Data dependent acquisition (DDA) mass spectrometry

Abstract

A data dependent acquisition (DDA) mass spectrometry method includes ionizing a sample to produce sample ions; analyzing the sample ions by performing one or more MS1 mass analysis scans in order to obtain MS1 data; identifying one or more precursor ions from the MS1 data; and then analyzing the sample ions by performing one or more MS2 mass analysis scans. Each MS2 scan targets a precursor ion of the one or more precursor ions identified from the MS1 data. The method further includes, for each precursor ion of one or more precursor ions identified from the MS1 data: determining a value indicative of a collision cross-section (CCS) of the precursor ion from the MS1 data; and selecting which precursor ion (s) the one or more MS2 mass analysis scans target based on the CCS indicator and / or determining an order in which the one or more MS2 mass analysis scans target precursor ions based on the CCS indicator.

Description

TECHNICAL FIELD

[0001] The present invention relates generally to the field of mass spectrometry, and more specifically to data dependent acquisition (DDA) mass spectrometry. BACKGROUND

[0002] Proteins are analyzed and studied by mass spectrometry (referred to as proteomics), primarily to identify and quantify proteins that exist in a certain biological state within a cell. Proteins are composed of chains of amino acids (referred to as primary sequence), which is the primary standard used for protein identification in proteomics analysis. However, proteins can also be decorated by modifications (such as phosphate moieties or glycans, referred to as post-translational modifications (PTMs)). These modifications have been shown to affect the biology associated with the modified protein, highlighting the necessity to identify these modified states as well as to characterize the extent of modification (e.g., site localization, modification structure, etc.).

[0003] Unfortunately, these modifications occur in sub-stoichiometry, resulting in lower relative abundance compared to their unmodified counterparts, which leads to difficulties in efficiently selecting and analyzing these species. To avoid the reduction in modified protein abundance, both online and offline enrichment techniques have been employed, whereby modified peptides derived from proteins are retained while most unmodified peptides are discarded.

[0004] In the field of structural biology, scientists study the higher order structure of proteins and their complexes. There are multiple analytical tools available for such studies. Within the field of mass spectrometry, one such tool is cross-linking mass spectrometry. In cross-linking, a cross-linking reagent (a small molecule with two reactive ends at the two ends of the molecule) is introduced into the sample, and these reactive ends bind to reactive amino acids along the protein backbone, covalently binding to the amino acid and the cross-linking reagent. The other reactive end of the cross-linking reagent is able to move within a space defined by the length of the molecule. If there is another reactive amino acid within this spatial distribution, that amino acid also binds to the molecule, linking two locations of the protein, providing the scientist with a spatial map of the structure of the protein / protein complex.

[0005] While providing important structural information, this technique is also fraught with analytical challenges. That is, when the protein is digested by enzymes for analysis through a proteomics workflow, the cross-linked peptides exist in sub-stoichiometric amounts and are therefore difficult to analyze, similar to PTMs. Analysis of cross-linked samples shows that most of the precursors selected for fragmentation are unmodified peptides and not cross-linked peptides. Recently, the chemistry of the cross-linking reagent has been adjusted to allow scientists to enrich for cross-linked peptides, similar to the enrichment step for phosphorylated peptides. However, this requires additional online or offline sample processing on the mass spectrometer.

[0006] For both examples, the analytical challenge faced is how to identify these modified precursors and selectively target them for mass spectrometric analysis.

[0007] It is believed that there is still room for improvement in apparatus and methods for mass spectrometry. SUMMARY

[0008] A first aspect provides a mass spectrometry method, the method comprising:

[0009] ionising a sample to produce sample ions;

[0010] (i) analysing the sample ions by performing one or more MS1 mass analysis scans to obtain MS1 data;

[0011] (ii) identifying one or more precursor ions from the MS1 data; and

[0012] (iii) analysing the sample ions by performing one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data;

[0013] wherein the method further comprises:

[0014] for each of the one or more precursor ions identified from the MS1 data: determining from the MS1 data a value indicative of the collision cross section (CCS) of the precursor ion;

[0015] and

[0016] selecting which precursor ion or precursor ions the one or more MS2 mass analysis scans target based on the CCS indicative value(s) and / or determining an order in which the one or more MS2 mass analysis scans target precursor ions based on the CCS indicative value(s).

[0017] Embodiments provide a data dependent acquisition (DDA) mass spectrometry method in which one or more precursor ions are identified from MS1 data and one or more MS2 mass analysis scans are then triggered to target the identified precursor ions of interest.

[0018] In embodiments, the precursor ions of interest are identified based on a value indicative of the collision cross section (CCS) of the precursor ion of interest as determined from the MS1 data (“CCS indicative value”) and / or an order in which the MS2 scan analyses the precursor ions is determined based on their CCS indicative values. This contrasts with conventional DDA methods, such as the “Top-N” method, in which the MS2 scans analyse the precursor ions in an order determined based on the intensity of the precursor ions in the MS1 data.

[0019] The embodiments build on previous work (e.g. as described in UK patent application GB 2,612,580, the entire contents of which are incorporated herein by reference), in which it has been demonstrated that CCS-indicative values can be obtained directly from MS1 data. This is in contrast to conventional methods for determining CCS values, which use dedicated ion mobility analyzers (e.g. drift cell, travelling wave ion guides, trapped ion mobility separators (TIMS), etc.) to separate ions in the CCS domain. The present inventors have now recognized that the ability to determine CCS-indicative values directly from MS1 data, without the need for ion mobility separation, enables a DDA method in which precursor ions of interest are selected and / or prioritized based on their CCS-indicative values. Thus, in the embodiments described herein, CCS values or values indicative of CCS values as determined from MS1 mass measurements themselves are used to distinguish between precursor ions of interest and drive an intelligent DDA workflow.

[0020] In some embodiments, for each precursor ion for which a CCS-indicative value is determined from MS1 data, this value is compared to one or more values indicative of the expected collision cross section (CCS) or expected collision cross section (CCS) range of the precursor ion, and the precursor ion to be targeted by the MS2 scan is selected based on this comparison. Precursor ions for which the CCS-indicative value deviates from the expected CCS-indicative value or range are allowed to be identified and prioritized for targeting, such as in particular modified and cross-linked peptides and / or proteins. This in turn increases the amount of instrument analysis time used to target such ions of interest, which can be present in low abundance and sub-stoichiometric amounts, and reduces or minimizes the need for sample enrichment prior to MS analysis.

[0021] Thus, it will be appreciated that the embodiments provide an improved mass spectrometry method.

[0022] In the method, a sample is ionized (i.e. ionized by an ion source) to produce sample ions. The sample ions can be, for example, peptide ions and / or protein ions, etc. The sample can be provided to the ion source from a separation device such as a capillary electrophoresis separation device or a chromatographic separation device (e.g. a liquid chromatography (LC) separation device or a gas chromatography (GC) separation device), etc. Additionally or alternatively, the ions can be separated by a separation device such as an ion mobility separation device, a micro-separation ion mobility separation device, or a device configured to separate ions according to their mass-to-charge ratio (m / z).

