Classifications

• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/06—Electron- or ion-optical arrangements
  • H01J49/062—Ion guides

Definitions

• Mass spectrometry has firmly established itself as the primary technique for identifying proteins due to its unparalleled speed, sensitivity and specificity. Strategies can involve either analysis of the intact protein or more commonly digestion of the protein using a specific protease that cleaves at predictable residues along the peptide backbone. This provides smaller stretches of peptide sequence which are more amenable to analysis via mass spectrometry.
• the mass spectrometry technique providing the highest degree of specificity and sensitivity is Electrospray Ionisation ("ESI") interfaced to a tandem mass spectrometer allowing fragmentation studies by low energy MS/MS.
• ESI Electrospray Ionisation
• These experiments involve separation of the complex digest mixture by microcapillary liquid chromatography with on-line mass spectral detection using automated acquisition modes whereby conventional MS and MS/MS spectra are collected in a data dependant manner.
• This information can be used directly to search databases for matching sequences leading to identification of the parent protein.
• This approach has recently allowed the identification of proteins that are present at low endogenous concentrations.
• the limiting factor for identification of the protein is not the quality of the MS/MS spectrum produced but is the initial identification of the multiply charged peptide precursor ion in the MS mode.
• Fig. 1 shows a conventional mass spectrum and shows how doubly charged species may be obscured in a singly charged background.
• the voltage pulse of a single ion must be high enough to trigger the discriminator and so register the arrival of an ion.
• the detector producing the voltage may be an electron multiplier or MicroChannel Plate detector ("MCP"). These detectors are charge sensitive so the size of signal they produce increases with increasing charge state. Discrimination in favour of higher charge states may therefore be accomplished by either increasing the discriminator voltage level of the TDC and/or by lowering the detector gain or a combination of both.
• Fig. 2A shows a conventional mass spectrum obtained with an orthogonal acceleration Time of Flight mass spectrometer and Fig. 2B shows a corresponding mass spectrum obtained by lowering the gain of the ion detector. As can be seen from comparing Figs.
• one of the disadvantages of this technique is that lowering the gain and/or increasing the discriminator level decreases the detection efficiency for the desired charge state and hence the sensitivity is reduced. Furthermore, it is impossible pick out an individual charge state according to this method. All that can be done is to reduce the efficiency of detection of lower charge states with respect to higher charge states.
• a method of mass spectrometry comprising: trapping a plurality of ions in an AC or RF ion guide in the presence of a gas at a pressure P for a period of time T, wherein the product P x T is at least 1 mbar-ms.
• the product P x T is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 mbar-ms.
• the product P x T is less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10000 mbar-ms.
• T falls within a range selected from the group consisting of: (i) 50-100 ⁇ s; (ii) 100-150 ⁇ s; (iii) 150-200 ⁇ s; (iv) 200-250 ⁇ s; (v) 250-300 ⁇ s; (vi) 300-350 ⁇ s; (vii) 350-400 ⁇ s; (viii) 400-450 ⁇ s; (ix) 450-500 ⁇ s; (x) 500-550 ⁇ s; (xi) 550- 600 ⁇ s; (xii) 600-650 ⁇ s; (xiii) 650-700 ⁇ s; (xiv) 700-750 ⁇ s; (xv) 750-800 ⁇ s; (xvi) 800-850 ⁇ s; (xvii) 850-900 ⁇ s; (xviii) 900- 950 ⁇ s; and (xix) 950-1000 ⁇ s .
• T falls within a range selected from the group consisting of: (i) 1-2 ms; (ii) 2-3 ms; (iii) 3-4 ms; (iv) 4-5 ms; (v) 5-6 ms; (vi) 6-7 ms; (vii) 7-8 ms; (viii) 8-9 ms; and (ix) 9-10 ms .
• T falls within a range selected from the group consisting of: (i) 10-15 ms; (ii)
• T falls within a range selected from the group consisting of: (i) 100-110 ms; (ii) 110-120 ms; (iii) 120-130 ms; (iv) 130-140 ms; (v) 140-150 ms; (vi) 150-160 ms; (vii) 160-170 ms; (viii) 170-180 ms; (ix) 180-190 ms; and (x) 190-200 ms.
• T falls within a range selected from the group consisting of: (i) 200-250 ms; (ii) 250-300 ms; (iii) 300-350 ms; (iv) 350-400 ms; (v) 400-450 ms; (vi) 450-500 ms; (vii) 500-550 ms; (viii) 550-600 ms; (ix) 600-650 ms; (x) 650-700 ms; (xi) 700- 750 ms; (xii) 750-800 ms; (xiii) 800-850 ms; (xiv) 850-900 ms; (xv) 900-950 ms; and (xvi) 950-1000 ms .
• T is at least than: (i) 50 ⁇ s; (ii) 60 ⁇ s (iii) 70 ⁇ s; (iv) 80 ⁇ s; (v) 90 ⁇ s; or (vi) 100 ⁇ s .
