Method of Charge State Selection

Abstract

A method of mass spectrometry or ion mobility spectrometry is disclosed in which analyte ions of a desired charge state are isolated. The method comprises: separating analytes according to their electrophoretic mobility; ionising the analytes; and mass filtering the resulting analyte ions, wherein the mass to charge ratios of the ions transmitted by a mass filter are varied as a function of the electrophoretic mobility and according to a predetermined relationship such that substantially only ions having said desired charge state are transmitted by the mass filter.

Claims

1. A method of mass spectrometry or ion mobility spectrometry comprising: separating analytes in a separator such that analytes having different electrophoretic mobilities elute from the separator at different times; ionising the separated analytes as they elute from the separator so as to form analyte ions that are separated from each other; transmitting the analyte ions to at least one device that manipulates the ions; and varying the operation of the at least one device based on the elution time of the analytes from the separator.

2. The method of claim 1, wherein the at least one device comprises a gas phase ion mobility separator that accumulates the analyte ions and periodically releases them into an ion mobility separation region of the ion mobility separator as a packet of ions; and wherein the frequency and/or duty cycle at which the packets of ions are released into the ion mobility separator is varied as a function of elution time of the electrophoretic mobility separator.

3. The method of claim 2, wherein for a given packet of ions that is released into the separation region of the ion mobility separator, one or more further packet of ions is subsequently released into the separation region whilst at least some of the ions from said given packet of ions are still travelling through and/or being separated in the ion mobility separator.

4. The method of claim 1, wherein the at least one device comprises a gas phase ion mobility separator, wherein one or more potential well or hill is periodically conveyed along the ion mobility separator so as to drive analyte ions through the device, and wherein the amplitude and/or velocity of the one or more potential well or hill is varied as a function of the elution time from the electrophoresis separator.

5. The method of claim 1, wherein the at least one device comprises a fragmentation device that fragments the analyte ions and wherein a fragmentation condition in the fragmentation device is varied based on: (i) the elution time from the electrophoretic mobility separator; or (ii) a combination of the elution time from the electrophoretic mobility separator and the mass to charge ratio of the analyte ions being fragmented; or (iii) the ratio of the elution time from the electrophoretic mobility separator to the mass to charge ratio of the analyte ions being fragmented.

6. The method of claim 5, wherein the fragmentation device fragments the analyte ions by collisionally induced dissociation (CID) and wherein the collision energy with which the ions are fragmented is varied as a function of elution time from the electrophoretic mobility separator.

7. The method of claim 5, wherein the fragmentation technique is varied as a function of elution time from the electrophoretic mobility separator such that a first type of fragmentation technique is used to fragment analyte ions of analyte that elutes from the separator during a first time period and a second, different type of fragmentation technique is used to fragment analyte ions of analyte that elutes from the separator during a second time period.

8. The method of claim 7, wherein analyte ions generated from analytes that elute with an elution time representative of a relatively high electrophoretic mobility are fragmented by electron transfer dissociation (ETD) or electron capture dissociation (ECD), whereas analyte ions generated from analytes that elute with an elution time representative of a lower electrophoretic mobility are fragmented by collisionally induced dissociation (CID).

9. The method of claim 1, wherein the at least one device comprises a time of flight mass analyser having an ion trap that accumulates analyte ions and periodically releases packets of analyte ions into or towards an extraction region of the mass analyser, wherein the extraction region pulses each of said packets of ions into a time of flight region of the mass analyser, wherein there is a delay period between the time at which any given packet of ions is released into or towards the extraction region and the time at which the extraction region pulses that packet of ions into the time of flight region, and wherein the delay period is varied in duration as a function of the elution time from the electrophoretic mobility separator.

10. The method of claim 1, wherein the at least one device comprises a time of flight mass analyser having an ion trap that accumulates analyte ions and periodically releases packets of analyte ions into or towards an extraction region of the mass analyser, wherein the extraction region pulses each of said packets of ions into a time of flight region of the mass analyser, and wherein the frequency at which the packets of ions are released into or towards the extraction region is varied as a function of elution time of the electrophoretic mobility separator.