[0023] The method can comprise performing a plurality of repeated cycles, wherein each cycle comprises performing steps (i), (ii) and (iii) as described above. Thus, steps (i)-(iii) can be repeatedly performed in a cyclical manner. The method can comprise repeatedly performing the cycle during a separation run of the separation device.

[0024] In the first step (i) of each cycle, the sample ions are analysed by performing one or more MS1 mass analysis scans, i.e. by mass analysing the sample ions using a mass analyser. The mass analyser can be any suitable mass analyser, such as an orbitrap mass analyser (e.g. an Orbitrap TM mass analyser), a time-of-flight (ToF) mass analyser such as a multi-reflecting time-of-flight (MR-ToF) mass analyser, an ion cyclotron resonance (ICR) mass analyser, etc.

[0025] In particular embodiments, the mass analyser is an orbitrap mass analyser, wherein the orbitrap mass analyser is operated at a relatively high pressure, e.g. such that the timescale of ion gas collisions within the mass analyser is similar to the timescale required for a mass analysis scan. For example, orbitrap mass analysers are typically operated at a pressure of around 10 -10 mbar or 10 -11 mbar (i.e. so as to minimise ion gas collisions during each mass analysis scan), but in embodiments, when obtaining MS1 data, the orbitrap mass analyser is operated at a pressure of > 10 -9 mbar (e.g. between 1 x 10 -9 mbar and 9 x 10 -9 mbar (such that ion gas collisions are significant during each mass analysis scan). As described in UK patent application GB2,612,580, operating an orbitrap mass analyser at this pressure regime allows the CCS value of an ion to be estimated from the width of the ion peak in the MS1 data.

[0026] In each cycle, one or more precursor ions are identified from the MS1 data, and then the sample ions are analysed by performing one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data. The precursor ions can be initially identified from the MS1 data in any suitable manner (e.g. using suitable data processing and / or peak detection).

[0027] The step (ii) of identifying one or more precursor ions from the MS1 data can comprise identifying a plurality of different precursor ions in the MS1 data. The step (iii) of then analysing the sample ions by performing one or more MS2 mass analysis scans can comprise performing a plurality of MS2 mass analysis scans, wherein each MS2 mass analysis scan of the plurality of MS2 mass analysis scans targets a different one of the plurality of different precursor ions identified in the MS1 data.

[0028] In each MS2 mass analysis scan, the method can comprise: isolating a targeted precursor ion from the sample ions; fragmenting the isolated precursor ion so as to generate fragment ions; and mass analysing the fragment ions using the mass analyser. The step of isolating a targeted precursor ion from the sample ions can comprise using a mass filter (e.g. a quadrupole mass filter) to select the targeted precursor ion according to its mass-to-charge ratio (m / z). Sample ions having m / z within a narrow (e.g. < 5 Da, such as about 2 Da) isolation window centred on the m / z of the targeted precursor ion can be transmitted, while sample ions having m / z outside the isolation window can be attenuated. By varying the centre m / z of the isolation window for each different targeted precursor ion, each of the plurality of MS2 mass spectral analysis scans can be targeted to each of a plurality of different precursor ions.

[0029] In some embodiments, for example where the mass analyser is an orbitrap mass analyser, the method can comprise performing a single MS1 mass analysis scan in each cycle so as to obtain MS1 data, followed by performing a single MS2 mass analysis scan in respect of each of one or more targeted precursor ions. However, this is not essential, and for example where a different type of mass analyser is used (such as a ToF mass analyser which forms individual mass spectra by averaging the results of multiple scans) and / or where a more complex analysis method is used, more than one MS1 mass analysis scan can be used in a cycle to obtain MS1 data, and / or more than one MS2 mass analysis scan can be performed in respect of each of one or more targeted precursor ions.

[0030] In the method, for each of one or more precursor ions identified from the MS1 data, a value indicative of the collision cross section (CCS) of that precursor ion is determined from the MS1 data (i.e. a “CCS indicative value”). The CCS indicative value can be determined for some, most or all of the precursor ions apparent in the MS1 data.

[0031] The CCS indicative value can be the CCS value itself, or some other value indicative of the CCS value of the precursor ion. Each CCS indicative value is determined (directly) from the MS1 data, for example from a single MS1 spectrum (and without any reference to ion mobility drift times).

[0032] In particular embodiments, the value indicative of the collision cross section (CCS) of the precursor ion is the width of the ion peak in the MS1 data associated with that precursor ion (and the subsequent steps using the CCS indicative value are performed using this width). Alternatively, the actual collision cross section (CCS) value or some other value indicative of the collision cross section (CCS) of the precursor ion can be determined from the width of the ion peak in the MS1 data associated with that precursor ion (and the subsequent steps using the CCS indicative value can be performed using this value). In embodiments, any suitable measure of ion peak width can be used, such as for example the full width at half maximum (FWHM). These embodiments are particularly suitable, but not exclusively, for embodiments such as described in UK patent application GB 2,612,580, in which the mass analyser is an orbitrap mass analyser operating under suitable pressure regimes to allow estimation of the CCS value of an ion from the width of the ion peak in the MS1 data.

[0033] However, other methods of determining or estimating a CCS indicative value from MS1 data can be used (without using ion mobility separation). For example, UK patent application GB 2303690.8 (the entire contents of which are incorporated herein by reference) describes a method in which a time-of-flight (ToF) mass analyser is used to determine a CCS indicative value. This is done by obtaining two sets of data (i.e. by performing two scans (or two sets of scans)), in which one or both of (i) the ion path length and (ii) the gas pressure in the ion path is changed between the two sets of data. By comparing the intensities of corresponding ion peaks in the two sets of data, the CCS of the ion producing that corresponding ion peak can be determined.

[0034] Thus, the mass analyser can be a time-of-flight (ToF) mass analyser (such as a multi-reflecting time-of-flight (MR-ToF) mass analyser) configured to determine the mass-to-charge ratio (m / z) of an ion by determining the time of flight of the ion along an ion path, and step (i) of analysing sample ions by performing one or more MS1 mass analysis scans so as to obtain MS1 data can comprise:

[0035] operating the mass analyser in a first operating mode, and analysing the sample ions by determining the time of flight of the sample ions along an ion path so as to obtain a first set of MS1 data, wherein in the first operating mode (i) the ion path has a first path length, and (ii) the ion path is maintained at a first pressure; and

[0036] operating the mass analyser in a second mode of operation and analysing the sample ions by determining a time of flight of the sample ions along the ion path in order to obtain a second set of MS1 data, wherein in the second mode of operation (i) the ion path has a second path length, and (ii) the ion path is maintained at a second pressure, wherein the second path length is different from the first path length and / or the second pressure is different from the first pressure.

[0037] In these embodiments, the second path length can be greater than the first path length and / or the second pressure can be greater than the first pressure. In particular, the time-of-flight mass analyser can comprise one or more ion reflectrons (e.g. various types of ion reflectrons described in GB2303690.8), and in the first mode of operation the ions can be caused to undergo n reflections in the one or more ion reflectrons, where n is an integer > 0, and in the second mode of operation the ions can be caused to undergo m reflections in the one or more ion reflectrons, where m is an integer > n.