• T is at least: (i) 200 ⁇ s; (ii) 300 ⁇ s (iii) 400 ⁇ s; (iv) 500 ⁇ s; (v) 600 ⁇ s; (vi) 700 ⁇ s; (vii) 800 ⁇ s; (viii) 900 ⁇ s; or (ix) 1000 ⁇ s .
• T is at least: (i) 2 ms; (ii) 3 ms (iii) 4 ms; (iv) 5 ms; (v) 6 ms; (vi) 7 ms; (vii) 8 ms; (viii) 9 ms; or (ix) 10 ms .
• T is at least: (i) 20 ms; (ii) 30 ms (iii) 40 ms; (iv) 50 ms; (v) 60 ms; (vi) 70 ms; (vii) 80 ms; (viii) 90 ms; or (ix) 100 ms.
• T is at least: (i) 100 ms; (ii) 200ms (iii) 300 ms; (iv) 400 ms; (v) 500 ms; (vi) 600 ms; (vii) 700 ms; (viii) 800 ms; or (ix) 900 ms .
• T is at least: (i) Is; (ii) 2s; (iii) 3s; (iv) 4s; (v) 5s; (vi) 6s; (vii) 8s; (viii) 9s; or (ix) 10s.
• T is less than: (i) 10s; (ii) 9s; (iii) 8s; (iv) 7s; (v) 6s; (vi) 5s; (vii) 4s; (viii) 3s; or (ix) 2s.
• T is less than: (i) 1000 ms; (ii) 900ms (iii) 800 ms; (iv) 700 ms; (v) 600 ms; (vi) 500 ms; (vii) 400 ms; (viii) 300 ms; or (ix) 200 ms .
• T is less than: (i) 100 ms; (ii) 90ms (iii) 80 ms; (iv) 70 ms; (v) 60 ms; (vi) 50 ms; (vii) 40 ms; (viii) 30 ms; or (ix) 20 ms .
• T is less than: (i) 10 ms; (ii) 9 ms (iii) 8 ms; (iv) 7 ms; (v) 6 ms; (vi) 5 ms; (vii) 4 ms; (viii) 3 ms; or (ix) 2 ms .
• T is less than: (i) 1000 ⁇ s; (ii) 900 ⁇ s (iii) 800 ⁇ s; (iv) 700 ⁇ s; (v) 600 ⁇ s; (vi) 500 ⁇ s; (vii) 400 ⁇ s; (viii) 300 ⁇ s; or (ix) 200 ⁇ s .
• T is less than: (i) 100 ⁇ s; (ii) 90 ⁇ s (iii) 80 ⁇ s; (iv) 70 ⁇ s; (v) 60 ⁇ s; or (vi) 50 ⁇ s .
• P falls within a range selected from the group consisting of: (i) 0.01-0.02 mbar; (ii) 0.02-0.03 mbar; (iii) 0.03-0.04 mbar; (iv) 0.04-0.05 mbar; (v) 0.05-0.06 mbar; (vi) 0.06-0.07 mbar; (vii) 0.07-0.08 mbar; (viii) 0.08-0.09 mbar; and (ix) 0.09-0.10 mbar.
• P falls within a range selected from the group consisting of: (i) 0.1-0.2 mbar; (ii) 0.2-0.3 mbar; (iii) 0.3-0.4 mbar; (iv) 0.4-0.5 mbar; (v) 0.5-0.6 mbar; (vi) 0.6- 0.7 mbar; (vii) 0.7-0.8 mbar; (viii) 0.8-0.9 mbar; and (ix) 0.9- 1.0 mbar.
• P falls within a range selected from the group consisting of: (i) 1-2 mbar; (ii) 2-3 mbar; (iii) 3-4 mbar;
• P falls within a range selected from the group consisting of: (i) 10-20 mbar; (ii) 20-30 mbar; (iii) 30-40 mbar; (iv) 40-50 mbar; (v) 50-60 mbar; (vi) 60- 70 mbar; (vii) 70-80 mbar; (viii) 80-90 mbar; and (ix) 90-100 mbar.
• P is at least: (i) 0.01 mbar; (ii) 0.02 mbar; (iii) 0.03 mbar; (iv) 0.04 mbar; (v) 0.05 mbar; (vi) 0.06 mbar; (vii) 0.07 mbar; (viii) 0.08 mbar; or (ix) 0.09 mbar.
• P is at least: (i) 0.1 mbar; (ii) 0.2 mbar; (iii) 0.3 mbar; (iv) 0.4 mbar; (v) 0.5 mbar; (vi) 0.6 mbar; (vii) 0.7 mbar; (viii) 0.8 mbar; or (ix) 0.9 mbar.
• P is at least: (i) 1 mbar; (ii) 2 mbar; (iii) 3 mbar; (iv) 4 mbar; (v) 5 mbar; (vi) 6 mbar; (vii) 7 mbar; (viii) 8 mbar; or (ix) 9 mbar.
• P is at least: (i) 10 mbar; (ii) 20 mbar; (iii) 30 mbar; (iv) 40 mbar; (v) 50 mbar; (vi) 60 mbar; (vii) 70 mbar; (viii) 80 mbar; (ix) 90 mbar; or (x) 100 mbar.
