EP2499655A1

EP2499655A1 - Detection and/or quantification of a compound in a sample

Info

Publication number: EP2499655A1
Application number: EP10798370A
Authority: EP
Inventors: Timothy Riley; James Landridge
Original assignee: Micromass UK Ltd
Current assignee: Micromass UK Ltd
Priority date: 2009-11-13
Filing date: 2010-11-15
Publication date: 2012-09-19
Also published as: GB0919870D0; WO2011058381A1

Abstract

The invention relates to a method of screening and/or detecting and/or quantifying the amount of one or more compounds of interest in a sample. The invention also relates to a control system and mass spectrometer programmed to carry out the method. The method involves identifying one or more parent and/or daughter ion masses of interest, acquiring a first data set from the sample, determining the presence of candidate parent ions of interest and/or a first set of candidate daughter ions of interest in the first data set, selecting the candidate parent ions of interest, fragmenting the selected candidate parent ions of interest to produce a second set of candidate daughter ions of interest and creating a mass spectrum of at least some of the second set of candidate daughter ions of interest. One embodiment of the invention involves determining or calculating the quantity of at least a first compound of interest from the mass spectrum. Another embodiment of the invention involves confirming the presence or absence of a first compound of interest in the mass spectrum.

Description

DETECTION AND/OR QUANTIFICATION OF A COMPOUND IN A SAMPLE

This invention relates generally to a method of detecting and/or quantifying the amount of a compound in a sample using mass spectrometry (MS). More specifically, although not exclusively, this invention relates to a method of screening a sample in order to detect the presence and/or to quantify the amount of one or more compounds of interest.

Over the past two decades, liquid chromatography in combination with tandem quadrupole MS has been used very successfully to quantify targeted small molecules, peptides, and proteins contained in complex sample mixtures. The tandem quadrupole MS system is typically operated in a multiple reaction monitoring (MRM) mode of data acquisition such that, at an appropriate time in the chromatographic separation of the mixture when a component of interest elutes into the mass spectrometer, the parent ion of that component is selected with nominal mass accuracy using the first mass filter of the tandem quadrupole instrument.

This precursor ion is subsequently dissociated in the tandem quadrupole collision cell and one or more of the product ions produced in this collisional induced dissociation process is selected with nominal mass accuracy for detection using the second quadrupole mass filter. The intensity of these detected MRM product ion signal(s) is used as a quantitative measure of the amount of the component present in the mixture. This MRM selection process can be repeated at high data acquisition rates thus allowing many targeted analytes to be quantified in a single chromatographic separation of a mixture.

This method of quantification is generally recognised as being very sensitive and selective. However, there are situations when the complexity of the sample can define the lower limit of detection and dynamic range of this strategy.

The present invention seeks to provide improved selectivity and/or substantially improved detection limits and dynamic range compared to conventional low resolution, nominal mass accuracy tandem quadrupole MRM analysis for targeted quantitative analysis of complex mixtures. The invention builds on LC-MSE alternating scan high and low energy acquisition methodology developed by the present applicants, which can be used to qualitatively and quantitatively characterize small molecules and proteins contained in complex mixtures. This methodology is explained in detail in US6717130 and US71 12784, the entire contents are incorporated herein by reference. A more enabling implementation of the LC-MSE data acquisition strategy may be accomplished by acquiring the alternating high and low energy LC-MS full scan data at elevated resolution (> 5,000 RP FWHM) and high mass accuracy (<30 ppm). When appropriately processed, this data provides accurate mass and intensity information on all detectable precursor and chromatographically aligned product ions and can be used to identify and quantify the level of components in the analyzed mixture. In many cases, this LC-MSE methodology may provide selectivity, limits of detection and dynamic range that are superior to that provided by LC-tandem quadrupole MRM analysis.

The present invention provides a method and apparatus for the detection and profiling of organic species, (in the preferred embodiment biomolecules), with the approach easily applied for a wide range of organic compounds, such as proteins, peptides, glycopeptides, endogenous small molecules such as lipids and sugars, small molecule metabolites or environmental contaminants.

