures and/or molecular conformations; temporally separating at least some first ions of interest in an ion mobility spectrometer or separator; fragmenting said first ions of interest which emerge from said ion mobility spectrometer or separator in a collision, fragmentation or reaction device to produce a plurality of second ions; analysing one or more characteristics of one or more of said second ions; and determining or confirming based upon the analysis of said one or more characteristics of said one or more second ions whether or not said first ions of interest have or are likely to have either: (i) a single structure and/or molecular conformation; or (ii) multiple structures and/or molecular conformations.
2. A method as claimed in claim 1, wherein either: (i) said first ions in said ion mobility fraction comprise ions having a single structure and/or molecular conformation; or (ii) said first ions in said ion mobility fraction comprise * *, ions having multiple structures and/or molecular conformations which are substantially unresolved by said ion mobility **** spectrometer or separator.

.. :
3. A method as claimed in claim 1 or 2, wherein: (i) said first ions comprise parent or precursor ions or first or subsequent generation fragment ions; and/or (ii) said second ions comprise first generation fragment ions or second or subsequent generation fragment ions and/or (iii) said first ions of interest comprise first ions in an ion mobility fraction which are estimated, predicted, recognised or determined to comprise multiple structures and/or molecular conformations; and/or -23 - (iv) said first ions of interest comprise first ions in an ion mobility fraction which are estimated, predicted, recognised or determined to comprise a single structure and/or molecular conformation.
4. A method as claimed in claim 1, 2 or 3, wherein said step of analysing one or more characteristics of one or more of said second ions comprises: (1) measuring or determining the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of said second IOnS; and/or (ii) comparing the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of said second ions with other second ions and/or with the arrival time distributions, mean or average arrival times and/or arrival time profiles of one or more of said first ions; and/or (iii) determining whether or not said one or more second ions have substantially different arrival time distributions, mean or average arrival times and/or arrival time profiles.
5. A method as claimed in claim 4, wherein: (i) if it is determined that two or more of said second ions have substantially different arrival time distributions, mean or average arrival times and/or arrival time profiles then it is determined or confirmed that said first ions of interest have multiple structures and/or molecular conformations; and/or (ii) if it is determined that two or more of said second ions have only one arrival time distribution, mean or average * *. arrival time and/or arrival time profile then it is determined " 30 that said first ions of interest have a single structure and/or S...molecular conformation. *..

:
6. A method as claimed in any preceding claim, wherein said first ions and/or said second ions comprise: (i) one or more biopolymers, proteins, peptides, :.:. polypeptides, oligionucleotides, oligionucleosides, amino acids, carbohydrates, sugars, lipids, fatty acids, vitamins, hormones, portions or fragments of DNA, portions or fragments of cDNA, portions or fragments of RNA, portions or fragments of mRNA, portions or fragments of tRNA, polyclonal antibodies, monoclonal antibodies, ribonucleases, enzymes, metabolites, polysaccharides, -24 -phosphorolated peptides, phosphorolated proteins, glycopeptides, glycoproteins or steroids and/or fragments thereof; and/or (ii) biomolecules bound to polyethelene glycol (PEG) and/or fragments thereof; and/or (iii) antibodies and/or fragments thereof; and/or (iv) pegylated proteins and/or fragments thereof; and/or (v) antibodies encapsulated in or protected by polyethelene glycol (PEG) and/or fragments thereof; and/or (vi) polyethelene glycol (PEG) and/or fragments thereof; and/or (vii) multiple conformations of polyethelene glycol (PEG) and/or fragments thereof; and/or (viii) PEG modified proteins, lipsomes or nanoparticles and/or fragments thereof.
7. A method as claimed in any preceding claim, wherein said first ions correspond with a first sample and either: (i) said first sample is taken from a diseased organism and/or a non-diseased organism; (ii) said first sample is taken from a treated organism and/or a non-treated organism; or (iii) said first sample is taken from a mutant organism and/or from a wild type organism.
