Patent Grant
10317362
The invention relates to methods for the mass spectrometric analysis of complex substance mixtures and in particular for the identification of isomers and isobars by means of time-of-flight mass spectrometers which are equipped with a trapped ion mobility spectrometer, a mass filter and a fragmentation cell.
The U.S. Pat. No. 7,838,826 B1 (M. A. Park, 2008) discloses a trapped ion mobility spectrometer (TIMS), which is also called a trapped ion mobility separator hereinafter. In a trapped ion mobility spectrometer, ions inside a multipolar ion guide system are typically driven through a gas stream against an electric field barrier, which preferably has a ramp with increasing electric field strength. The ions gather along the ramp at positions where the gas friction for one ion species is in equilibrium with the restoring electric force in each case. The ions therefore gather, spatially separated according to their ion mobility. When sufficient ions have been stored, after a predetermined accumulation time, for example, the counter-acting electric field of the electric field barrier is lowered in a “scan”, which causes the ions to be released sequentially from lower to higher mobility over the end of the ramp and to be transferred to a mass spectrometer, preferably a time-of-flight mass spectrometer with orthogonal ion injection.
The lowering of the counter-acting electric field (scan) typically takes between 20 and 250 milliseconds. The trapped ion mobility separator can be very compact. The tube (11) as the main part can have a length of only five centimeters, for example.
This type of mobility spectrometer is unique in that the mobility resolving power depends on the scan speed.
The U.S. patent application Ser. No. 14/931,163 (M. A. Park et al.; “Acquisition of Fragment Ion Mass Spectra of Ions Separated by their Mobility”), which is incorporated herein by reference, describes a method to identify organic substances in a complex substance mixture, such as the enzymatic digest of a proteome. The method disclosed there is preferably conducted with a mass spectrometric system which is schematically depicted in
The substances dissolved in a liquid are fed to an ion source, preferably an electrospray ion source, and ionized. Depending on the complexity of the substance mixture, the substances may already be chromatographically separated or may originate in unseparated form from a syringe pump.
First a mass-mobility map, as shown schematically in
For complex substance mixtures, the mass-mobility map comprises a large number of ion signals, which typically extend along the mobility axis for around one to three milliseconds. The mobility resolution here is much smaller than the mass resolution. The mass of the ion signals decreases along the mobility axis, and singly and multiply charged ions form strongly dispersed, partially overlapping bands (see
A first group of ion signals is then selected from the mass-mobility map. This group contains a number of ion signals which are arranged in temporal sequence, i.e. they do not overlap in the direction of the mobility axis.
After the acquisition of the mass-mobility map and the selection of a first group of ion signals, a mobility scan is subsequently carried out to identify the substances of the ion signals of the group. The mass filter, the fragmentation cell and the time-of-flight mass spectrometer are needed for this purpose. After the trapped ion mobility separator has been filled and the mobility scan has started, the mass filter, usually a quadrupole mass filter, is switched to allow ions of the first ion signal from the group to pass through. It is preferable for all ions of the isotopic distribution to be included here in order to obtain fragment ion spectra whose isotopic distributions agree with those of reference spectra from spectral libraries. The mass filter is therefore set to a transmission window of a few daltons. The ions of the first ion signal thus selected according to mobility (scan time) and mass are fragmented in the fragmentation cell; the fragment ions are measured in the time-of-flight mass spectrometer as a fragment ion spectrum. As a rule, the substance can be identified from this fragment ion spectrum, usually via comparisons with reference spectra of a spectral library. In the further course of the same mobility scan, the ions of further ion signals are selected according to mobility and mass in the same way and fragmented. The time intervals between the ions of the group are chosen such that the mass filter can be switched to the mass of the ions of the subsequent ion signal. The further substances of the selected ion signals of this group are identified by acquiring the corresponding fragment ion spectra. The trapped ion mobility separator is then filled with a further batch of ions, and the identification of a second group of ion signals from the mass-mobility map can start.