11. The method of claim 1, wherein the device is a mass selective ion trap that accumulates said analyte ions and ejects analyte ions having a defined range of mass to charge ratios at any given time, and wherein the range of mass to charge ratios ejected from the ion trap is varied as a function of the elution time from the electrophoretic mobility separator.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0177] Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which:

[0178] FIG. 1 shows a plot of mass to charge ratio value versus capillary electrophoresis retention time for a mixture of peptides ionised by electrospray, wherein the ions elute in order of high to low electrophoretic mobility;

[0179] FIG. 2 shows a similar plot to FIG. 1, except wherein the ions elute in order of low to high electrophoretic mobility;

[0180] FIG. 3 shows a plot of intensity versus the average charge state of peptides separated by nano-flow UPLC and ionized by positive ion electrospray;

[0181] FIG. 4 shows an apparatus illustrating an embodiment of the invention;

[0182] FIG. 5 shows a mass to charge ratio versus capillary electrophoresis retention time plot similar to FIG. 2, except after allowing only the doubly charged ions to be transmitted; and

[0183] FIG. 6 shows the mass to charge ratio versus capillary electrophoresis retention time plot as described in FIG. 5 after fragmenting the transmitted ions.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

[0184] Capillary electrophoresis (“CE”) is a known technique which is used to separate ionic species in a conductive medium in the presence of an electric field. Capillary electrophoresis causes ions in solution to migrate with velocities based on their solution charge (i.e. charge when in solution), hydrodynamic radius and frictional forces.

[0185] The velocity of an ion under conditions of capillary electrophoresis separation is given by:

ν.sub.a=E.Math.μ  (1)

wherein μ is the electrophoretic mobility, ν.sub.a is the velocity of the analyte and E is the electric field strength.

[0186] The electrophoretic mobility of an analyte depends on the viscosity of the medium, the size and shape of the analyte, and the solution charge. For peptides, the electrophoretic mobility can be related to the size of the peptide and may be estimated from its molecular weight. The Offord model (as detailed in: “Electrophoretic Mobilities of Peptides on Paper and their Use in the Determination of Amide Groups”, R. E. Offord, Nature 211, 591-593, 6 Aug. 1966) estimates the electrophoretic mobility as:

[00001]$\begin{array}{cc}\mu \propto \frac{q}{M.Math..Math.W^{2/3}}& \left(2\right)\end{array}$

wherein μ is the electrophoretic mobility and MW is the molecular weight of the analyte. When coupling a capillary electrophoresis device to a mass spectrometer, the correlation between mass to charge ratio value, charge state and capillary electrophoresis retention time can be clearly demonstrated. An example of a capillary electrophoresis device coupled to a mass spectrometer is described in “Utility of CE-MS Data in Protein Identification”, Brad J. Williams, William K. Russell, and David H. Russell, Anal. Chem. 2007, 79, 3850-3855. An arrangement is disclosed wherein capillary electrophoresis separation is followed by Matrix Assisted Laser Desorption Ionisation (“MALDI”). MALDI ionisations almost exclusively yield singly charged ions. Clear trends in the mass to charge ratio versus retention time are shown. These correlations follow trends in the capillary electrophoresis separation related to the solution charge state and size (correlated to the molecular weight) of the analytes.

[0187] Electrospray ionization (“ESI”) ion sources may be coupled to a mass spectrometer in order to ionise polar compounds such as peptides. The electrospray ionisation of peptides may result in ions with several different charge states. There is a strong correlation between the charge state in solution and the dominant charge state produced by the electrospray process. For peptides, the charge in solution and the charge state in the gas phase are both strongly related to the number of basic residues on the peptide. In particular, ions which are singly or doubly charged in solution appear predominantly as these charge states in the gas phase. Ions of higher charge state in solution may exist in more than one charge state in the gas phase. The observed correlation between solution phase and gas phase charge state and the strong correlation between molecular weight and solution phase hydrodynamic radius results in a capillary electrophoresis elution order correlated to both charge state and mass to charge ratio value observed in the electrospray mass spectrum of peptides.