[0038] In these embodiments, the step of determining a value indicative of the collision cross section (CCS) of the precursor ions from the MS1 data can comprise comparing the intensity of the precursor ion peak in the first set of data to the intensity of the corresponding precursor ion peak in the second set of data, and determining the value indicative of the CCS of the precursor ions based on the comparison. The method can comprise determining a ratio of the intensity of the precursor ion peak in the first set of data to the intensity of the corresponding precursor ion peak in the second set of data, and using the ratio to determine the CCS indicative value for the precursor ions. For example, in some embodiments the value indicative of the collision cross section (CCS) of the precursor ions is the ratio (and the subsequent steps using the CCS indicative value are performed using the ratio). Alternatively, the actual collision cross section (CCS) value can be determined from the ratio, e.g. by comparing the ratio to a calibration to determine the CCS of the precursor ions (and the subsequent steps using the CCS indicative value can be performed using the CCS value).

[0039] In general, in the methods described herein, the CCS indicative value (e.g. as determined for a plurality of precursor ions identified in the MS1 data) is used to select which of the plurality of precursor ions identified in the MS1 data one or more MS2 mass analysis scans should target and / or to determine an order in which the precursor ions should be targeted by the one or more MS2 mass analysis scans. Then, in step (iii), the one or more MS2 mass analysis scans are performed, wherein each MS2 mass analysis scan targets one of the selected precursor ions and / or wherein the precursor ions are targeted in the determined order.

[0040] The method can include: (a) selecting which of the identified precursor ions the MS2 mass analysis targets based on the CCS-indicative values (without ordering); or (b) determining the order in which the MS2 mass analysis scans target the precursor ions based on the CCS-indicative values (without selecting only some of the identified precursor ions to target); or (c) both selecting which of the identified precursor ions the MS2 mass analysis targets based on the CCS-indicative values (i.e., selecting some but not all of the identified precursor ions) and determining the order in which the MS2 mass analysis scans target the selected precursor ions based on the CCS-indicative values.

[0041] Other factors can be considered (in combination with the CCS-indicative values) when selecting the precursor ions and / or when determining the order. Thus, the method can include selecting which precursor ion or precursor ions one or more MS2 mass analysis scans target based on the CCS-indicative values and one or more other factors, and / or determining the order in which the one or more MS2 mass analysis scans target the precursor ions based on the CCS-indicative values and one or more other factors. The one or more other factors can include, for example, the intensities of the precursor ions in the MS1 data and / or the m / z of the precursor ions in the MS1 data, etc. The one or more other factors can also or instead include the number of times a precursor ion has been targeted by an MS2 mass analysis scan and / or the time elapsed since a precursor ion was previously targeted by an MS2 mass analysis scan. For example, precursor ions that have been targeted by a desired number of MS2 scan(s) can be added to an exclusion list, which can have a time limit. Thus, it should be noted that a precursor ion that is selected (only) based on its CCS-indicative value in the manner described above, below, and elsewhere herein can in fact be excluded from targeting by an MS2 mass analysis scan (i.e., due to one or more other factors).

[0042] Selecting which of the identified precursor ions the MS2 mass analysis scans target based on the CCS-indicative values can be done in any suitable manner. For example, those precursor ions whose CCS-indicative values are greater than a threshold value and / or less than a threshold value and / or within or outside a range can be selected, or a certain fixed number or proportion of the identified precursor ions with the highest or lowest CCS-indicative values or closest to a target CCS-indicative value can be selected.

[0043] Similarly, determining the order in which the MS2 mass analysis scans target the precursor ions based on the CCS-indicative values can be done in any suitable manner. For example, the precursor ions can be ordered according to highest to lowest CCS-indicative values, lowest to highest CCS-indicative values, etc.

[0044] In some embodiments, the precursor ions selected are precursor ions of a particular chemical class. For example, where the sample is a heterogeneous mixture of different chemical classes (e.g., containing two or more of peptides, lipids, steroids, etc.), each of the different chemical classes can exhibit a different trend line in the CCS indicator value - m / z space. In this regard, it has been recognized that there is a relationship between the CCS indicator value and the m / z for ions of various different chemical classes. These relationships can take the form of a “trend line” for each different chemical class. In practice, there will be some spread in the CCS indicator value for ions of a particular chemical class and a particular m / z, but generally the spread is small enough that ions of different chemical classes can be distinguished in most of the CCS indicator value - m / z space. Thus, in embodiments, precursor ions from only one chemical class can be selected for analysis and / or can be preferentially targeted by one or more MS2 mass analysis scans, e.g., by selecting the precursor ions based on their CCS indicator value and m / z and / or determining an order based on the CCS indicator value and m / z.

[0045] In particular embodiments, the method includes, for each of one or more of the precursor ions for which a CCS indicator value is determined: comparing the determined CCS indicator value to one or more values indicative of an expected collision cross section (CCS) or expected collision cross section range for the precursor ion. This comparison can be made for one or more or most or all of the precursor ions for which a CCS indicator value is determined. The step of selecting which precursor ion or precursor ions for an MS2 mass analysis scan to target can then include selecting which of the identified precursor ions for one or more MS2 mass analysis scans to target based on the comparison. Likewise, the step of determining an order for MS2 mass analysis scans to target precursor ions can include determining an order for the precursor ions for one or more MS2 mass analysis scans to target based on the comparison.

[0046] In some embodiments, the method can include, for each of one or more or most or all of the precursor ions for which a CCS indicator value is determined: calculating a difference between the determined CCS indicator value and an expected CCS indicator value. Then, only those precursor ions for which the calculated difference is greater than (or less than) a threshold value can be selected, or a certain fixed number or proportion of the identified precursor ions with the highest or lowest calculated difference can be selected. Additionally or alternatively, an order can be determined by ordering the precursor ions from highest to lowest calculated difference or from lowest to highest calculated difference.

[0047] In particular embodiments, the comparing step can comprise, for each precursor ion for which a CCS-indicative value is determined, determining whether the determined CCS-indicative value is (a) greater than a maximum expected CCS-indicative value for that precursor ion. The selecting step, based on the comparison, can then comprise: when it is determined that (a) the determined CCS-indicative value is greater than the maximum expected CCS-indicative value: selecting that precursor ion for targeting by one of the MS2 mass analysis scans. Then, in step (iii), one of the MS2 mass analysis scans can target the selected precursor ion. Additionally or alternatively, the determining an order step, based on the comparison, can comprise: when it is determined that (a) the determined CCS-indicative value is greater than the maximum expected CCS-indicative value: giving that precursor ion a relatively high priority in the order. The method can optionally comprise, when it is determined that (a) the determined CCS-indicative value is less than the maximum expected CCS-indicative value: not selecting that precursor ion (and the MS2 mass analysis scan does not target that precursor ion) and / or giving that precursor ion a relatively low priority in the order.