8. A mass spectrometer comprising: an ion mobility spectrometer or separator; a collision, fragmentation or reaction device arranged downstream of said ion mobility spectrometer or separator; and a control system arranged and adapted: (i) to pass first ions to said ion mobility spectrometer or * ,* separator so that said first ions are separated into one or more ion mobility fractions; S...(ii) to estimate, predict, recognise or determine whether or not first ions in an ion mobility fraction may comprise multiple : structures and/or molecular conformations; *. (iii) to cause at least some first ions of interest to be separated temporally in said ion mobility spectrometer or separator; *....: (iv) to cause first ions of interest which emerge, in use, * from said ion mobility spectrometer or separator to be fragmented in said collision, fragmentation or reaction device to produce a plurality of second ions; -25 - (v) to analyse one or more characteristics of one or more of said second ions; and (vi) to determine or confirm based upon the analysis of said one or more characteristics of said one or more second ions whether or not said first ions of interest have or are likely to have either: (a) a single structure and/or molecular conformation; or (b) multiple structures and/or molecular conformations.
9. A mass spectrometer as claimed in claim 8, wherein said control system is arranged and adapted: (i) to measure or determine the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of said second ions; and/or (ii) to compare the arrival time distributions, mean or average arrival times and/or arrival time profiles of at least some of said second ions with other second ions and/or with the arrival time distributions, mean or average arrival times and/or arrival time profiles of one or more of said first ions; and/or (iii) to determine whether or not said one or more second ions have two or more different arrival time distributions, mean or average arrival times and/or arrival time profiles.
10. A mass spectrometer as claimed in claim 8 or 9, wherein said ion mobility spectrometer comprises: (i) a gas phase electrophoresis device; and/or (ii) a drift tube wherein an axial DC voltage gradient is maintained, in use, along at least a portion of said drift tube; and/or (iii) one or more multipole rod sets; and/or (iv) a plurality of electrodes through which ions are transmitted in use; and/or (v) a plurality of plate or mesh electrodes and wherein at * * S * least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of said plate or mesh electrodes are arranged generally in the plane in which ions travel in use. * S. * S * *.. .

*..
11. A mass spectrometer as claimed in claim 8, 9 or 10, wherein said ion mobility spectrometer or separator is axially segmented.
12. A mass spectrometer as claimed in any of claims 8-11, further comprising: -26 - (i) DC voltage means arranged and adapted to maintain a substantially constant DC voltage gradient along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said ion mobility spectrometer or separator in order to urge ions along the axial length of said ion mobility spectrometer or separator; and/or (ii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes forming said ion mobility spectrometer or separator in order to urge at least some ions along at least a portion of the axial length of said ion mobility spectrometer or separator; and/or (iii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said ion mobility spectrometer or separator.
13. A mass spectrometer as claimed in any of claims 8-12, further comprising means arranged and adapted to maintain at least a portion of said ion mobility spectrometer or separator at a pressure selected from the group consisting of: (i) > 1.0 x 10 mbar; (ii) > 1.0 x 102 mbar; (iii) > 1.0 x l01 mbar; (iv) > 1 mbar; (v) > 10 mbar; (vi) > 100 nthar; (vii) > 5.0 x i0 mbar; (viii) > 5.0 x 10 mbar; (ix) 103_102 mbar; and (x) 10-10' rnbar. * ** 30
14. A mass spectrometer as claimed in any of claims 8-13, **.* wherein said fragmentation, collision or reaction device comprises either: **** : (i) a multipole rod set; and/or (ii) plurality of electrodes through which ions are transmitted in use; and/or : (iii) a plurality of plate or mesh electrodes and wherein at *....: least 50%, 555, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of * * said plate or mesh electrodes are arranged generally in the plane in which ions travel in use.-27 -
15. A mass spectrometer as claimed in any of claims 8-14, wherein said fragmentation, collision or reaction device is axially segmented.
16. A mass spectrometer as claimed in any of claims 7-15, further comprising: (1) a DC voltage means arranged and adapted to maintain a substantially constant DC voltage gradient along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said fragmentation, collision or reaction device in order to urge ions along the axial length of said fragmentation, collision or reaction device; and/or (ii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes forming said fragmentation, collision or reaction device in order to urge ions along the axial length of said fragmentation, collision or reaction device; and/or (iii) transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to electrodes along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said fragmentation, collision or reaction device.