If the mobility scan takes 20 milliseconds, for example, and an ion signal typically lasts 2.5 milliseconds on leaving the trapped ion mobility separator, then up to eight signals can be selected for each group in a single mobility scan; for a mobility scan lasting 50 milliseconds, each group can comprise around 20 signals. If a mass-mobility map comprises around 200 ion signals which are reasonably uniformly distributed, they can be split up into around 10 groups with a scan lasting 50 milliseconds. If they are not distributed uniformly, it may be necessary to form more than 10 groups in order to cover all signals of the mass-mobility map.
Several mass-mobility maps can be acquired and summed to improve the quality. It may furthermore be expedient, for larger molecules, to only select ion signals of ions which are at least doubly charged, since they can be fragmented more easily than singly charged ions and with a high yield.
Assuming a scan duration of 50 milliseconds each, around 20 of these measuring cycles can be completed, until after around one second a new mass-mobility map is measured, in order to take account of a change during a chromatographic separation, for example. Theoretically, around 400 ion species per second can be identified in this way. In reality, this number of measurements is not completely achieved, even when so many ion species are present. Even in very complex substance mixtures, it is usually not the case that so many ion species are available, however. The excess measuring capacity can then be used to measure weak ion signals in successive measurement cycles several times and to sum the fragment ion spectra in order to achieve a better quality of spectra and thus a more certain identification.
This method can be modified in a variety of ways depending on the analytical requirements. A higher mobility resolution can be achieved by longer scan durations, while at the same time larger numbers of ion signals can be selected for each group. All ions of an ion source can be utilized by a trapped ion mobility separator with parallel accumulation of ions (U.S. patent application Ser. No. 14/614,456; “Trapping Ion Mobility Spectrometer with Parallel Accumulation”, M. A. Park and M. Schubert) without ion losses occurring during the mobility scan.
“Isomers” are molecules which have the same elemental composition, i.e. the same molecular formula, but different primary structures (“structural isomers”) or different secondary or tertiary structures (“conformational isomers”). Isomers have precisely the same mass. “Isobars” are different molecules which are composed of different elements such that their mass has the same number of atomic mass units (daltons), but not exactly the same mass. Isobars are then said to have the “same nominal mass”. Molecular ions always have isotopic distributions which span several daltons. If the molecular ions of two molecules whose masses differ by only one, two or three daltons interfere with each other, it is not possible to separate the isotopic distributions of these molecules from each other with a mass filter. These molecules are called “near isobars” here. When the term “isomers” is used here, the expression is often to include the “isobars” or “near isobars” as well.
There is a need to have methods for analyzing complex substance mixtures which can be used to identify isomeric and isobaric organic substances in complex substance mixtures, in particular pharmacological substances of particular analytical interest, and to determine the mobility K0 or the collision cross-section a of the substances as accurately as possible.
Experience has shown that not all ion signals of a mass-mobility map belong to single homogeneous ion species. Each ion signal can contain one ion species, but can occasionally also contain several isomers or isobars which are not resolved according to their mobility, and whose isotopic distributions overlap. Ion mixtures with sufficiently different mobility have already been resolved according to mobility by the known methods. Other ion mixtures contain ion species which are not resolved according to mobility.
The invention provides a method for analyzing a substance mixture in which a first mass-mobility map is acquired using a mass spectrometer which comprises a trapped ion mobility separator and a time-of-flight analyzer. The method is characterized by the fact that (a) an ion signal of interest is selected in the first mass-mobility map and examined as to whether it is a superimposition of signals from different ion species which are not sufficiently resolved according to mass or mobility, and that (b) a second mass-mobility map is acquired wherein at least one parameter of the trapped ion mobility separator is changed such that the region around the ion signal of interest is measured in the second map with a higher mobility resolution and/or mobility accuracy compared to the first map.
An “ion signal” is understood to be the signal of those ions which are released almost simultaneously from the trapped ion mobility separator during the mobility scan and transferred to the downstream mass analyzer.