[0048] Additionally or alternatively, the comparing step can comprise, for each precursor ion for which a CCS-indicative value is determined, determining whether the determined CCS-indicative value is (b) less than a minimum expected CCS-indicative value for that precursor ion. The selecting step, based on the comparison, can then comprise: when it is determined that (b) the determined CCS-indicative value is less than the minimum expected CCS-indicative value: selecting that precursor ion for targeting by one of the MS2 mass analysis scans. Then, in step (iii), one of the MS2 mass analysis scans can target the selected precursor ion. Additionally or alternatively, the determining an order step, based on the comparison, can comprise: when it is determined that (b) the determined CCS-indicative value is less than the minimum expected CCS-indicative value: giving that precursor ion a relatively high priority in the order. The method can optionally comprise, when it is determined that (b) the determined CCS-indicative value is greater than the minimum expected CCS-indicative value: not selecting that precursor ion (and the MS2 mass analysis scan does not target that precursor ion) and / or giving that precursor ion a relatively low priority in the order.

[0049] Additionally or alternatively, the comparing step can comprise, for each precursor ion for which a CCS-indicative value is determined, determining whether (c) the determined CCS-indicative value falls outside an expected range of CCS-indicative values for that precursor ion. Then, the step of selecting based on the comparison can comprise: when it is determined (c) that the determined CCS-indicative value falls outside the expected range of CCS-indicative values: selecting that precursor ion for targeting by one of the MS2 mass analysis scans. Then, in step (iii), one of the MS2 mass analysis scans can target the selected precursor ion. Additionally or alternatively, the step of determining an order based on the comparison can comprise: when it is determined (c) that the determined CCS-indicative value falls outside the expected range of CCS-indicative values: giving that precursor ion a relatively high priority in the order. The method can optionally comprise, when it is determined (c) that the determined CCS-indicative value falls within the expected range of CCS-indicative values: not selecting that precursor ion (and the MS2 mass analysis scan does not target that precursor ion) and / or giving that precursor ion a relatively low priority in the order.

[0050] In these embodiments, in step (iii), the MS2 scans will target precursor ions with a relatively high priority in the order, and then target precursor ions with a relatively low priority.

[0051] As described above and elsewhere herein, identifying precursor ions in this way whose CCS-indicative values deviate from expected CCS-indicative values or ranges allows for identification and preferential targeting of modified sample ions, such as modified and cross-linked peptides and / or proteins. For example, in particular embodiments, precursor ions whose CCS-indicative values are higher than expected can be cross-linked peptides or glycosylated peptides. Precursor ions whose CCS-indicative values are lower than expected can be phosphorylated peptides.

[0052] In embodiments, one or more values indicative of an expected collision cross section (CCS) or range of collision cross sections can be determined by calibration, such as a calibration curve. Then, the method can comprise, for each precursor ion for which a CCS-indicative value is determined, determining from the calibration one or more values indicative of an expected collision cross section (CCS) or range of collision cross sections for that precursor ion, and using the one or more values in the comparing step.

[0053] The calibration can include an expected CCS-indicative value and / or an expected range of CCS-indicative values as a function of mass-to-charge ratio (m / z). For example, the calibration can include a maximum expected CCS-indicative value as a function of mass-to-charge ratio (m / z) and a minimum expected CCS-indicative value as a function of mass-to-charge ratio (m / z), which together can form an expected range of CCS-indicative values as a function of mass-to-charge ratio (m / z), i.e., an expected region in the CCS-indicative value - m / z space. Then, when a determined CCS-indicative value of a precursor ion falls outside of the expected region, the precursor ion can be selected and / or prioritized in the order.

[0054] In embodiments, the selected precursor ion is a modified precursor ion, such as a modified or cross-linked peptide ion and / or protein ion. Then, the calibration can be generated by analyzing one or more samples of unmodified precursor ions, where the precursor ions are of the same type as the sample ions to be analyzed. For example, when the sample ions are peptide ions and / or protein ions, the calibration can be generated by analyzing one or more samples of unmodified peptide ions and / or protein ions. The calibration can be generated by measuring values indicative of the collisional cross-section of a number of unmodified precursor ions having varying m / z (e.g., peak width) and establishing the calibration from these measurements (e.g., by averaging, etc.).

[0055] As described above, in each MS2 mass analysis scan, the targeted precursor ion is fragmented. In some embodiments, the fragmentation energy used and / or the fragmentation method used in each MS2 mass analysis scan of the plurality of MS2 mass analysis scans is the same. Alternatively, one or both of the fragmentation energy and / or the fragmentation method can vary between some or all of the MS2 mass analysis scans of the plurality of MS2 mass analysis scans, and can be selected based on the CCS-indicative value of the targeted precursor ion, e.g., in order to obtain improved MS2 data.

[0056] Thus, the method can comprise, for one or more or each of the MS2 mass analysis scans: selecting a fragmentation energy to use when performing the MS2 mass analysis scan (and using the selected fragmentation energy when performing the MS2 mass analysis scan) based on the CCS-indicative value of the precursor ion targeted by the MS2 mass analysis scan. Additionally or alternatively, the method can comprise, for one or more or each of the MS2 mass analysis scans: selecting a fragmentation method to use when performing the MS2 mass analysis scan (and using the selected fragmentation method when performing the MS2 mass analysis scan) based on the CCS-indicative value of the precursor ion targeted by the MS2 mass analysis scan. The fragmentation method can be selected from a plurality of possible fragmentation methods, where the plurality of possible fragmentation methods can comprise any combination of two or more fragmentation methods, such as, for example, collision-induced dissociation (CID), electron-induced dissociation (EID), photodissociation, etc. Many other types of fragmentation are possible.

[0057] In these embodiments, the fragmentation energy and / or method can be selected according to a particular chemical class inferred from, for example, the CCS-indicative value and m / z of the precursor ion targeted, for example in order to obtain improved MS2 data.

[0058] Additionally or alternatively, the fragmentation energy and / or method can be selected based on a comparison (e.g. in a manner corresponding to that described above). For example, the fragmentation energy and / or method can be selected according to whether (and / or how much) the CCS-indicative value of the precursor ion deviates from an expected CCS-indicative value or range. This can allow improved MS2 data to be obtained for unmodified and modified / cross-linked peptides and / or proteins, etc.

[0059] These“decision-driven fragmentation” methods can be performed in conjunction with or independently of the CCS-indicative value-driven DDA methods described above.

[0060] Thus, a second aspect provides a mass spectrometry method, the method comprising:

[0061] ionising a sample to produce sample ions;

[0062] (i) analysing the sample ions by performing one or more MS1 mass analysis scans in order to obtain MS1 data;

[0063] (ii) identifying one or more precursor ions from the MS1 data; and

[0064] (iii) analysing the sample ions by performing one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data;

[0065] wherein the method further comprises, for each of the one or more precursor ions identified from the MS1 data: determining, from the MS1 data, a value indicative of a collision cross section (CCS) of the precursor ion; and

[0066] for each of one or more of the MS2 mass analysis scans:

[0067] selecting a fragmentation energy to be used in performing the MS2 mass analysis scan based on the CCS-indicative value of the precursor ion targeted by the MS2 mass analysis scan; and / or

[0068] selecting a fragmentation method to be used in performing the MS2 mass analysis scan based on the CCS-indicative value of the precursor ion targeted by the MS2 mass analysis scan.