17. A mass spectrometer as claimed in any of claims 8-16, further comprising a device arranged and adapted to maintain at least a portion of said fragmentation, collision or reaction device at a pressure selected from the group consisting of: (i) > 1.0 x l0 mbar; (ii) > 1.0 x 102 mbar; (iii) > 1.0 x 10' mbar; (iv) > 1 mbar; (v) > 10 mbar; (vi) > 100 mbar; (vii) > 5.0 x i0 S. mbar; (viii) > 5.0 x l02 mbar; (ix) i03_i02 mbar; and (x) 10-10 mbar.
18. A mass spectrometer as claimed in any of claims 8-17, wherein said control system is arranged and adapted to switch or repeatedly switch said fragmentation, collision or reaction device between a first mode of operation wherein ions are substantially fragmented and a second mode of operation wherein substantially fewer or no ions are fragmented.-28 -
19. A mass spectrometer as claimed in claim 18, wherein: (a) in said first mode of operation ions exiting said ion mobility spectrometer or separator are accelerated through a potential difference selected from the group consisting of: (1) �= V; (ii) �= 20 V; (iii) �= 30 V; (iv) �= 40 V; (v) �= 50 V; (vi) �= V; (vii) �= 70 V; (viii) �= 80 V; (ix) �= 90 V; and (x) �= 100 V; and/or (b) in said second mode of operation ions exiting said ion mobility spectrometer or separator are accelerated through a potential difference selected from the group consisting of: (i) �= V; (ii) �= 15 V; (iii) �= 10 V; (iv) �= SV; and (v) �= 1V; and/or (c) said control system is arranged and adapted to switch said fragmentation, collision or reaction device between said first mode of operation and said second mode of operation at least once every 1 ins, 5 ms, 10 ins, 15 ms, 20 ins, 25 ms, 30 ins, 35 ins, ins, 45 ins, 50 ins, 55 ins, 60 ms, 65 ms, 70 ms, 75 ins, 80 ins, 85 ins, 90 ins, 95 ins, 100 ins, 200 ins, 300 ins, 400 ins, 500 ins, 600 ins, 700 ms, 800 ms, 900 ms, 1 s, 2 s, 3 S1 4 s, 5 s, 6 S1 7 s, 8 S, 9 s or 10 S.
20. A mass spectrometer as claimed in any of claims 8-19, wherein said collision, fragmentation or reaction device is 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 : 35 fragmentation device; (xii) an ion-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; -29 - (xix) an ion-atom reaction fragmentation device; (xx) an ion- Inetastable ion reaction fragmentation device; (xxi) an ion- metastable molecule reaction fragmentation device; (xxii) an ion-metastable atom reaction fragmentation device; (xxiii) an ion-ion reaction device for reacting ions to form 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 lonisation Dissociation ("EID") fragmentation device.
21. A mass spectrometer as claimed in any of claims 8-20, further comprising either: (a) an ion source selected from the group consisting of: (i) an Electrospray ionisation ("ESI") ion source; (ii) an Atmospheric Pressure Photo lonisation ("APPI") ion source; (iii) an Atmospheric Pressure Chemical lonisation ("APCI") ion source; (iv) a Matrix Assisted Laser Desorption lonisation ("MALDI") ion source; (v) a Laser Desorption lonisation ("LDI") ion source; (vi) an Atmospheric Pressure lonisation ("API") ion source; (vii) a Desorption lonisation on Silicon ("DIOS") ion source; (viii) an Electron Impact ("El") ion source; (ix) a Chemical lonisation ("CI") ion source; Cx) a Field lonisation ("Fl") ion source; (xi) a Field Desorption ("FD") ion source; (xii) an Inductively Coupled Plasma ("ICE") ion source; (xiii) a Fast Atom Bombardment ("FAD") ion source; (xiv) a Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xv) a Desorption Electrospray lonisation ("DESI") ion source; (xvi) a Nickel-63 radioactive ion source; : (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption lonisation ion source; and (xviii) a Thermospray ion source; and/or (b) a continuous or pulsed.ion source; and/or (c) one or more ion guides arranged upstream and/or downstream of said ion mobility spectrometer or separator and/or said collision, fragmentation or reaction device; and/or (d) one or more ion traps or one or more ion trapping regions arranged upstream and/or downstream of said ion mobility -30 -spectrometer or separator and/or said collision, fragmentation or reaction device; and/or (e) a mass analyser arranged upstream and/or downstream of said ion mobility spectrometer or separator and/or said collision, fragmentation or reaction device, said mass analyser being 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 (f) one or more energy analysers or electrostatic energy analysers arranged upstream and/or downstream of said ion mobility spectrometer or separator and/or said collision, fragmentation or reaction device; and/or (g) one or more ion detectors arranged upstream and/or downstream of said ion mobility spectrometer or separator and/or said collision, fragmentation or reaction device; and/or (h) one or more mass filters arranged upstream and/or downstream of said ion mobility spectrometer or separator and/or said collision, fragmentation or reaction device, wherein said one or more mass filters are selected from the group consisting of: (i) a quadrupole mass filter; (ii) a 2D or linear quadrupole ion * *.**** 30 trap; (iii) a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; Cv) an ion trap; (vi) a magnetic sector mass filter; (vii) a Time of Flight mass filter; and (viii) a Wein filter; and/or (1) a device or ion gate for pulsing ions into said ion mobility spectrometer or separator and/or said collision, : 35 fragmentation or reaction device; and/or *** * (j) a device for converting a substantially continuous ion beam into a pulsed ion beam.