The second, more detailed mass-mobility map is also called a “zoom map” hereinafter; it no longer contains all ion signals, but only the ion signals of the ion species selected in narrow mass and mobility ranges.
The invention provides a further method in which a first mass-mobility map is acquired using a mass spectrometer which comprises a trapped ion mobility separator, a mass separator, a fragmentation cell and a time-of-flight analyzer. Ion signals are selected from the first mass-mobility map and then fragment ion spectra of ions of the selected ion signals are acquired, wherein the ions are selected according to mobility and mass. The method is characterized by the fact that, in a first step, one of the selected ion signals, its fragment ion spectrum and/or the substance identification, is investigated as to whether this ion signal of interest is a superposition of signals of different ion species, which are not sufficiently resolved according to mass or mobility. In a second step, a second mass-mobility map is acquired wherein at least one parameter of the trapped ion mobility separator is changed for the analysis of the ion signal of interest such that the region around the ion signal of interest is measured in the second map with a higher mobility resolution and/or mobility accuracy compared to the first map. In the second step, additionally or alternatively, an additional fragment ion spectrum of ions of the ion signal of interest is acquired, wherein at least one parameter of the trapped ion mobility separator is changed such that the ions, selected according to mobility and mass, of the additionally acquired fragment ion spectrum have a smaller mobility bandwidth ΔK compared to those of the fragment ion spectrum acquired before.
The mass-mobility map from which the mobility or the collision cross-section and the mass of the ion signal are determined, and/or the fragment ion spectrum, can be used for the substance identification of the ion signals. The additional fragment ion spectrum is preferably used for the substance identification of the ion signal of interest. The mobility or the collision cross-section of the ion signal of interest can additionally or alternatively be determined from the second mass-mobility map and used for the substance identification of the ion signal of interest.
The ion signal of interest can be analyzed with regard to the signal width or signal form, or both, as to whether it is a superimposition of signals of different ion species. The signal width and/or the signal form of the ion signal of interest are preferably compared with the signal width or signal form of a single ion species and investigated in particular for a deviation along the mobility direction.
The at least one parameter of the trapped ion mobility separator which is changed in order to measure the region around the ion signal of interest with increased mobility resolution and/or increased mobility accuracy in the second map, is preferably the scan speed of the trapped ion mobility separator, in particular a reduction in the scan speed during the scan around the ion signal of interest. The scan speed of the trapped ion mobility separator can be reduced there as well in order to select ions with a smaller mobility bandwidth ΔK for the additional fragment ion spectrum.
The ion signal of interest is selected from the ion signals of the first map, using a selection list containing known isomers or isomers of particular analytical interest. The selection list here is preferably dependent on the type of substance mixture. The selection can be done by a user as well, however, who marks the ion signal of interest in a visual representation of the first map. The extent of the marking in the direction of mobility here can set the mobility resolution of the second map in the region of the ion signal of interest and specify the parameter(s) of the trapped ion mobility separator. It is preferable that only those ion signals are selected which have not already been selected and identified in a previous map.
The acquisition of the first and/or second map can involve a calibration of the mobility axis so that the mobility or the collision cross-section of the ion signal of interest can be determined. For the calibration, it is preferable if the known mobilities or collision cross-sections of identified substances of the substance mixture or of reference substances are used, the reference substances being added to the substance mixture. In particular, at least two reference substances whose mobilities encompass the mobility of the ion signal of interest can be added to the substance mixture.
The substance mixture can be fed to the ion source in dissolved form with a syringe pump, with the mass-mobility maps remaining essentially the same from measurement to measurement.
The substance mixture can also be first separated in a chromatograph and fed to the ion source as a separated substance mixture, however. The chromatography system is preferably a liquid chromatography (LC) system and the separated substance mixture is fed to the ion source in liquid form. The chromatography system (substance separator) can also be a gas chromatography system or carry out an electrophoretic separation, e.g. in the form of a capillary electrophoresis. For complex mixtures, a plurality of substances can still be superimposed even after a separation.