[0069] selecting a fragmentation method to be used in performing the MS2 mass analysis scan based on the CCS-indicative value of the precursor ion targeted by the MS2 mass analysis scan.

[0070] The aspect can and in embodiments does include any one or more, or each, of the optional features described herein.

[0071] Another aspect provides a non-transitory computer readable storage medium storing computer software code which, when executed on a processor, performs the method described above.

[0072] Another aspect provides a control system for an analytical instrument such as a mass spectrometer, the control system being configured to cause the analytical instrument to perform the method described above.

[0073] Another aspect provides an analytical instrument such as a mass spectrometer, the analytical instrument comprising the control system described above.

[0074] Another aspect provides an analytical instrument such as a mass spectrometer, the analytical instrument comprising:

[0075] an ion source configured to ionise a sample to produce sample ions;

[0076] a mass filter configured to filter ions using an isolation window;

[0077] a fragmentation device configured to fragment sample ions so as to produce fragment ions;

[0078] a mass analyser; and a mass analyser; and

[0079] a control system configured to:

[0080] (i) cause the instrument to perform one or more MS1 mass analysis scans to obtain MS1 data;

[0081] (ii) identify one or more precursor ions from the MS1 data; and

[0082] (iii) cause the instrument to perform one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data;

[0083] wherein the control system is further configured to:

[0084] for each of the one or more precursor ions identified from the MS1 data: determine from the MS1 data a value indicative of a collision cross section (CCS) of the precursor ion;

[0085] and

[0086] select which precursor ion or precursor ions the one or more MS2 mass analysis scans target based on the CCS indicative value and / or determine an order in which the one or more MS2 mass analysis scans target precursor ions based on the CCS indicative value.

[0087] Another aspect provides an analytical instrument, such as a mass spectrometer, comprising:

[0088] an ion source configured to ionize a sample to produce sample ions;

[0089] a mass filter configured to filter ions using an isolation window;

[0090] at least one fragmentation device configured to fragment sample ions to produce fragment ions;

[0091] a mass analyzer; and

[0092] a control system configured to:

[0093] (i) cause the instrument to perform one or more MS1 mass analysis scans to obtain MS1 data;

[0094] (ii) identify one or more precursor ions from the MS1 data; and

[0095] (iii) cause the instrument to perform one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data;

[0096] wherein the control system is further configured to:

[0097] for each precursor ion of the one or more precursor ions identified from the MS1 data: determine, from the MS1 data, a value indicative of a collision cross section (CCS) of the precursor ion;

[0098] and

[0099] for each MS2 mass analysis scan of the one or more MS2 mass analysis scans:

[0100] select, based on the CCS indicative value of the precursor ion targeted by the MS2 mass analysis scan, a fragmentation energy to be used in performing the MS2 mass analysis scan; and / or

[0101] select, based on the CCS indicative value of the precursor ion targeted by the MS2 mass analysis scan,

[0102] select, based on the CCS indicative value of the precursor ion targeted by the MS2 mass analysis scan, a fragmentation method to be used in performing the MS2 mass analysis scan.

[0103] These aspects can and in some embodiments do include any one or more or each of the optional features described herein. BRIEF DESCRIPTION OF DRAWINGS

[0104] Various embodiments will now be described in greater detail in relation to the accompanying drawings, in which:

[0105] Figure 1 a mass spectrometer according to an embodiment is schematically illustrated;

[0106] Figure 2 simulated data showing observed mass resolution of unmodified and modified precursor ions measured during proteomic analysis using an Orbitrap mass analyser operated with standard ultra-high vacuum (UHV); TM simulated data showing observed mass resolution of unmodified and modified precursor ions measured during proteomic analysis using an Orbitrap mass analyser operated with standard ultra-high vacuum (UHV);

[0107] Figure 3 simulated data showing observed mass resolution of unmodified and modified precursor ions measured during proteomic analysis using an Orbitrap mass analyser operated with standard ultra-high vacuum (UHV); TM simulated data showing observed mass resolution of unmodified and modified precursor ions measured during proteomic analysis using an Orbitrap mass analyser operated with standard ultra-high vacuum (UHV);

[0108] Figure 4Simulated data of observed mass resolution of ions using standard pressure (labeled "precursor, standard pressure") are shown, as well as real data measured using the CCS workflow of the implementation scheme (labeled "precursor, increased pressure"), where it can be seen that as UHV increases, the various charge states present in the precursor resolve into charge state-related trend lines due to the inherently higher CCS values ​​of the higher charge states.

[0109] Figure 5 Showing from Figure 4 Data on ions in the 2+ charge state, and simulated data of observed mass resolution for precursors modified to reduce CCS values ​​(labeled "modification, CCS decrease") and precursors modified to increase CCS values ​​(labeled "modification, CCS increase").

[0110] Figure 6 Simulated MS1 mass spectra (ion isotopes not shown) of precursors containing 12 different intensities and m / z values ​​are presented;

[0111] Figure 7 It shows Figure 6 The observed mass resolution of each precursor is given as a function of m / z, along with the “expected resolution” corresponding to the 2+ trend line of the unmodified standard and the “unmodified region” representing the extended mass resolution value of the 2+ ions in the unmodified standard.

[0112] Figure 8 Examples based on Figure 7 The data shows two selection strategies, in which ions falling below the "unmodified region" are targeted for MS2 analysis. Figure 8 B), or ions falling above the "unmodified region" are targeted for MS2 analysis. Figure 8 A); and

[0113] Figure 9 The DDA mass spectrometry method according to the implementation scheme is illustrated schematically. Detailed Implementation

[0114] Figure 1 An analytical instrument, such as a mass spectrometer, that can be used in conjunction with the methods described herein is illustrated. Figure 1 As shown, the instrument includes an ion source 10, a mass filter 20, a fragmentation device 30, and a mass analyzer 40.

[0115] The ion source 10 is configured to generate ions from a sample. The ion source 10 can be coupled to a separation device (not shown), such as a liquid chromatography (LC) separation device, a gas chromatography (GC) separation device, or a capillary electrophoresis separation device, among others, such that the sample is ionized in the ion source 10 comes from the separation device. The ion source 10 can be any suitable ion source, such as an electrospray ionization (ESI) ion source, an atmospheric pressure ionization (API) ion source, a chemical ionization ion source, an electron impact (El) ion source, or the like.

[0116] The analytical instrument can additionally or alternatively include an ion separation device arranged downstream of the ion source and configured to separate sample ions according to a physical-chemical property. For example, the instrument can include an ion mobility (IM) separator, a differential ion mobility separator, or a device configured to separate ions according to their mass-to-charge ratio (m / z).