One aspect of the present invention provides a method of detecting and/or quantifying the amount of one or more compounds of interest in a sample, the method comprising the steps of:

i) identifying one or more parent and/or daughter ion masses of interest;

ii) acquiring a first data set from the sample;

iii) determining the presence of candidate parent ions of interest and/or a first set of candidate daughter ions of interest in the first data set;

iv) selecting the candidate parent ions of interest;

v) fragmenting the selected candidate parent ions of interest to produce a second set of candidate daughter ions of interest;

vi) creating a mass spectrum of at least some of the second set of candidate daughter ions of interest; and

vii) determining or calculating the quantity of a first compound of interest from the mass spectrum.

A second aspect of the invention provides a method of screening a sample for the presence of one or more compounds of interest, the method comprising the steps of: i) identifying one or more parent and/or daughter ion masses of interest;

ii) acquiring a first data set from the sample;

iv) selecting the candidate parent ions of interest; v) fragmenting the selected candidate parent ions of interest to produce a second set of candidate daughter ions of interest;

vii) confirming the presence or absence of a first compound of interest in the mass spectrum.

The first data set may comprise a set of fragmented data, e.g. a set of data derived or measured from fragmented ions, and/or a set of unfragmented data, e.g. a set of data derived or measured from unfragmented ions. Advantageously, the acquisition of the first set of data may comprise the use of an alternating scan, high and low energy acquisition method, e.g. as described above.

The method is preferably carried out using a mass spectrometer, for example which comprises a filtering device that may be used in the selection step, e.g. to select the candidate parent ions of interest, and/or a fragmentation device that may be used in the fragmentation step, e.g. to fragment the selected candidate parent ions of interest, and/or one or more mass analysers that may be used in the acquisition and/or mass spectrum creation step or steps, e.g. to acquire the first data set and/or to create the mass spectrum, e.g. of at least some of the candidate daughter ions of interest.

The filtering device and/or mass analyser may comprise a quadrupole, for example the selection step and/or acquisition step and/or mass spectrum creation step or steps may involve the use of a resolving quadrupole. Additionally or alternatively, the filtering device may comprise a two dimensional or three dimensional ion trap or time-of-flight (ToF) mass analyser. The mass analyser or mass analysers may comprise or further comprise one or more of a time-of-flight mass analyser and/or an ion cyclotron resonance mass analyser and/or an orbitrap mass analyser and/or a two dimensional or three dimensional ion trap.

Filtering by means of selection based upon mass-to-charge ratio (m/z) can be achieved by means of a first mass analyser. This mass analyser can be used to serially select ions based upon m/z, for example a quadrupole; or to transmit a wide m/z range, separate ions according to their m/z, and then select the ions of interest by means of their m/z value. An example of the latter would be a time-of-flight mass analyser combined with a timed ion selector(s). As such, one embodiment of the invention provides a filtering window set to allow the parent ion of interest and isotopically labelled analogues to be selected, e.g. during the selection step. Advantageously, the method may further comprise isolating and/or separating the one or more compounds of interest, for example from two or more of a plurality of compounds, e.g. using a chromatographic technique such as liquid chromatography, which may be carried out using a liquid chromatograph. The method may further comprise measuring an elution time for the compound of interest and/or comparing the measured elution time with an expected elution time.

In some embodiments, the method of the first aspect of the invention further comprises determining or calculating the quantity of a second compound of interest from the / or a further mass spectrum. Similarly, the method of the second aspect of the invention may further comprise confirming the presence or absence of a second compound of interest in the or a further mass spectrum.

The first and second compounds of interest may be separated using a chromatographic technique such as liquid chromatography, which may be carried out using a liquid chromatograph. Additionally, the first and second compounds of interest may be selected by order or time of elution, e.g. from a liquid chromatograph. It will be appreciated that the aforementioned methods may be used to determine or calculate the quantity or confirm the presence of multiple or a plurality of compounds of interest. The method may further comprise measuring an elution time for the first and/or second compounds of interest and/or comparing the measured elution time with an expected elution time.