• P is less than: (i) 100 mbar; (ii) 90 mbar; (iii) 80 mbar; (iv) 70 mbar; (v) 60 mbar; (vi) 50 mbar; (vii) 40 mbar; (viii) 30 mbar; or (ix) 20 mbar.
• P is less than: (i) 10 mbar; (ii) 9 mbar; (iii) 8 mbar; (iv) 7 mbar; (v) 6 mbar; (vi) 5 mbar; (vii) 4 mbar; (viii) 3 mbar; or (ix) 2 mbar.
• P is less than: (i) 1 mbar; (ii) 0.9 mbar; (iii) 0.8 mbar; (iv) 0.7 mbar; (v) 0.6 mbar; (vi) 0.5 mbar; (vii) 0.4 mbar; (viii) 0.3 mbar; or (ix) 0.2 mbar.
• P is less than: (i) 0.10 mbar; (ii) 0.09 mbar; (iii) 0.08 mbar; (iv) 0.07 mbar; (v) 0.06 mbar; (vi) 0.05 mbar; (vii) 0.04 mbar; (viii) 0.03 mbar; or (ix) 0.02 mbar.
• P is selected from the group consisting of: (i) > 0.01 mbar; (ii) > 0.05 mbar; (iii) > 0.1 mbar; (iv) > 0.2 mbar; (v) > 0.5 mbar; (vi) > 1 mbar; (vii) > 2 mbar; (viii) > 5 mbar; and (ix) > 10 mbar.
• the sample of ions preferably comprises at least some ions having similar or substantially the same mass to charge ratios but different charge states.
• the at least some ions may have similar or substantially the same mass to charge ratios preferably wherein the mass to charge ratios differ by less than: (i) 20 mass to charge units; (ii) 15 mass to charge units; (iii) 10 mass to charge units; (iv) 5 mass to charge units; (v) 4 mass to charge units; (vi) 3 mass to charge units; (vii) 2 mass to charge units; and (viii) 1 mass to charge unit, wherein 1 mass to charge unit equals 1 dalton per unit of electronic charge.
• the plurality of ions may comprise a plurality of ionised molecules, the molecules comprising a plurality of different 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 mR A, portions or fragments of tRNA, polyclonal antibodies, monoclonal antibodies, ribonucleases, enzymes, metabolites, polysaccharides, phosphorolated peptides, phosphorolated proteins, glycopeptides, glycoproteins or steroids.
• a method of enhancing the relative proportion or abundance of multiply charged ions to singly charged ions in a sample of ions comprising: trapping the sample of ions in an AC or RF ion guide in the presence of a gas at a pressure P for a period of time T, wherein the product P x T is at least 1 mbar-ms .
• a method of separating analyte ions having a first charge state from background ions having a second charge state comprising : trapping a sample of ions in an AC or RF ion guide in the presence of a gas at a pressure P for a period of time T, wherein the product P x T is at least 1 mbar-ms .
• the first charge state comprises doubly charged ions and/or triply charged ions and/or quadruply charged ions and/or ions having a higher charge state.
• the second charge state comprises singly charged ions .
• At least some analyte ions preferably have a first mass to charge ratio and at least some background ions have a second mass to charge ratio, wherein the first mass to charge ratio differs from the second mass to charge ratio by less than 20, 15, 10, 5, 4, 3, 2 or 1 mass to charge units.
• a method of mass spectrometry comprising: providing a sample of singly charged ions and doubly charged ions having similar mass to charge ratios; onwardly transmitting doubly charged ions whilst at least partially relatively attenuating singly charged ions by trapping the sample of ions in an AC or RF ion guide in the presence of a gas at a pressure P for a period of time T, wherein the product P x T is at least 1 mbar-ms; and mass analysing the doubly charged ions .
• a method of discriminating against singly charged ions in favour of doubly charged ions and/or ions of higher charge states comprising: transmitting a sample of ions comprising singly charged ions and doubly charged ions and/or ions of higher charge state into an AC or RF ion guide; maintaining the AC or RF ion guide at a pressure P; and trapping the ions within the ion guide for a period of time T; wherein the product P x T is at least 1 mbar-ms.
• a method of separating ions having similar or substantially the same mass to charge ratios (m/z) on the basis of their charge state (z), comprising: trapping the ions within an AC or RF ion guide at a pressure P and for a period of time T, wherein the product P x T is at least 1 mbar-ms.
• the AC or RF ion guide comprises electrodes and the AC or RF ion guide has a central longitudinal axis, and wherein the combination of pressure and trapping time is such that singly charged ions are forced radially outwards from the central longitudinal axis whereas multiply charged ions are caused to forced towards the central longitudinal axis.
• the singly charged ions are preferably substantially ejected from or lost from the AC or RF ion guide, whereas at least some preferably a majority of the multiply charged ions are substantially retained within the AC or RF ion guide.