[0117] The mass filter 20 is arranged downstream of the ion source 10 and is configured to receive ions from the ion source 10 (optionally via an ion separation device). The mass filter 20 is configured to filter the received ions according to their mass-to-charge ratio (m / z). The mass filter 20 can be configured such that received ions having m / z within a m / z transmission window (or “isolation window”) of the mass filter are transmitted forward by the mass filter, while received ions having m / z outside the m / z transmission window are attenuated by the mass filter, i.e. not transmitted forward by the mass filter. The width and / or center m / z of the transmission window is controllable (variable), e.g. by suitably controlling the RF voltage and / or DC voltage applied to the electrodes of the mass filter 20. Thus, for example, the mass filter 20 is capable of operating in a transmission mode of operation, whereby most or all ions within a relatively wide m / z window are transmitted forward by the mass filter 20, and is capable of operating in a filtering mode of operation, whereby only ions within a relatively narrow m / z window (centered at a desired m / z) are transmitted forward by the mass filter 20. The mass filter 20 can be any suitable type of mass filter, such as a quadrupole mass filter.

[0118] The fragmentation device 30 is arranged downstream of the mass filter 20, and is configured to receive most or all of the ions transmitted by the mass filter 20. The fragmentation device 30 can be configured to selectively fragment some or all of the received ions, i.e. so as to generate fragment ions. The fragmentation device 30 can be operable in a fragmentation mode of operation, whereby most or all of the received ions are fragmented so as to generate fragment ions (which fragment ions can then be transmitted onwards from the fragmentation device 30), and can be operable in a non-fragmentation mode of operation, whereby most or all of the received ions are transmitted onwards without being (intentionally) fragmented. It is also possible to achieve the non-fragmentation mode of operation by causing the ions to bypass the fragmentation device 30. The fragmentation device 30 can also be operable in one or more intermediate modes of operation, e.g. whereby the degree of fragmentation is controllable (variable). The fragmentation device 30 can also be operable in a higher order (MS N ) fragmentation mode of operation, e.g. whereby fragment ions are further fragmented one or more times by the fragmentation device 30.

[0119] The fragmentation device 30 can be any suitable type of fragmentation device, such as for example a collision-induced dissociation (CID) fragmentation device, an electron-induced dissociation (EID) fragmentation device, a photodissociation fragmentation device, etc. Many other types of fragmentation are possible.

[0120] In some embodiments, the fragmentation device 30 can perform more than one type of fragmentation method, or provide more than one type of fragmentation device, so that the instrument can select between performing different fragmentation methods. Similarly, the fragmentation device 30 can use different fragmentation energies to fragment ions.

[0121] In some embodiments, the fragmentation device 30 is a collision-induced dissociation (CID) fragmentation device. A CID fragmentation device can comprise a collision cell which can be filled with a collision gas, e.g. maintained at a relatively high pressure. Ions can be selectively fragmented in the collision cell by controlling (varying) the kinetic energy with which the ions enter the collision cell. In a fragmentation mode of operation, ions can be accelerated so that the ions enter the collision cell with a relatively high kinetic energy, which can fragment most or all of the accelerated ions. In a non-fragmentation mode of operation, ions can be entered into the collision cell with a relatively low kinetic energy, which can be insufficient to fragment most or all of the ions. In an intermediate mode of operation, ions can be entered into the collision cell with an intermediate kinetic energy.

[0122] A mass analyzer 40 is arranged downstream of the fragmentation device 30 and is configured to receive ions from the fragmentation device 30. Thus, depending on the mode of operation of the fragmentation device 30, the mass analyzer 40 can receive unfragmented precursor ions and / or fragment ions. The mass analyzer 40 is configured to analyze the received ions in order to determine their mass-to-charge ratio (m / z) and / or mass, i.e. to generate a mass spectrum of the ions. The mass analyzer 40 can be any suitable type of mass analyzer, such as an ion trap mass analyzer, an electrostatic orbitrap mass analyzer (such as an Orbitrap mass analyzer manufactured by Thermo Fisher Scientific), or a time-of-flight (ToF) mass analyzer such as a multi-reflecting time-of-flight (MR-ToF) mass analyzer. TM FT mass analyzer) or a time-of-flight (ToF) mass analyzer such as a multi-reflecting time-of-flight (MR-ToF) mass analyzer.

[0123] It is noted that, Figure 1 is merely schematic and that the instrument can and in embodiments does include any number of additional components in addition to the one or more additional components. In general, the instrument can include one or more ion transfer stages arranged between any of the components shown, e.g. including an atmospheric pressure interface and / or one or more ion guides, lenses, and / or other ion optical devices configured such that some or all of the ions can be suitably transmitted through the instrument. The ion transfer stages can include any suitable number and configuration of ion optical devices, e.g. optionally including one or more ion guides, lenses, and / or other ion optical devices.

[0124] In some embodiments, the instrument can include more than one mass analyzer. For example, the instrument can be a dual mass analyzer hybrid mass spectrometer of the type described in EP 3,410,463, the contents of which are incorporated herein by reference.

[0125] Also as Figure 1 shown, the instrument is under the control of a control unit 50, such as a suitably programmed computer, which controls the operation of the various components of the instrument and, for example, sets the voltages to be applied to the various components of the instrument. The control unit 50 can also receive and process data from the various components of the instrument, in various embodiments.

[0126] The instrument is capable of operating in various modes of operation. In particular, the instrument can be a tandem mass spectrometer capable of operating in a MS1 mode of operation and a MS2 mode of operation.

[0127] In MS1 ​​(or “full mass scan”) operating mode, mass filter 20 operates in its transport operating mode, and fragmentation device 30 operates in its non-fragmentation operating mode, for example, such that a wide range of m / z (e.g., full mass range) of undfragmented (“precursor” or “parent”) ions is analyzed by analyzer 40 to generate MS1 spectra.

[0128] In MS2 operating mode, mass filter 20 operates in its filtration operating mode, and fragmentation device 30 operates in its fragmentation operating mode, for example, causing precursor ions in a selected narrow m / z range to be fragmented, and the resulting fragment ("product" or "daughter") ions are analyzed by analyzer 40 to generate MS2 spectra.

[0129] The instrument can also operate in one or more higher-order fragmentation modes (such as, for example, MS3 mode), where the bulk ion is fragmented, at least some of the resulting fragment ions are themselves fragmented, and second-generation fragment ions (“grandchild ions”) are analyzed by analyzer 40 to produce an MS3 spectrum. Typically, the instrument can operate in any order of fragmentation mode, i.e., at MS... N Operation mode operation, where N≥2.

[0130] As mentioned above, one analytical challenge in proteomics is how to identify and select modified peptide ions (such as PTMs and cross-linked peptides) in proteomics workflows while minimizing additional sample handling requirements. Solutions based on implementation schemes utilize peptide-level structural changes induced by protein modifications. It has been demonstrated in the literature that modifications to peptide ions can affect their structure in the gas phase by either compression (i.e., a reduction in structural area / volume compared to unmodified peptides) or elongation (i.e., an increase in structural area / volume compared to unmodified peptide ions). These structural differences can be addressed using structure-sensitive techniques. One widely used technique of this kind is ion mobility, where ions are separated relative to their collision cross-section (CCS) value, which is related to the rotationally averaged structure adopted by the ions in the gas phase.