If the substances of the substance mixture are separated by means of a substance separator and fed to the ion source, the ion signals belonging to a substance only appear in mass-mobility maps being acquired in temporal sequence when the concentration of the substance has exceeded a sensitivity threshold. The concentration then increases initially in further mass mobility maps, reaches a maximum and decreases again. It is particularly favorable that an ion signal is only displayed in a visual representation of a mass-mobility map, or an ion signal of interest is only selected, when the ion signal has reached the maximum during the substance separation. Furthermore, an approximately constant concentration of the substance exists for a short time near the maximum.
If several samples with similar composition are to be separated and analyzed, for example always in the same way regarding the presence of specific isomers, then the optimum device parameters of the substance separator and/or the mass spectrometer can be determined for substances of interest in one or more cycles of a first sample and used for the samples subsequently analyzed. For a series of similar samples, the method according to the invention is therefore worked out in detail with the aid of a first sample and then used for the further samples in the same way. It is therefore expedient to save the device parameters which change during the separation process of the first sample and to re-use them later for further similar samples. At the known retention times of specific substances of interest, the corresponding device parameters of the mass spectrometer are used in particular, especially the corresponding device parameters of the trapped ion mobility separator, the mass filter, the fragmentation cell and/or the time-of-flight analyzer.
After the trapped ion mobility separator (TIMS cell) has been filled with sufficient ions, a mobility scan is started to identify selected ion signals. When the first ion signal appears at the exit of the trapped ion mobility separator, the mass filter is set to the mass of this ion signal and the ions are allowed to pass through the mass filter. The filtered ions are then fragmented in the fragmentation cell and the fragment ions are measured in the time-of-flight mass analyzer. It is usually possible to determine the substance of the ion signal from the fragment ion spectrum. The acquisitions of the fragment ion spectra of the second, third substance and so on then follow. When all eight ion signals of this signal group have been measured, the TIMS cell can be refilled and a second group of ion signals can be measured in a second measuring cycle.
The invention relates to a method for the detailed analysis of organic substances in complex mixtures which is conducted in a mass spectrometer comprising an ion source, a trapped ion mobility separator (TIMS), a mass separator, a fragmentation cell and a time-of-flight analyzer. In principle, mobility spectrometers can separate both isomers and isobars. They can usually separate near isobars as well.
In a first cycle, at least one mass-mobility map is acquired and selected ion signals from these maps are identified by the acquisition and evaluation of fragment ion spectra of the ions of these ion signals (first cycle).
The ion signals, their fragment ion spectra, their substance identifications and/or the identified substance structures are examined as to whether ion signals from these maps are possibly superpositions of different ion species which are not sufficiently resolved according to mass or mobility, and whether this means a mixture of isomers, isobars or near isobars is possibly present.
In a second cycle, further maps and/or fragment ion spectra are acquired, wherein detailed methods with changed method parameters are used in order to measure ion signals of analytical interest with increased mobility resolution or mobility accuracy, in particular in order to measure or select ions of analytical interest separately from other ion species. Detailed methods of the trapped ion mobility separator are particularly the temporal or spatial zoom. The mobility resolution or mobility separation selectivity around an ion signal of analytical interest can be higher than 100, preferably higher than 300, in particular higher than 500 here.
With many analytical methods it is not only important to detect groups with several isomers. If it is known that several isomers can be present for one substance of a substance group, and if only one isomer is found for this substance, it can be of particular analytical interest which isomer is present. For conformational isomers, in particular, the fragment ion spectra often only differ so slightly that a reliable identification of the isomer with the aid of this spectrum is not possible. If a complete group of isomers is found, then it is usually possible to determine the isomers by means of the structure of the isomeric group; this is usually not possible for an isomer that is individually present, however. If a fragment ion analysis does not give any indication of the identity of the isomer, the isomer can often be identified via a precise determination of the mobility or the collision cross-section.