• one or more of the following groups of ions are substantially ejected from or lost from the AC or RF ion guide: (i) ions having 2 charges; (ii) ions having 3 charges; (iii) ions having 4 charges; (iv) ions having 5 charges; (v) ions having 6 charges; (vi) ions having 7 charges; (vii) ions having 8 charges; (viii) ions having 9 charges; (ix) ions having 10 charges; (x) ions having 11 charges; (xi) ions having 12 charges; (xii) ions having 13 charges; (xiii) ions having 14 charges; (xiv) ions having 15 charges; (xv) ions having 16 charges; (xvi) ions having 17 charges; (xvii)
• one or more of the following groups of ions are substantially retained with the AC or RF ion guide: (i) ions having 2 charges; (ii) ions having 3 charges; (iii) ions having 4 charges; (iv) ions having 5 charges; (v) ions having 6 charges; (vi) ions having 7 charges; (vii) ions having 8 charges; (viii) ions having 9 charges; (ix) ions having 10 charges; (x) ions having 11 charges; (xi) ions having 12 charges; (xii) ions having 13 charges; (xiii) ions having 14 charges; (xiv) ions having 15 charges; (xv) ions having 16 charges; (xvi) ions having 17 charges; (xvii) ions having 18 charges; (xviii) ions having 19 charges; (xix) ions having 20 charges; (xx) ions having 21 charges; (xxi) ions having 22 charges; and (xxii) ions having more than 22 charges.
• a method of removing unwanted singly charged background ions from a mixture of singly charged background ions and multiply charged analyte ions comprising: transmitting the mixture of ions to an AC or RF ion guide; trapping the ions within the AC or RF ion guide maintained at a pressure P; setting the period of time during which the ions are trapped within the AC or RF ion guide at a value such that at least 50%, 60%, 70%, 80%, 90% or more than 90% of said singly charged ions will be substantially ejected from or lost from the AC or RF ion guide whereas at least 50%, 60%, 70%, 80%, 90% or more than 90% of said multiply charged ions will be substantially maintained within the AC or RF ion guide.
• the product P x T is at least 1 mbar-ms .
• a method of removing or attenuating singly and/or doubly charged ions from a mixture of at least singly, doubly and triply charged ions comprising: trapping the mixture of ions within an AC or RF ion guide or ion trap maintained at a pressure P for a period of time T, wherein P x T is at least 1 mbar-ms.
• a mass spectrometer comprising: an ion trap comprising an AC or RF ion guide wherein in a mode of operation a plurality of ions are trapped in or otherwise prevented from leaving the ion guide in the presence of a gas at a pressure P for a period of time T, wherein the product P x T is at least 1 mbar-ms.
• the mass spectrometer preferably further comprises an ion source for generating mainly molecular or pseudo-molecular ions .
• the ion source may comprise an atmospheric pressure ionization source such as an ion source selected from the group comprising: (i) an Electrospray ionisation (“ESI”) ion source; (ii) an Atmospheric Pressure Chemical Ionisation (“APCI”) ion source; (iii) an Atmospheric Pressure Photo Ionisation (“APPI”) ion source; (iv) an atmospheric pressure Matrix Assisted Laser
• the ion source may comprise a non-atmospheric pressure ionization source such as an ion source selected from the group consisting of: (i) a Fast Atom Bombardment (“FAB”) ion source; (ii) a Liquid Secondary Ions Mass Spectrometry (“LSIMS”) ion source; (iii) a Matrix Assisted Laser Desorption Ionisation (“MALDI”) ion source; (iv) a Matrix Assisted Laser Desorption (“MALDI”) ion source in combination with a collision cell for collisionally cooling ions; (v) a Laser Desorption Ionisation (“LDI”) ion source; (vi) an Electron Impact (“El”) ion source; and (vii) a Chemical Ionisation (“Cl”) ion source.
• FAB Fast Atom Bombardment
• LIMS Liquid Secondary Ions Mass Spectrometry
• MALDI Matrix Assisted Laser Desorption Ionisation
• MALDI Matr
• the AC or RF ion guide comprises a multipole rod set e.g. a quadrupole rod set, a hexapole rod set, an octopole rod set or a rod set having ten or more rods.
• a multipole rod set e.g. a quadrupole rod set, a hexapole rod set, an octopole rod set or a rod set having ten or more rods.
• the AC or RF ion guide may comprise a plurality of electrodes having apertures through which the ions are transmitted.
• the AC or RF ion guide may comprise an ion tunnel having a plurality of electrodes each having substantially the same size aperture or an ion funnel having a plurality of electrodes wherein the size of the apertures becomes progressively smaller or larger.
• the AC or RF ion guide may comprise a double helix arrangement of electrodes.
• the AC or RF ion guide may comprise a plurality of plates stacked adjacent to each other.
• the mass spectrometer preferably comprises a mass analyzer such as a Time of Flight mass analyzer, a quadrupole mass analyzer, a 2D or 3D ion trap, a Fourier Transform mass spectrometer or a Fourier Transform Ion Cyclotron Resonance mass spectrometer.
• a mass analyzer such as a Time of Flight mass analyzer, a quadrupole mass analyzer, a 2D or 3D ion trap, a Fourier Transform mass spectrometer or a Fourier Transform Ion Cyclotron Resonance mass spectrometer.