[0131] Recently, the inventors of this invention have published research results, such as those described in UK patent application GB 2,612,580, whereby these CCS values ​​are based on Orbitrap. TM The quality is measured using the quality measurement steps in the quality analyzer. This work shows that, in Orbitrap... TM Increased UHV pressure in the mass analyzer (e.g., approximately 10) -9At mbar, the decay of ions in the transient domain is correlated with their CCS value. For overall measurements, the decay rate of a single ion species (i.e., ions with a specific m / z and charge state) causes a change in the mass resolution (i.e., peak width) of that ion observed in the mass spectrometer. Here, this work is extended to develop a real-time, data-driven targeting workflow where precursors showing deviations from the expected mass resolution indicate that they are modified ions. These ions are then selectively targeted for MS2 analysis, increasing the instrument duty cycle utilization for modified peptide analysis and reducing or minimizing the need for pre-analytical sample enrichment.

[0132] Figure 2 The Orbitrap, which operates using standard ultra-high vacuum (UHV), is shown. TM Simulated data of observed mass resolution of precursor ions measured during proteomics analysis using a mass analyzer. For example... Figure 2 As shown, the mass resolution of ions is proportional to the reciprocal of the square root of the m / z value. The UHV used by the mass analyzer is maintained at a certain value (e.g., approximately 10). - 10 mbar or 10 -11 The mean free path of peptide ions (mbar) ensures that the ion population does not significantly decay during measurement, thus minimizing losses due to collisions with the background gas. Therefore, ions with different structures and inherently different CCS values ​​exhibit similar observed mass resolution values ​​as a function of m / z.

[0133] Figure 3 The corresponding simulation data for precursors with structurally influential modifications (such as glycans, crosslinks, or phosphate groups) are shown, superimposed on... Figure 2 On the data. Here, due to the lack of ion decay within the measurement time frame, structural differences do not cause separation in the mass resolution domain. Therefore, as Figure 3 As shown, even though the two ionic groups are structurally unique, due to the Orbitrap TM There are no collisions during the mass analyzer measurement, so the unmodified precursor and the modified precursor overlap in terms of mass resolution m / z.

[0134] To measure Orbitrap TM The CCS value in the mass analyzer increases the UHV to the point where the ions begin to show a perceptible decay on the transient acquisition timescale (e.g., approximately 10). -9 mbar). For example Figure 4 As shown, this attenuation is confirmed in the observed quality resolution.

[0135] from Figure 4It can be seen that the various charge states start to bunch up in the mass resolution - m / z space because higher charge state ions typically take on large CCS values. This leads to the ions of higher charge states decaying faster, resulting in lower observed mass resolution and separation of the various charge states in that dimension. By controlling the UHV pressure against known standards (e.g., Hela) and identifying unmodified precursor ions, these charge state dependent trends can be mapped and calibrated for later use in targeted workflows analyzing unknown compounds.

[0136] Furthermore, if only the individual charge states of Figure 4 are of interest, such as for example the 2+ charge state as shown in Figure 5 , it can be demonstrated that the modified ions can be distinguished from the unmodified ions either with higher CCS values and lower observed mass resolution (i.e. ions lower than the “no modification” bin in Figure 5 ), or with lower CCS values and higher observed mass resolution (i.e. ions higher than the “no modification” bin in Figure 5 ). In embodiments, this separation is used in targeted real-time workflows.

[0137] As discussed above, protein modifications occur at sub-stoichiometric levels and thus will result in a small fraction of the instrument analysis time being dedicated to protein analysis using conventional random acquisition. Figure 6 An example simulated MS1 mass spectrum is shown, for example as acquired during a proteomics workflow. In this example, the MS1 scan shows 12 precursor signals with various m / z and intensity values. In a standard proteomics workflow such as DDA, the “Top N” precursors with the highest intensity (isotopes not shown) of these precursors are selected for fragmentation, ignoring their modification status.

[0138] However, according to embodiments, elevated UHV pressure can be utilized to map the observed mass resolution in real-time.

[0139] Figure 7 The observed mass resolution of each precursor of Figure 6 is shown as a function of m / z. Additionally, Figure 5 The 2+ trend line of the unmodified standard presented in is shown by the “expected resolution” and the “unmodified region” which represents the spread observed in the mass resolution values of the 2+ ions of the unmodified standard.

[0140] Using the previously established "expected resolution" line for unmodified peptide ions (from known compounds such as HeLa), it can be seen that 6 precursor ions exhibit mass resolution outside of the expected "unmodified region". This data suggests that these 6 precursors are structurally unique, likely due to the presence of modifications (such as phosphorylation or cross-linking).

[0141] Building this knowledge into a data-driven workflow allows the user to selectively target higher CCS value precursors, for example for cross-linking analysis or glycan analysis, or target lower CCS value precursors for modifications that result in compression, such as phosphorylation. In this workflow, the precursors targeted would be those falling outside of the "unmodified region", so a significantly higher percentage of instrument analysis time would be directed at precursors that can exhibit modifications of interest.

[0142] Figure 8 An example of a method of operating an analytical instrument according to embodiments is illustrated in FIG. 1. Figure 7 Two selection strategies are illustrated based on the data shown. If the user is interested in studying modifications that are known to increase the CCS value of an ion, then ions falling below the "unmodified region" can be targeted (B), while all other ions can be excluded. Conversely, if the user is interested in modifications that traditionally exhibit CCS compression, such as phosphorylation, then the method can target values falling above the "unmodified region" for subsequent MS2 analysis (A). Figure 8 Figure 8 A).

[0143] Figure 9 An example of a method of operating an analytical instrument according to embodiments is illustrated in FIG. 1. Figure 1 A sample is provided to a separation device (e.g., an LC), such that the sample is separated, and eluent from the separation device is ionized in an ion source 10 (step 100). The resulting ions are analyzed by the instrument operating in a data-dependent acquisition (DDA) mode of operation. Additionally or alternatively, the sample ions can be separated by a separation device, such as an ion mobility separation device.

[0144] The data-dependent acquisition (DDA) mode of operation involves the instrument repeatedly performing the following steps during a separation run of the separation device: (i) obtaining an MS1 spectrum across a range of m / z of interest (step 102); (ii) identifying one or more precursor ions of interest in the MS1 spectrum; and (iii) obtaining an MS2 (or MS3) spectrum for each of the identified precursor ions of interest (step 104). N ) spectrum for each of the identified precursor ions of interest (step 104).

[0145] ​Step (i) involves the instrument performing one or more MS1 mass analysis scans to generate MS1 spectra. Step (iii) involves, for each of the identified precursor ions of interest: separating the precursor ion using mass filter 20, fragmenting the isolated precursor ion in fragmentation device 30, and performing mass analysis on the fragment ions using mass analyzer 40. Thus, during each cycle, multiple MS2 spectra (or more generally, multiple MS1 spectra) are obtained by successively changing the center of the (narrow) m / z window of the mass filter between each of a plurality of different target m / z values. N (Spectrum), for example, to sequentially select (and fragment) each of a plurality of different precursor ions having correspondingly different m / z values. The plurality of different m / z values ​​correspond to a plurality of different targeted precursor ions.