Additionally or alternatively, the first and second compounds of interest may be separated using an ion mobility technique, which may be carried out using an ion mobility cell. Additionally, the first and second compounds of interest may be selected by order or time of ion mobility drift. It will be appreciated that the aforementioned methods may be used to determine or calculate the quantity or confirm the presence of multiple or a plurality of compounds of interest. The method may further comprise measuring a drift time for the first and/or second compounds of interest and/or comparing the measured drift time with an expected drift time. An optional feature of the invention provides the use of one or more collisional cross section calculation techniques, for example to enable a user to predict the order of elution of the candidate parent ions of interest, e.g. to facilitate the selection of the first and second compounds of interest.

Additionally or alternatively, the method may involve the use of an ion mobility technique after the selection step and/or before the fragmentation step, for example to separate between two parent ions of the same or similar mass and/or with the same or similar elution time. Additionally or alternatively, the method may involve the use of an ion mobility technique for the selection step, e.g. the step of selecting the candidate parent ions of interest. The ion mobility technique may be carried out using an ion mobility cell.

The method may further comprise selecting the candidate parent ions of interest based on the first set of candidate daughter ions of interest.

One embodiment of the invention relates to determining the presence or quantity of ions of a particular class or species. This method could comprise identifying a daughter ion indicative of the particular class or species of interest and may subsequently comprise selecting candidate parent ions to determine the presence and/or identity of the ions of the class or species.

Advantageously, the compounds of interest may be identified after quantification, e.g. using the mass spectrum and/or data relating to the selected parent ions of interest.

Quantification can be divided into label-free and isotope labelling methods.

In the case of the label-free method, quantification can be achieved by comparison of the peak intensity, or area under the mass spectral peak for the precursor or fragment m/z values of interest between injections and across samples. The use of an internal standard normalisation factor to account for any associated analytical error is known. Another label-free method of quantification, spectral counting, involves summing the number of fragment ion spectra, or scans, that are acquired for each given peptide, in a non-redundant or redundant fashion. The associated peptide mass spectra for each protein are then summed, providing a measure of the number of scans per protein with this being proportional to a proteins abundance. Comparison can then be made between samples.

In the case of stable isotope labelling studies (e.g. ICAT, iTRAQ, TMT, metabolic labelling and SI LAC) analogue peptides are typically produced by incorporating stable isotopes into the peptide or protein sequences, making the peptide either different in mass (isotope labelling), or having a different reporter ion mass in the MS/MS spectrum (isobaric labelling). Quantification can subsequently be performed through comparison of the mass spectral peak intensity, or area, of the light and heavy labelled peptides. Preferably, the ion source is selected from the group comprising or consisting of: (i) an Electrospray lonisation ("ESI") ion source; (ii) an Atmospheric Pressure Photo lonisation ("APPI") ion source; (iii) an Atmospheric Pressure Chemical lonisation ("APCI") ion source; (iv) a Matrix Assisted Laser Desorption lonisation ("MALDI") ion source; (v) a Laser Desorption lonisation ("LDI") ion source; (vi) an Atmospheric Pressure lonisation ("API") ion source; (vii) a Desorption lonisation On Silicon ("DIOS") ion source; (viii) an Electron Impact ("El") ion source; (ix) a Chemical lonisatjon ("CI") ion source; (x) a Field lonisation ("Fl") ion source; (xi) a Field Desorption ("FD") ion source; (xii) an Inductively Coupled Plasma ("ICP") ion source; (xiii) a Fast Atom Bombardment ("FAB") ion source; (xiv) a Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xv) a Desorption Electrospray lonisation ("DESI") ion source; (xvi) a Nickel- 63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption lonisation ion source; and (xviii) a Thermospray ion source.

Further aspects of the invention provide an apparatus and/or control system configured or adapted or operative or programmed to execute the method, a computer program element comprising computer readable program code means for causing a processor to execute a procedure to implement the method and/or a computer readable medium embodying such a computer program element. A yet further aspect of the invention provides a computer readable medium having a program stored thereon, where the program is to make a computer execute a procedure to implement the method.

Embodiments of the invention will now be described by way of example only.