• a mass spectrometer comprising a device for substantially removing unwanted singly charged ions from a mixture of singly charged ions and multiply charged ions, the device comprising an AC or RF ion guide which in a mode of operation is operated as an ion trap so that ions are trapped within the AC or RF ion guide for a period of time T, the AC or RF ion guide being maintained in use at a pressure P and wherein the product P x T is at least 1 mbar-ms .
• a mass spectrometer comprising: an ion source; a vacuum chamber housing an AC or RF ion guide maintained in use at a pressure P; an electrode, wherein in a first mode of operation the potential applied to the electrode causes ions to be substantially trapped within the AC or RF ion guide and wherein in a second mode of operation the potential applied to the electrode allows ions to be released from the ion guide; a further vacuum chamber housing a mass analyzer; and control means arranged to control the period of time T that ions are trapped within the AC or RF ion guide, wherein in a mode of operation the control means arranges that the trapping time T is such that the product P x T is at least 1 mbar-ms .
• the mass spectrometer preferably further comprises a further AC or RF ion guide arranged in a further vacuum chamber.
• a quadrupole mass filter and/or a collision cell may be arranged in a yet further vacuum chamber intermediate the vacuum chamber (s) housing the AC or RF ion guide (s) and the vacuum chamber housing the mass analyzer.
• the ion source may comprise an atmospheric pressure ion source and the mass analyzer may comprise a Time of Flight mass analyzer.
• the AC or RF ion guide and/or the further AC or RF ion guide comprises: (i) a multipole rod set; (ii) an ion funnel comprising a plurality of electrodes having apertures therein through which ions are transmitted, wherein the diameter of the apertures becomes progressively smaller or larger; (iii) an ion tunnel comprising a plurality of electrodes having apertures therein through which ions are transmitted, wherein the diameter of the apertures remains substantially constant; (iv) a double helix arrangement of electrodes; and (v) a stack of plates wherein adjacent electrodes are connected to opposite phases of an AC or RF supply.
• a mass spectrometer comprising: an ion source; a first AC or RF ion guide disposed in an upstream ion guide vacuum chamber, the first AC or RF ion guide being maintained at a pressure PI; a second AC or RF ion guide disposed in a downstream ion guide vacuum chamber, the second AC or RF ion guide being maintained at a pressure P2; and a mass analyser disposed in a further vacuum chamber, the further vacuum chamber being disposed downstream of the upstream ion guide vacuum chamber and the downstream ion guide vacuum chamber; wherein, in use, ions are arranged to be trapped in the first AC or RF ion guide for a time TI and/or ions are arranged to be trapped in the second AC or RF ion guide for a time T2 wherein PI x TI is at least 1 mbar-ms and/or P2 x T2 is at least 1 mbar-ms.
• the AC or RF ion guide and/or the further AC or RF ion guide comprises: (i) a multipole rod set; (ii) an ion funnel comprising a plurality of electrodes having apertures therein through which ions are transmitted, wherein the diameter of the apertures becomes progressively smaller or larger; (iii) an ion tunnel comprising a plurality of electrodes having apertures therein through which ions are transmitted, wherein the diameter of the apertures remains substantially constant; (iv). a double helix arrangement of electrodes; and (v) a stack of plates wherein adjacent electrodes are connected to opposite phases of an AC or RF supply.
• a method of mass spectrometry comprising: operating an AC or RF device in a first mode wherein the AC or RF device acts as an ion guide to substantially transmit ions received at an entrance to the device through to an exit of the device; and operating the AC or RF device in a second mode wherein the AC or RF device acts as an ion trap to substantially trap ions within the device and to substantially prevent the ions from exiting the device, wherein in the second mode the AC or RF device is maintained at a pressure P and ions are trapped within the AC or RF device for a period of time T, wherein the product P x T is at least 1 mbar-ms .
• the period of time T is a continuous or substantially continuous period of time.
• the period of time T is an accumulative period of time.
• a method of mass spectrometry comprising ejecting background ions from a mixture of ions by trapping the ions at a pressure > 0.01 mbar and for a time > 50 ⁇ s .
• a mass spectrometer comprising a device for ejecting background ions from a mixture of ions, the device being arranged to trap the ions at a pressure > 0.01 mbar and for a time > 50 ⁇ s .
• ions having a chosen charge state may be selected from a mixture of ions having differing charge states by trapping the ions in an RF device for a period of time and in the presence of a buffer gas at a particular pressure .
• Ions generated from an Electrospray Ionisation source typically contain a mixture of charge states.
• ions are usually generated at atmospheric pressure and admitted to the mass spectrometer through means of a pumping aperture that forms part of a differentially pumped vacuum system. In normal operation these ions continually stream through an RF device into regions of lower pressure by means of further dif erentially pumped regions which lead in turn to a mass analyser housed in an analyser vacuum chamber. The resulting mass spectrum therefore contains ions of all the charge states generated in the ionisation region of the instrument.
• ions can be trapped by raising the potential of this gate electrode higher than the body or reference DC potential of the AC or RF device.