The search for ion signals which are possibly unresolved signal superpositions of different ion species can be carried out in a variety of ways:
(a) Computer programs can automatically analyze the ion signals in the mass mobility maps, in particular their temporal width (signal width) and intensity distribution (signal form), and also overlaps of isotopic distributions.
(b) A user can examine the ion signals visually, and can be supported here by databases, which contain in particular those substance groups which frequently contain isomers. The databases here can in particular contain substance groups of particular analytical interest and be specific to the type of substance mixture under investigation.
(c) The fragment ion spectra should differ from each other, particularly in the case of isobars and near isobars, and facilitate the recognition of a mixture of different ion species. It is thus possible to identify ion mixtures with different numbers of double bonds.
(d) Substances which are identified in the map of the first cycle can also be examined as to the probability of the presence of isomers with the aid of databases, even without an analysis of the ion signals. Here as well, the databases can particularly contain substance groups, where the measurement of isomers is of particular analytical interest, or can be specific to the type of substance mixture under investigation. This means that ion signals whose signal forms and widths exhibit no peculiarities can also be included in the measurement of a second cycle. Some substance groups have a tendency to form isomers whose mobilities differ only very slightly, for example because with two isomers, a double bond can be located at two adjacent carbon atoms. These distinctions can particularly be of pharmacological importance. If only one isomer is present, it is often of particular interest to determine accurately which isomer it is.
(e) The at least approximately measured collision cross-sections a can be compared with library-type collections of substance data with their collision cross-sections in an automated way by computer programs, for example, and lead to more accurate measurements in the event of discrepancies.
It should be noted here that it does not always make sense for all ion signals which could contain isomers or isobars to be measured with increased mobility resolution in the second cycle also. Priority here should be given to the analytical significance.
It is not necessarily always only isomers or isobars which lead to ion signals which are a superposition of ion signals of different ion species and appear without being cleanly resolved according to mass and mobility. The mass filter is usually set such that the whole isotopic group is transmitted for one ion species; for heavier ions, the transmission width can optimally be around five daltons or more. This means that mixtures of ion species whose molecular masses differ by only a few daltons are no longer cleanly separated, however. If the molecules are very different, the mixture can be identified from the fragment ion spectra. They can also have nearly the same structure, however, for example when they have slightly different numbers of double bonds. Their molecular masses then each differ only by two, four or six daltons (for singly charged ions). Those mixtures of ion species with molecular masses which differ by only a few daltons can be separated by the method according to the invention in a second cycle with a correspondingly increased mobility resolution.
If ion signals are found for which a particular interest in a detailed analysis exists, the identification method can be performed a second time with the same substance mixture, but for each ion signal of interest, detailed methods are specifically set-up with specifically determined method parameters.
A temporal zoom is usually set here, which expands the mobility scan for the ion signals of analytical interest such that an enhanced mobility resolving power is achieved around these ion signals (see U.S. Pat. No. 8,766,176 B2; “Acquisition Modes for Ion Mobility Spectrometers using Trapped Ions”, D. A. Kaplan et al.). How high the resolution has to be can often only be specified by experience or from information in suitable libraries. It is preferable if mobility resolutions of Rmob=150 to 250 are set.
If the ion signals can be superpositions, and if these ion signals are of sufficiently high analytical interest, then, after a renewed filling of the TIMS cell, further maps and/or fragment ion spectra are acquired, for which detailed methods with changed method parameters are used in order to measure the ion signals of analytical interest with increased mobility resolution or mobility accuracy, in particular in order to measure or select ions of analytical interest separately from other ion species The mobility resolution can be higher than 100, higher than 300, or even higher than 500, depending on the requirement. This second mass mobility map with increased mobility resolution will be called “zoom map” hereinafter.