One preferred basis of the approach is to initially use an alternating low and elevated energy (LC-MSE) scan function in combination with an LC separation of a mixture. In combination with this a target list of molecules is supplied that contains information such as, but is not limited to, m/z of precursor and fragment ions, retention time, ion mobility drift time and rate of change of mobility (e.g. FAIMS). During the course of the LC separation and as the target ions elute into the mass spectrometer (and as either low energy precursor ions, or elevated energy fragment ions are detected, or the retention time window is activated) the first mass analyzer (MS1 ) selects a narrow m/z range (of a variable and changeable width) to pass ions through to the gas cell. The ions are subsequently fragmented (or not) and product ions are recorded with the second mass analyzer. The instrument then switches back to the LC-MSE mode of operation after the ion finishes eluting or the retention time window ends. This approach builds upon the methodology described in US6717130 and US71 12784 and numerous combinations or analytical derivations of this technique can be applied. This invention provides a method to qualitatively profile mixtures via LC-MSE, with the target mode providing specific fragment ion information with enhanced signal to noise and increased specificity for target compound quantification. The distinguishing features of this approach are the ability to profile samples with a variety of prior knowledge in a highly specific manner. Compared to MRM it has wider profiling or open platform capabilities. Compared to LC-MSE it provides a mechanism to remove interfering species with MS1 (or IMS stage) and hence remove noise and increase signal for quantitative studies. The variable band width nature of the first mass analyzer allows for multiple species to be selected simultaneously for fragmentation and quantification (such as isotope labelled peptide or modified peptides).

The method and apparatus of the present invention improves on the LC-MSE strategy through the addition of another stage of mass filtering. The invention can be illustrated on a quadrupole time-of-flight type mass spectrometer system although other geometry mass spectrometry systems may also be employed. In a typical LC-MSE experiment, sample ions created in the ion source of a quadrupole time-of-flight type instrument are transmitted through the quadrupole analyzer which is operated continuously in broad band pass mode. These ions are then transmitted into a collision cell which alternately and repeatedly switches between a first mode where the sample precursor ions are substantially fragmented to product ions and a second mode wherein the sample precursor ions are not substantially fragmented. Typically high resolution, accurate mass spectra are acquired in both modes and at the end of the experiment associated precursor and product ions are recognized by the closeness in fit of their chromatographic elution times and optionally other physicochemical properties. The signal intensity of either the parent or the product ion associated with a targeted component(s) of interest can be used to determine the quantity of the component in the mixture.

In a first embodiment this invention improves the specificity of the LC-MSE analysis for targeted quantitative analysis by using the quadrupole time-of-flight type instrument or a functionally equivalent mass spectrometer that provides an ion source, a first mass analyzer capable of low or high resolution m/z selection, a capability to dissociate the ions selected in the first mass analyzer and a second mass analyzer capable of mass measuring the ions produced in this dissociation process preferably at elevated resolution (>5000 FWHM and high mass accuracy (<30ppm).

In a less preferred embodiment a Trap-ToF mass spectrometer can be used in a similar way instead of a quadrupole time-of-flight type instrument. Further embodiments of the invention could include the use of a Quadrupole followed by a linear trap and a ToF, a linear Trap followed by an orbitrap, a Quadrupole followed by a linear trap and an Orbitrap, or any other type of selective analyser (that may itself be a collisional device i.e. a trap) or filter followed by a collision cell and then a full scan analyser. In the instance that the selective analyser is also a collisional device the following collision cell may not be essential.

It will be appreciated that the ion source may comprise any suitable ion source. However, the ion source is preferably one of: an Electrospray lonisation ("ESI") ion source; an Atmospheric Pressure Photo lonisation ("APPI") ion source; an Atmospheric Pressure Chemical lonisation ("APCI") ion source; a Matrix Assisted Laser Desorption lonisation ("MALDI") ion source; a Laser Desorption lonisation ("LDI") ion source; an Atmospheric Pressure lonisation ("API") ion source; a Desorption lonisation On Silicon ("DIOS") ion source; an Electron Impact ("El") ion source; a Chemical lonisatjon ("CI") ion source; a Field lonisation ("Fl") ion source; a Field Desorption ("FD") ion source; an Inductively Coupled Plasma ("ICP") ion source; a Fast Atom Bombardment ("FAB") ion source; a Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; a Desorption Electrospray lonisation ("DESI") ion source; a Nickel- 63 radioactive ion source; an Atmospheric Pressure Matrix Assisted Laser Desorption lonisation ion source; and a Thermospray ion source.