• ions are preferably still able to enter the device at the upstream end through the differential pumping aperture and hence ions can build up in concentration. If the electrode voltage is reduced then the accumulated ions will be released.
• Fig. 1 shows how doubly charged ions may be obscured amongst a background of singly charged ions in a typical mass spectrum
• Fig. 2A shows a conventional mass spectrum and Fig. 2B shows a corresponding mass spectrum obtained by lowering the detector gain;
• Fig. 3A shows a schematic drawing of a collisional trapping charge state selector device according to the preferred embodiment and Fig. 3B shows a timing diagram for the voltage applied to an electrode adjacent the exit of the AC or RF device;
• Fig. 4A shows a mass spectrum of ions obtained by guiding ions through the AC or RF device without trapping the ions when the AC or RF device was maintained at a pressure of 1.4 mbar
• Fig. 4B shows a mass spectrum of ions obtained by guiding ions through the AC or RF device without trapping the ions when the AC or RF device was maintained at a pressure of 2.7 bar
• Fig. 4C shows a mass spectrum obtained wherein ions were trapped at a pressure of 1.4 mbar for 60 ms
• Fig. 4D shows a mass spectrum obtained according to the preferred embodiment wherein ions were trapped within the AC or RF device at a pressure of 2.7 mbar for 60 ms
• Fig. 5A is an expansion of Fig. 4B
• Fig. 5B is an expansion of Fig. 5D;
• Fig. 6A shows a plot of trapping time against pressure for which the ratio of the intensity of doubly charged ions from Gramacidin-S (m/z 571) to that of singly charged ions from Leucine Enkephalin (m/z 556) was doubled over that for no trapping
• Fig. 6B shows a plot of trapping time against pressure for which the ratio of the intensity of triply charged ions from Renin Substrate (m/z 586) to that of singly charged ions from Leucine Enkephalin (m/z 556) was doubled over that for no trapping;
• Fig. 7 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.64 mbar;
• Fig. 8 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.64 mbar;
• Fig. 9 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 1.64 mbar;
• Fig. 10 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.95 mbar;
• Fig. 11 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.95 mbar;
• Fig. 12 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 1.95 mbar;
• Fig. 13 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.23 mbar;
• Fig. 14 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.23 mbar;
• Fig. 15 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 2.23 mbar;
• Fig. 16 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.51 mbar;
• Fig. 17 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.51 mbar;
• Fig. 18 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 2.51 mbar;
• Fig. 19 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.86 mbar;
• Fig. 20 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.86 mbar; and Fig.
• 21 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 2.86 mbar.
• a preferred AC or RF ion guide/ion trap 5 will now be described in relation to Fig. 3A.
• Ions from an ion source 1 enter an upstream vacuum chamber 2 which may have an optional RF ion guide 3 arranged therein. However, such an ion guide 3 is not essential and may be omitted.
• the upstream vacuum chamber 2 is pumped by a pump. Ions pass through a differential pumping aperture 9 into an intermediate vacuum chamber 4.
• Another RF ion guide 5 is preferably provided in the intermediate vacuum chamber 4 and according to one embodiment this AC or RF ion guide 5 may be operated in one mode of operation as an ion trap.
• Ions may, for example, be trapped in the guide 5 by raising the potential of a differential pumping aperture 6 which separates the intermediate vacuum chamber from a downstream vacuum chamber 7 preferably housing another RF ion guide 8.
• the electric field resulting from the voltage applied to the differential pumping aperture 6 preferably extends into the downstream region of the intermediate ion guide 5 and hence has the effect of preventing ions from exiting the ion guide 5.
• a voltage may or may not be applied to an electrode adjacent an upstream end of the intermediate AC or RF ion guide 5.
• the differential pumping aperture 9 is preferably maintained at a higher DC potential than the reference DC potential of the intermediate AC or RF ion guide 5 then ions are effectively prevented from exiting the AC or RF ion guide 5 via the entrance.
• Ions entering the AC or RF ion guide 5 quickly become thermalised i.e. lose their kinetic energy and when a trapping voltage is removed the ions preferably exit the intermediate AC or RF ion guide 5 by the repulsive space-charge effect of further ions entering the ion guide 5.
• Other embodiments are also contemplated especially in relation to ion tunnel ion guides wherein an axial voltage gradient is used to encourage ions to travel through and/or leave the ion guide (s).
• ions When the potential applied to the differential pumping aperture 6 is lowered, ions may exit the ion guide 5 and pass through the differential pumping aperture 6 into the downstream vacuum chamber 7 which preferably houses a downstream AC or RF ion guide 8. Ions are preferably guided through the downstream vacuum chamber 7 by the ion guide 8 and may then pass through a further differential pumping aperture 10 into an analyser vacuum chamber (not shown) housing a mass analyser (not shown) .
• a timing diagram of the voltage applied to the differential pumping aperture 6 or more generally to an exit electrode of the AC or RF ion trap 5 is shown in Fig. 3B.
• V tra When the differential pumping aperture 6 or the exit to the ion trap 4 is at a voltage V tra then ions are unable to exit the AC or RF ion trap 5 and hence accumulate in the device 5.