[0188] Separation in capillary electrophoresis also depends on the electroosmotic flow of the buffer solution. The direction of the electroosmotic flow with relation to the direction of electrophoretic migration of the analyte ions is governed by the composition or coating applied to the walls of the capillary electrophoresis capillary. Electroosmotic flow can be used to govern the elution order of analytes. For example, if the electroosmotic flow is opposed to the electrophoretic migration then analytes with low mobility will elute before analytes with higher electrophoretic mobility.

[0189] A preferred embodiment of the present invention will now be described with reference to FIG. 1. According to the preferred embodiment, a mixture of peptides is subjected to capillary electrophoresis and the peptides are then ionised by an electrospray ioniser. The resulting ions are then mass analysed in a mass spectrometer so as to determine their mass to charge ratios. FIG. 1 shows a plot of the mass to charge ratios of the ions as a function of their capillary electrophoresis retention time. It can be observed that the analyte ions are separated into three enclosed areas that represent singly (1+), doubly (2+) and triply (3+) charged gas-phase ions. In this example, ions elute in order of high to low electrophoretic mobility. The ions which elute first are ions with the highest solution charge state and lowest mass to charge ratio.

[0190] FIG. 2 shows a plot corresponding to that in FIG. 1, except wherein the ions elute in order of low to high electrophoretic mobility. The order of elution is reversed by reversing the electroosmotic flow.

[0191] It will be observed that in the embodiments of both FIGS. 1 and 2 there is correlation between elution time and mass to charge ratio within each band. The analytes having different gas phase charge states are separated from each other.

[0192] FIG. 3 shows a plot of intensity versus the average charge state for peptides from a tryptic digest of a mixture of four proteins separated by nano-flow high pressure liquid chromatography and which have been ionised by positive ion electrospray. The average charge state is an intensity weighted value of the charge states of the mass spectral peaks associated with each peptide. It is clear that most of the ions formed cluster around and close to an average charge state of 2+ or 3+, representing doubly charged and triply charged ions. This demonstrates that the electrospray process generates predominantly a single charge state for a given peptide. This is most clearly seen for the doubly charged ions, which are shown by peaks distributed closely and fairly evenly about the 2+ charge state. However, the peaks are less evenly distributed about the 3+ charge state and FIG. 3 shows that many peaks are distributed just below the 3+ value. This indicates that some of the peptides that form triply charged ions also form doubly charged ions. The dominant charge states produced by electrospray ionisation correlate with the charge states in solution that have been separated by the capillary electrophoresis.

[0193] Referring again to FIG. 1 or 2, at a given time during capillary electrophoresis separation, doubly charged ions (2+) will form within a distinct range of mass to charge ratios that lies between the upper and lower dashed lines. Very little, if any, signal from peptides of lower or higher gas phase charge states will be present within this range. In FIG. 1, the mass to charge ratios of the ions that have a charge state of 2+ increase as the capillary electrophoresis retention time increases. Conversely, in FIG. 2 the mass to charge ratios of the ions that have a charge state of 2+ decrease as the capillary electrophoresis retention time increases.

[0194] According to a preferred embodiment a mass filter, such as a quadrupole mass filter, may be located downstream of a capillary electrophoresis separation device and an ion source. The mass filter is preferably located upstream of another mass analyser, such as a Time of Flight mass spectrometer. In operation, the mass filter may be operated in a bandpass mode such that only ions having mass to charge ratios within the ranges bounded by the dotted lines in FIG. 1 or 2 are transmitted. The scan law of the quadrupole mass filter is preferably synchronised with the capillary electrophoresis elution time. In this way, the final mass spectrum may be enriched with ions of particular charge states.

[0195] FIG. 4 shows a mass spectrometer according to an embodiment of the present invention. Analytes are preferably first separated in solution by a capillary electrophoresis device 1. The analytes are then ionised by an ion source 2. The ion source 2 preferably comprises an Atmospheric Pressure Ionization (“API”) ion source. An analytical filter 3 such as a quadrupole mass filter 3 is preferably provided downstream of the ion source 2 and is preferably scanned or stepped continuously or discontinuously in synchronisation with the capillary electrophoresis device 1 such that only ions having a combination of mass to charge ratios within a desired range and a capillary electrophoresis retention time within a desired range are transmitted at any instant in time. The ions transmitted may be passed directly to a Time of Flight mass analyser 5 or may be caused to dissociate into product ion species in a fragmentation device 4.