22. A mass spectrometer as claimed in any of claims 8-21, wherein said mass spectrometer further comprises: a C-trap; and -31 -an orbitrap mass analyser; wherein in a first mode of operation ions are transmitted to said C-trap and are then injected into said orbitrap mass analyser; and wherein in a second mode of operation ions are transmitted to said C-trap and then to a collision cell wherein at least some ions are fragmented into fragment ions, and wherein said fragment ions are then transmitted to said C-trap before being injected into said orbitrap mass analyser.
23. A computer program executable by the control system of a mass spectrometer comprising an ion mobility spectrometer or separator and a collision, fragmentation or reaction device, said computer program being arranged to cause said control system: (1) to pass first ions to said ion mobility spectrometer or separator so that said first ions are separated into one or more ion mobility fractions; (ii) to estimate, predict, recognise or determine whether or not first ions in an ion mobility fraction may comprise multiple structures and/or molecular conformations; (iii) to cause at least some first ions of interest to be separated temporally in said ion mobility spectrometer or separator; (iv) to cause first ions of interest which emerge, in use, from said ion mobility spectrometer or separator to be fragmented in said collision, fragmentation or reaction device to produce a plurality of second ions; (v) to analyse one or more characteristics of one or more of said second ions; and :.:: 30 (vi) to determine or confirm based upon the analysis of said one or more characteristics of said one or more second ions *:::: whether or not said first ions of interest have or are likely to have either: (a) a single structure and/or molecular conformation; S..* or (b) multiple structures and/or molecular conformations. : *.*
24. A computer readable medium comprising computer executable instructions stored on said computer readable medium, said instructions being arranged to be executable by a control system of a mass spectrometer comprising an ion mobility spectrometer or separator and a collision, fragmentation or reaction device, to cause said control system: -32 - (i) to pass first ions to said ion mobility spectrometer or separator so that said first ions are separated into one or more ion mobility fractions; (ii) to estimate, predict, recognise or determine whether or not first ions in an ion mobility fraction may comprise multiple structures and/or molecular conformations; (iii) to cause at least some first ions of interest to be separated temporally in said ion mobility spectrometer or separator; (iv) to cause first ions of interest which emerge, in use, from said ion mobility spectrometer or separator to be fragmented in said collision, fragmentation or reaction device to produce a plurality of second ions; (v) to analyse one or more characteristics of one or more of said second ions; and (vi) to determine or confirm based upon the analysis of said one or more characteristics of said one or more second ions whether or not said first ions of interest have or are likely to have either: (a) a single structure and/or molecular conformation; or (b) multiple structures and/or molecular conformations.
25. A computer readable medium as claimed in claim 24, wherein said computer readable medium is selected from the group consisting of: (i) a ROM; (ii) an EAROM; (iii) an EPROM; (iv) an EEPROM; (v) a flash memory; and (vi) an optical disk. * .* * * * * S. S... * * *.*. *SSS * . S S. * ** * S. * S S S.. *S*SSS*SS