• V extract When the voltage applied to the exit differential pumping aperture 6 or the exit electrode of the AC or RF ion trap 5 falls to V extract then ions are allowed to the exit the ion trap 5 and pass to the next stages and subsequently to the ion detector (not shown) .
• the AC or RF ion guide/ion trap 5 is maintained in the intermediate vacuum chamber 4 at a pressure in the range 1-3 mbar.
• the upstream AC or RF ion guide 3 and/or the downstream AC or RF ion guide 8 may also be used to trap ions therein.
• FIGs. 4A and 4B show the mass spectra obtained when the AC or RF device 5 is operated as an ion guide substantially without trapping ions therein (e.g. the voltage applied to the exit of the AC or RF device 5 is maintained at Ve x t r c -
• Fig. 4A shows the mass spectrum obtained when the AC or RF device 5 was maintained at a pressure of 1.4 mbar
• Fig. 4B shows the mass spectrum obtained when the AC or RF device 5 was maintained at a pressure of 2.7 mbar. In both cases the AC or RF device 5 acted as an ion guide without trapping ions.
• An ion tunnel comprises a plurality of electrodes having preferably circular apertures through which ions are transmitted in use.
• the ion tunnel may therefore be considered to comprise a plurality of stacked rings .
• the ion tunnel comprises two interleaved combed arrangements of electrodes. Adjacent electrodes in the ion tunnel device are supplied with opposite phases of an AC or RF voltage supply.
• the voltage supply is preferably sinusoidal but other embodiments are contemplated wherein, for example, a square wave or other non- sinusoidal waveform may be applied to the device.
• the ion tunnel device preferably comprises 10-20, 20-30, 30-40, 40-50, 50-60, 60- 70, 70-80, 80-90, 90-100 or more than 100 electrodes.
• the vast majority of the electrodes have substantially similar size apertures in contrast to an ion funnel.
• at least 75%, 80%, 85%, 90%, 95% or 99% of the electrodes forming the ion tunnel have substantially the same size and/or area internal apertures .
• the present invention is not limited to using an ion tunnel ion guide and other AC or RF devices are intended to fall within the scope of the present invention.
• An equimolar mixture of Leucine-Enkephalin (which exhibits a singly charged peak at m/z 556) and Gramacidin-S (which exhibits a doubly charged peak at m/z 571) was infused into the mass spectrometer. The slight difference in intensities between the two species is largely attributable to differing ionisation efficiencies and is normal in Electrospray mass spectrometry.
• Figs. 4C and 4D show mass spectra obtained when the RF device 5 was operated as an ion trap. Ions were trapped within the ion trap 2 for 60 ms in both cases.
• Fig. 4C shows the mass spectrum obtained when the ions were trapped for 60 ms in the ion trap 5 at a pressure of 1.4 mbar. As is apparent, Fig. 4C is substantially similar to the mass spectra shown in Figs. 4A and 4B.
• Fig. 4D illustrates an embodiment of the present invention and shows the mass spectrum which resulted from mass analysing the ions which emerged from the ion trap 5 when the ion trap 5 was maintained at a pressure of 2.7 mbar and ions were trapped within the ion trap 5 for 60 ms .
• Fig. 5A corresponds with the data shown in Fig. 4B and shows the mass spectrum for ions in the mass to charge ratio range 290- 580 (as opposed to ions having mass to charge ratios in the range 556-573 as shown in Fig. 4B) .
• Fig. 5B corresponds with the data shown in Fig. 4D and shows the mass spectrum for ions in the mass to charge ratio range 290-580 (as opposed to ions having mass to charge ratios in the range 556-573 as shown in Fig. 4D) .
• Figs. 5A corresponds with the data shown in Fig. 4B and shows the mass spectrum for ions in the mass to charge ratio range 290- 580 (as opposed to ions having mass to charge ratios in the range 556-573 as shown in Fig. 4D) .
• m is the mass of the ion
• z is the charge of the ion
• e is the charge of an electron
• ⁇ is the angular frequency of the RF supply.
• V 0 is the peak RF voltage applied to the rods
• R 0 in the inscribed radius of the rods R is the radial distance from the centre and 2N is the number of rods .
• R 0 is the inscribed radius of the rings
• ⁇ Z 0 is the ring centre to ring centre separation in the axial direction
• II is a first order modified Bessel function of the first kind
• 10 is a zeroth order modified Bessel function of the first kind.
• the authors go on to state that the ion micro-motion is not interrupted by the collisions, but only slightly modified in phase and amplitude, while any secular motion is damped out exponentially.
• the experimental results presented in the present application show that there is an abundance of doubly charged ions relative to that of singly charged ions following the accumulation of ions in a 2D stacked ring ion guide at a pressures of 2.7 mbar (2 torr) for a trapping period of 60 ms .
• the data shows enhancement of ions with higher charge states (z values) but with the same m/z values as the product of pressure and storage time is increased.
• This process by which ions arrange themselves into layers or bands is similar to that which takes place when DNA segments are centrifuged in a caesium chloride density gradient solution to separate out the DNA satellites.