Such a zoom map is shown in
Several ion signals, which now usually represent individual ion species, i.e. isomers, isobars or near isobars, can then be selected again from the zoom map. In a further step of the method, fragment ion spectra of these ion signals can be acquired. The fragment ion spectra can be used for the identification of the now separated isomers, isobars or near isobars, although an unequivocal identification is not always achieved, because the fragment ion spectra of isomers can be very similar. If it is known that several isomers can be present for one substance of a substance group, and if only one isomer is found for this substance, it can be of particular analytical interest which isomer is present.
A temporal zoom can also be supported by a spatial zoom.
The determination of the optimal method parameters for the ion signals of analytical interest can be done automatically, for example in the case when the ion signal reveals a superposition structure in a mass mobility map of the first cycle. The mobility resolutions required for the ion signals of analytical interest can also be taken from libraries of isomers, if available. The ion signals of analytical interest can be noted in a list of the ion signals which are obtained from the first cycle. It is also possible to select the ion signals of interest simply in a representation of the mass-mobility map on a computer screen by marking the ion signals there, e.g. framed with a box. The size of the box can also specify the desired resolution here.
If an accurate determination of the mobility K0 or the collision cross-section a of a substance is of interest, reference substances (calibration substances) of accurately known mobility (or accurately known collision cross-sections) with similar or equal mass and only slightly different mobility can be added to the substance mixture especially for the second (or any further) cycle of the method.
The ion source depicted in
The substances can also be fed directly to the ion source as dissolved substances by a syringe pump or after separation by means of liquid chromatography or electrophoresis. A chromatographic pre-separation can take an hour or longer and provide a plurality of mass mobility maps with a few hundred thousand ion signals.
If the substances of the complex mixture are fed in after separation by chromatography or electrophoresis, the ion signals belonging to a substance appear in the mass-mobility maps acquired roughly once a second only when the concentration of the substance has exceeded a sensitivity threshold.
In the case of a pre-separation by means of liquid chromatography or electrophoresis, a calibration is difficult, since calibration substances usually elute at different times to the analyte substances. A calibration solution can be added, however, ideally in an automated way.
It should be noted here that series of analyses are often conducted on substance mixtures which are known to have nearly the same composition and are all subject to the same analytical objective. If a detailed method (second cycle) with satisfactory fine analyses of isomers, isobars or near isobars has been established for this type of substance mixture, it is not absolutely necessary to carry out the first cycle. A first cycle carried out once can be used as the basis for analyses of a specific type of substance mixture. If large numbers of samples with a similar type of composition are to be analyzed with the aid of a chromatographic or an electrophoretic separation, for example always in the same way for the presence of specific isomers, the detailed methods obtained and developed in first separation cycles of a first sample can run automatically in separations of further samples, whereby the parameters belonging to the given retention times are used. The proposal is therefore to save the parameter changes during the cycles and to re-use them later for similar samples.
If the mass spectrometer is equipped with computers with sufficient capacity and sufficient speed, and if corresponding programs can be provided, then the fully automatic measurement of mass-mobility maps, selection of groups of ion signals, measurement of the fragment ion spectra, selection of ion signals of particular analytical interest, measurement of zoom maps, and measurement of the fragment ion spectra of the now separated ion species in a single LC run is possible. If the ion signals of interest are selected with the aid of the user, then at least two LC runs are required, with the zoom maps being measured in the second LC run.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2016 110 165 | Jun 2016 | DE | national |
| 10 2017 111 067 | May 2017 | DE | national |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7838826 | Park | Nov 2010 | B1 |
| 8766176 | Park et al. | Jul 2014 | B2 |
| 9546980 | Räther et al. | Jan 2017 | B1 |
| 20130299690 | Shvartsburg et al. | Nov 2013 | A1 |
| 20160231275 | Park et al. | Aug 2016 | A1 |
| 20170122906 | Mann | May 2017 | A1 |
| Number | Date | Country |
|---|---|---|
| 102012004398 | Oct 2012 | DE |
| Number | Date | Country | |
|---|---|---|---|
| 20170350861 A1 | Dec 2017 | US |