At a chromatographic retention time when a targeted analyte of interest is about to elute into the mass spectrometer ion source, the first mass analyzer of the mass spectrometer (MS1 ) is used to select a narrow m/z range (of a variable and changeable width) of sample precursor ions which includes the m/z of the targeted precursor ion. These selected ions are then transferred to an instrument stage capable of dissociating the ions by means of alternate and repeated switches between a first mode where the sample precursor ions are substantially fragmented to product ions and a second mode wherein the sample precursor ions are not substantially fragmented. Typically high resolution, accurate mass spectra are acquired in both modes and at the end of the experiment associated precursor and product ions are recognized by the closeness in fit of their chromatographic elution times and optionally other physicochemical properties. This detection process is sustained until the analyte of interest has completed its chromatographic elution into the mass spectrometer. However, in a less preferred embodiment only fragmented data are considered, wherein the sample precursor ions are substantially fragmented to product ions. This analysis cycle is then repeated as each targeted analyte of interest chromatographically elutes into the mass spectrometer. For each targeted analyte analysis cycle, the first mass filter is used to select a m/z range appropriate for the targeted analyte precursor ion, and alternating high resolution, accurate mass precursor and associated product ion information are collected.

In a second embodiment the invention improves the specificity of the first embodiment by complementing the targeted MS1 mass selection precursor selection and coupled high resolution accurate mass alternating scan parent and product ion data acquisition process with ion mobility selection. In this embodiment a mass spectrometer that is functionally equivalent to a quadrupole time-of-flight type instrument geometry capable of ion mobility separation such as the Synapt G2 HDMS system is used for the analysis. In addition to switching the MS1 mass filter to selectively transmit a m/z range which includes the targeted analyte precursor ion, only parent or product ion signal with ion mobility, accurate mass and chromatographic retention time attributes characteristic of the target analyte would be used for its quantitative measurement.

In a preferred embodiment information about at least some, preferably most, in some cases all, ions present may be extracted from the first data set, e.g. quantification of the quantities of ions or identification of the presence and identity of ions.

In one embodiment, it may be possible to combine and/or compare information from the first data set and the mass spectrum for the ions of interest, e.g. to confirm or compare the quantity of the ions of interest from the two different data sets.

The unique aspect to this invention is the combination of an open-platform discovery approach (LC-MSE), with a simultaneous quantitative approach that provides greater specificity and sensitivity. In particular the targeted strategy provides a means to reduce chemical noise from interfering species in the elevated energy (or MS/MS) mode. Combining these two approaches allows unknown compounds to be monitored, whilst specific target molecules can be accurately quantified.

The present invention relates to a method, utilizing a mass spectrometer that can be used to detect and quantitatively profile surrogate peptides, or metabolites in complex mixtures. The method incorporates analysis of the sample, by liquid chromatography in combination with mass spectrometry using a combination of different scan modes. In brief these include: a) Inject the sample and start the LC-MSE acquisition

b) Determine when a specific target molecule is present, (using any combination of peptide mass, fragment mass, retention time or ion mobility drift time)

c) Alternatively use information such as retention time from a previous experiment, or from empirical evidence to start the targeted experiment.

d) Target the said compound(s) of interest using a given transmission window of the first mass analyzer (MS1 ) to select a given window of m/z. It should be noted that this first mass analyzer might be an ion mobility spectrometer.

e) Confirm the presence of the compound using a combination of the fragment ion masses, summing their intensity to provide a quantitative profile of the given species.

In order to aid the illustrate of the invention, the process of the invention is shown in the attached figures which show flow diagrams of the method of the present invention.

Figure 1 is a schematic of one embodiment of the present invention

Figure 2 is a schematic of a second embodiment of the present invention

Figure 3 is a schematic of a third embodiment of the present invention

There may be variations in the invention from the schemes illustrated and described above which would fall within the invention. It would be feasible to mix, or merge parts of the schematics such that the invention may be performed by a method not specifically described in this disclosure.