• the DNA molecules separate into a number of bands - the main band and three additional bands (satellites) .
• the different satellite bands have different densities depending on whether they are AT- rich or CG-rich segments. This separation of DNA into bands is the result of the different centrifugal forces acting on the different classes of DNA molecules.
• ions with the same m/z value, but different z values will experience different effective radial forces as a result of the effective pseudo-potential well generated by the inhomogeneous RF fields, and will consequentially separate into different bands. Ions with the lower z values will occupy larger radial positions. Hence, these ions are more likely to be lost through collisions with the rods or rings of the ion guide, or not be transmitted through any small orifice arranged along the axis of the ion guide after its exit.
• a method for enhancing the signal from doubly, triply or more highly charged ions from that of background singly charged ions is particularly advantageous for the study of protein digests.
• the peptides from protein digests when ionised by electrospray, often yield an abundance of doubly charged, triply or more highly charged ions .
• the method, as described above, of first storing ions at elevated pressures in an ion guide or ion trap employing inhomogeneous RF fields provides a means of enhancing the relative abundance of multiply charged ions to that of singly charged ions having the same m/z values.
• 6A shows a plot of trapping time (ms) against pressure (mbar) for which the ratio of the intensity of doubly charged ions from Gramacidin-S (m/z 571) to that of the singly charged ions from Leucine Enkephalin (m/z 556) is doubled over that for no trapping.
• the particular data points are:
• FIG. 6B shows a plot of trapping time (ms) against pressure (mbar) for which the ratio of the intensity of the triply charged ions from Renin Substrate (m/z 586) to that of the singly charged ions from Leucine Enkephalin (m/z 556) is doubled over that for no trapping.
• the particular data points are:
• FIG. 7 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.64 mbar
• Fig. 8 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.64 mbar
• Fig. 7 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.64 mbar
• Fig. 8 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.64 mbar
• Fig. 7 shows the effect of storage
• Fig. 10 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.95 mbar
• Fig. 11 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.95 mbar
• Fig. 11 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.95 mbar
• Fig. 10 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 1.95 mbar
• Fig. 11 shows the effect of
• Fig. 13 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.23 mbar
• Fig. 14 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.23 mbar
• Fig. 14 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.23 mbar and Fig.
• Fig. 15 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 2.23 mbar.
• Fig. 16 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.51 mbar, Fig.
• FIG. 17 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.51 mbar and Fig. 18 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 2.51 mbar.
• Fig. 19 shows the effect of storage or trapping time on the intensity of doubly charged Gramacidin-S (m/z 571) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.86 mbar
• Fig. 20 shows the effect of storage or trapping time on the intensity of triply charged Renin Substrate (m/z 586) ions and singly charged Leucine Enkephalin (m/z 556) ions at 2.86 mbar and Fig.
• 21 shows the ratio of intensities of: (i) doubly charged Gramacidin-S ions (m/z 571) to singly charged Leucine Enkephalin (m/z 556) ions; and (ii) triply charged Renin Substrate (m/z 586) ions to singly charge Leucine Enkephalin (m/z 556) ions, as a function of storage or trapping time at 2.86 mbar.
• the ion signal can first increase before eventually decreasing as the trapping time is increased. This effect can be observed to a greater or lesser extent in Figs, 7, 10, 13, 14, 17, 19 and 20. It is thought that the increase in signal intensity is due to ions beginning to migrate towards the centre of the pseudo-potential well as a result of frequent collisions with the lighter gas molecules. This ion migration is likely to be the precursor to the process in which ions with higher values of z 2 /m eventually displace ions which have lower values of z/m and occupy the central space. Ions that accumulate in the central region are more likely to be transmitted through the exit of the ion guide and to the ion detection system.
• ions that initially collapse into the centre of the pseudo-potential well may be expected to show a corresponding increase in signal intensity.
• pressure and trapping time it is possible to enhance the ratio of the intensity of the multiply charged ions with respect to that of singly charged ions with similar m/z values and simultaneously increase the absolute intensity of the multiply charged ions .
• ions may advantageously be trapped in the AC or RF ion guide/ion trap and then released when the mass spectrometer is ready to accept these ions thereby gaining the advantage of the extra sensitivity that is observed when ions are trapped according to the preferred embodiment described above.
• the preferred embodiment also looks particularly useful for preferentially transmitting ions having a large number of charges .
• horse heart myoglobin has a molecular mass of 16951. 48 and ions may in some conditions have 8 or 9 charges or in other conditions the ions may have between 10-28 charges .
• Experimental data suggests that with highly charged ions preferentially transmission of multiply charged ions in favour of lower or singly charged ions occurs down to pressures P and trapping times T wherein the product P x T is 1 mbar-ms .
• Experimental data suggests that at or above the product of P x T equalling 1 mbar-ms the beneficial effect of the selective enhancement of multiply charged ions is observed .
• the preferred embodiment can be used for removing background ions from a mixture of ions , wherein the mixture of ions comprises a plurality of different 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, phosphorolated peptides , phosphorolated proteins , glycopeptides , glycoproteins or steroids .

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