[0196] FIG. 5 shows a plot of mass to charge ratio versus capillary electrophoresis retention time that is similar to that of FIG. 2, except that the ions have been filtered after separation in the capillary electrophoresis device 1 such that only the doubly charged ions (2+) have been transmitted by the filter 3.

[0197] FIG. 6 shows a plot of mass to charge ratio versus capillary electrophoresis retention time for the ions of FIG. 5 after they have been fragmented in fragmentation device 4. The properties of the resulting fragment ions are illustrated as the circles located in capillary electrophoresis retention time regions 6,7,8,9. The retention times associated with the fragment ions represent the retention times of their respective parent ions, as the fragment ions have been generated after the parent ions have been separated by capillary electrophoresis and the fragment ions themselves have not been subjected to separation by capillary electrophoresis. Filtering the doubly charged ions prior to fragmentation provides the advantage that the likelihood of the fragment ion intensity or mass to charge ratio information being corrupted due to mass interference is reduced.

[0198] According to an alternative embodiment the filter 3 may alternatively be used as a high-pass or low-pass mass to charge ratio filter, rather than a band-pass filter, in order to only transmit ions above or below a desired mass to charge ratio for a given retention time. For example, the filter 3 could be varied with retention time so as to only transmit ions above the upper dashed line in FIG. 1 or FIG. 2. In this manner, only ions having a gas phase charge state of 3+ would be transmitted for subsequent analysis or fragmentation etc. Similarly, the filter 3 could be varied with retention time so as to only transmit ions below the lower dashed line in FIG. 1 or FIG. 2 such that only ions having a gas phase charge state of 1+ would be transmitted for subsequent analysis or fragmentation etc. Alternatively, more than one mass to charge ratio transmission window may be scanned or set simultaneously. This may be achieved, for example, by applying a broadband resonance excitation waveform with frequency notches (i.e. with specific frequencies omitted) to a quadrupole mass filter. In this way, multiple mass to charge ratio regions may be transmitted simultaneously that track several mass to charge ratio trend lines in the capillary electrophoresis elution profile. For example, the ions having a gas phase charge state of 1+ and 3+ in FIG. 1 or 2 might be transmitted simultaneously and the ions of charge state 2+ might be filtered out.

[0199] Although the embodiments described above relate to a quadrupole mass filter that is scan linked with capillary electrophoresis separation, another type of filter may be used to achieve similar results. For example, a differential ion mobility filter or Field Asymmetric Ion Mobility Spectrometry filter (FAIMS) may be arranged to scan synchronously with the capillary electrophoresis elution. Alternatively, an ion mobility separator (IMS) filter such as a Differential Mobility Analyser (“DMA”) may be used. Both of these devices separate ions based on gas phase ion mobility. These devices may be operated at atmospheric pressure or at sub-atmospheric pressure.

[0200] In a second embodiment of the present invention the strong correlation between capillary electrophoresis elution time and mass to charge ratio for a given charge state may be used to optimise the operation of a downstream gas phase ion mobility separator (IMS). This may be a linear DC field drift cell or a travelling wave IMS device. In this mode, a nested capillary electrophoresis IMS acquisition may be performed producing a comprehensive CE-IMS two dimensional separation. At any instance in time only a narrow mass to charge ratio range or electrophoretic mobility range for a given charge state will be delivered to the IMS device. Based upon this knowledge, multiple packets of ions may be released into the IMS device sequentially while ions are being separated in the IMS device. The frequency and/or duty cycle at which these packets of ions are released into the gas phase IMS device may be arranged such that no overlapping of ion species occurs. This mode of operation is advantageous to minimize space-charge effects such as peak broadening or drift time shifting in the gas phase IMS device.