The method could be altered in several different ways and this may well provide additional utility. For example in the case of isotope labelled compounds (where a set mass difference indicates quantitative changes) the ability to broaden MS1 to pass both isotopes is beneficial. The fragment ions can then be used to identify which component is which.

An alternative embodiment of the invention may involve having a collision cell that may be bypassed in such a way that only ions desired to be fragmented to produce product ions are passed through the collision cell. It will be appreciated that other means of chromatographic separation, such as capillary electrophoresis and gas chromatography, may be used in an analogous manner to perform targeted quantification of analytes in complex mixtures. It will be appreciated by those skilled in the art that any number of combinations of the aforementioned features and/or those shown in the appended drawings provide clear advantages over the prior art and are therefore within the scope of the invention described herein.

Claims

1 . A method of detecting and/or quantifying the amount of one or more compounds of interest in a sample, the method comprising the steps of:

i) identifying one or more parent and/or daughter ion masses of interest; ii) acquiring a first data set from the sample;

iii) determining the presence of candidate parent ions of interest and/or a first set of candidate daughter ions of interest in the first data set; iv) selecting the candidate parent ions of interest;

2. A method of screening a sample for the presence of one or more compounds of interest, the method comprising the steps of:

3. Method according to claim 1 or claim 2, wherein the first data set comprises a set of fragmented data and a set of unfragmented data.

4. Method according to any preceding claim, wherein the acquisition of the first set of data comprises the use of an alternating scan high and low energy acquisition method.

5. Method according to any preceding claim carried out using a mass spectrometer that comprises a filtering device for selecting the candidate parent ions of interest, a fragmentation device for fragmenting the selected candidate parent ions of interest and one or more mass analysers for acquiring the first data set and/or for creating the mass spectrum.

6. Method according to any preceding claim, wherein the selection step and/or acquisition step and/or mass spectrum creation step or steps involve the use of a quadrupole filtering device.

7. Method according to claim 5, wherein the filtering device comprises a two dimensional or three dimensional ion trap.

8. Method according to claim 5, wherein the mass analyser or mass analysers comprise or further comprise one or more of a time-of-flight mass analyser and/or an ion cyclotron resonance mass analyser and/or an orbitrap mass analyser and/or a two dimensional or three dimensional ion trap.

9. Method according to any preceding claim further comprising separating the one or more compounds of interest from a plurality of compounds using a chromatographic technique.

10. Method according to claim 9, wherein the chromatographic technique comprises a liquid chromatographic technique.

1 1 . Method according to claim 10 further comprising measuring an elution time for the compound of interest and comparing the measured elution time with an expected elution time.

12. Method according to any preceding claim further comprises determining or calculating the quantity or confirming the presence or absence of a second compound of interest from the / or a further mass spectrum.

13. Method according to claim 12, wherein the first and second compounds of interest are selected by order or time of elution from a liquid chromatograph.

14. Method according to claim 13 further comprising measuring an elution time for each of the first and second compounds of interest and comparing each measured elution time with an expected respective elution time.

15. Method according to claim 12, wherein the first and second compounds of interest are separated using an ion mobility cell.

16. Method according to claim 15, wherein the first and second compounds of interest are selected by order or time of ion mobility drift.

17. Method according to claim 16 further comprise measuring a drift time for the first and/or second compounds of interest and comparing the measured drift time with an expected drift time.

18. Method according to any preceding claim, wherein the step of selecting the candidate parent ions of interest is carried out using an ion mobility cell.

19. A control system operative or programmed to execute a method according to any preceding claim.

20. An analytical instrument comprising a control system according to claim 10.

21 . Analytical instrument according to claim 20, wherein the instrument comprises a mass spectrometer.

22. A computer program element comprising computer readable program code means for causing a processor to execute a procedure to implement the method of any one of claims 1 to 18.

23. Computer readable medium embodying a computer program element according to claim 23.

24. A computer readable medium having a program stored thereon, where the program is to make a computer execute a procedure to implement the method of any one of claims 1 to 18.