[0201] One or more travelling wave may be conveyed along the IMS device as ions pass through the device, e.g. in order to drive ions through the IMS device. The travelling wave is preferably a DC potential hill or well that is conveyed along the device. The amplitude and/or velocity and/or DC field strength of the travelling wave may be varied so as to optimise the IMS conditions (e.g. resolution or elution time) for the range of mass to charge ratio values or solution phase mobility eluting from the capillary electrophoresis device. Additionally, or alternatively, other IMS conditions could be varied as a function of the elution time from the capillary electrophoresis device.

[0202] In another embodiment, a fragmentation device is provided downstream of the capillary electrophoresis device. The conditions of the fragmentation device may be changed during the capillary electrophoresis elution time in order to optimise the conditions for efficient fragmentation of ions eluting from the capillary electrophoresis device. This may comprise, for example, varying the collision energy for collisionally induced dissociation (CID) or varying the reaction time for electron transfer dissociation (ETD) or electron capture dissociation (ECD) in the fragmentation device.

[0203] Alternatively, the fragmentation technique may be varied as a function of elution time from the capillary electrophoresis device. For example, in the example shown in FIG. 1 multiply charged ions elute first from the capillary electrophoresis device and singly charged ions elute at later retention times. Different fragmentation techniques may be preferred for ions of different charge states. In this case, it may be advantageous to utilise a technique such as ETD at the start of the capillary electrophoresis separation in order to efficiently fragment ions of high charge state. However, ions of lower charge state elute from the capillary electrophoresis device at later retention times and it may therefore be desirable to switch to CID fragmentation.

[0204] Many other fragmentation methods exist which require different conditions for differing mass to charge ratio values or charge states which will benefit from this approach.

[0205] In another embodiment the performance of an orthogonal acceleration Time of Flight mass analyser may be optimized using the correlation between mass to charge ratio and capillary electrophoresis elution time. For example, a relatively narrow mass to charge ratio range eluting from a capillary electrophoresis device at a given time may be released to the Time of Flight orthogonal sampling region as a pulse. The orthogonal acceleration voltage pulse may be synchronised to the release of this pulse such that this mass to charge ratio range in transmitted into the Time of Flight analyser with high efficiency. As the capillary electrophoresis separation proceeds the mass to charge ratio range eluting from the device changes and hence the time delay between release of an ion pulse and the energising of the orthogonal acceleration pulse may be changed so as to maintain the highest sampling efficiency. As such, for a given charge state over the entire mass to charge ratio range, high sampling efficiency may be maintained.

[0206] In another embodiment a mass selective ion trap may be arranged downstream of the capillary electrophoresis device. The performance of the mass selective ion trap may be improved by changing the mass range over which the analytical ion trap scans to accommodate the mass to charge ratio range eluting from the capillary electrophoresis device. In this way, the speed of mass selective ejection (i.e. amu per unit time) may be minimised, thus maximising mass resolution.

[0207] Combinations of the above embodiments may be employed in order to optimise multiple performance attributes of multiple different downstream devices.

[0208] 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.

[0209] For example, separation techniques other than the capillary electrophoresis described may be used. Separation techniques which use solution phase electrophoretic mobility may be used such as, for example, Capillary Electrochromatography (“CEC”) or Micellar Electrokinetic Chromatography (“MEKC”). Alternatively, Size Exclusion Chromatography (“SEC”) may be used, which separates analytes based on size and also produces separations with a strong indicator weight dependence. As the separation has a molecular weight correlation, this separation may also be linked with optimisation of downstream devices in the mass spectrometer.

[0210] Although there is far weaker correlation between mass to charge ratio, charge state and retention time for other liquid chromatography techniques, some correlation does exist and similar linked experiments using other liquid chromatography techniques to optimise performance can be envisaged. For example pH gradient chromatography or ion exchange chromatography etc may be used.

[0211] The techniques described can be applied to comprehensive two dimensional (2D) chromatography. For example, with normal phase liquid chromatography (LC) as the first dimension and rapid profiling of the LC peaks with fast capillary electrophoresis separation in the second dimension. With the correlated mass to charge ratio and or charge state output of the capillary electrophoresis used to optimize the conditions of the downstream devices.