[0146] like Figure 9 As shown, according to the implementation scheme, a list of target precursor ions is generated based on the CCS indication values ​​determined for each ion peak in the MS1 data. Therefore, the instrument identifies peaks in the MS1 spectrum (step 110) and calculates the CCS indication value for each identified peak in the MS1 spectrum (step 111). Then, the calculated CCS indication value for each peak is compared with the expected CCS indication value or expected CCS indication value range for that peak (step 112). As described above, this can be done based on a calibration curve (e.g., such as...). Figure 7 (As shown) to determine the expected CCS indication value, this calibration curve can be created by analyzing one or more samples of unmodified precursor ions.

[0147] In step 113, if a peak has a CCS indication value sufficiently similar to the expected value, the peak can be ignored or assigned a relatively low priority. If a peak has a CCS indication value sufficiently different from the expected value, the peak can be added to the target list (in step 114) and / or assigned a relatively high priority. Once all peaks in the MS1 spectrum have been considered, a complete list of MS2 targets (optionally along with their priority order) can be compiled and used to guide the MS2 scan in step 104. This process can then be repeated for the next MS1 spectrum, and so on.

[0148] It should be pointed out that, Figure 9 A simplified approach is shown, and in practice, additional steps can be provided in the workflow. Specifically, additional logical steps, such as charge state determination / inclusion checks, intensity grading (“Top-N”) steps, and / or the use of one or more exclusion lists to exclude MS2 targets that have been analyzed or recently analyzed, can be provided, for example, in the manner of a standard DDA workflow.

[0149] It will therefore be appreciated that in embodiments, the difference in observed mass resolution driven by CCS is used to target precursors that fall above or below the expected unmodified region. The CCS driven decision on which ions to target can increase the amount of instrument analysis time used to target ions of interest, which can exist in low abundance and substoichiometric amounts. This can beneficially reduce or minimize the need for sample enrichment prior to MS analysis.

[0150] While specific embodiments have been described in detail above, various alternative embodiments are possible. For example, the CCS indicative value can be determined from MS1 data using the method described in UK patent application GB 2303690.8. In this method, the CCS indicative value is determined using a time-of-flight (ToF) mass analyser, wherein two sets of data are obtained (i.e. by performing two scans (or two sets of scans)), wherein one or both of (i) ion path length and (ii) gas pressure in the ion path are varied between the two sets of data. By comparing the intensities of corresponding ion peaks in the two sets of data, the CCS of the ion that produced that corresponding ion peak can be determined.

[0151] In some embodiments, the selected precursor ions can be precursor ions of a particular chemical class. For example, where the sample is a heterogeneous mixture of different chemical classes (e.g. comprising two or more of peptides, lipids, steroids, etc.), each of the different chemical classes can show a different trend line in the CCS indicative value - m / z space. Then, by selecting precursor ions based on their CCS indicative value and m / z and / or determining an order based on the CCS indicative value and m / z, precursor ions from only one chemical class can be selected for analysis and / or can be preferentially targeted by one or more MS2 mass analysis scans.

[0152] The methods described herein can also be used to trigger different MS2 fragmentation energies or methods. That is, different fragmentation strategies can be selected depending on the CCS indicative value. For example, again referring to Figure 7 If there are both peptides that fall into the unmodified region and peptides with lower CCS indicative values and thus can be phosphorylated, then the phosphorylated peptides can be selected and fragmented with one fragmentation method and / or energy (e.g. EID at a certain electron energy), and subsequently the unmodified peptides can be selected and fragmented with a different fragmentation method and / or energy (e.g. HCD or EID at a different electron energy) than the phosphorylated case. Similarly, for the case of a heterogeneous mixture of different chemical classes (e.g. peptides, lipids, steroids as described above), different fragmentation methods and / or energies can be used for different chemical classes, e.g. one fragmentation strategy can be used for peptides and another fragmentation strategy for steroids, etc.

[0153] While the application has been described with reference to various embodiments, it will be understood that various changes can be made and equivalents can be substituted for elements thereof without departing from the scope of the application as expressed in the claims.

Claims

1. A mass spectrometry method, the method comprising: ionising a sample to produce sample ions; (i) analysing the sample ions by performing one or more MS1 mass analysis scans so as to obtain MS1 data; (ii) identifying one or more precursor ions from the MS1 data; and (iii) analysing the sample ions by performing one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data; wherein the method further comprises: for each of the one or more precursor ions identified from the MS1 data: determining a value indicative of the collision cross section (CCS) of the precursor ion from the MS1 data; and selecting which precursor ion or precursor ions the one or more MS2 mass analysis scans target based on the CCS indicative value and / or determining an order in which the one or more MS2 mass analysis scans target precursor ions based on the CCS indicative value.

2. The method of claim 1, wherein the method comprises: for each of one or more of the precursor ions for which a CCS indicative value is determined: comparing the determined CCS indicative value to one or more values indicative of an expected collision cross section (CCS) or range of collision cross sections for the precursor ion; and selecting which precursor ion or precursor ions the one or more MS2 mass analysis scans target based on the comparison and / or determining the order in which the one or more MS2 mass analysis scans target precursor ions based on the comparison.

3. The method of claim 2, wherein: the comparing step comprises, for each of one or more of the precursor ions for which a CCS indicative value is determined: determining whether (a) the determined CCS indicative value is greater than a maximum expected CCS indicative value for the precursor ion; and / or (b) the determined CCS indicative value is less than a minimum expected CCS indicative value for the precursor ion; and / or (c) the determined CCS indicative value falls outside an expected range of CCS indicative values for the precursor ion; and the step of selecting based on the comparison comprises: when it is determined that (a) the determined CCS indicative value is greater than the maximum expected CCS indicative value; and / or (b) the determined CCS indicative value is less than the minimum expected CCS indicative value; and / or (c) the determined CCS indicative value falls outside the expected range of CCS indicative values: selecting the precursor ion; and / or the step of determining the order based on the comparison comprises: when it is determined that (a) the determined CCS indicative value is greater than the maximum expected CCS indicative value; and / or (b) the determined CCS indicative value is less than the minimum expected CCS indicative value; and / or (c) the determined CCS indicative value falls outside the expected range of CCS indicative values: giving the precursor ion a relatively high priority in the order.

4. The method of claim 3, wherein: ​ The step of selecting based on the comparison comprises: when it is determined that (a) the determined CCS indicative value is less than the maximum expected CCS indicative value; and / or (b) the determined CCS indicative value is greater than the minimum expected CCS indicative value; and / or (c) the determined CCS indicative value falls within the expected range of CCS indicative values: not selecting the precursor ion; and / or The step of determining the order based on the comparison comprises: when it is determined that (a) the determined CCS indicative value is less than the maximum expected CCS indicative value; and / or (b) the determined CCS indicative value is greater than the minimum expected CCS indicative value; and / or (c) the determined CCS indicative value falls within the expected range of CCS indicative values: giving the precursor ion a relatively low priority in the order.

5. The method of any preceding claim, wherein the precursor ion that is selected and / or given a relatively high priority in the order is a modified precursor ion.

6. The method of claim 5, wherein the sample ions are peptide ions and / or protein ions, and wherein the precursor ion that is selected and / or given a relatively high priority in the order is a modified or cross-linked peptide ion and / or protein ion.

7. The method of claim 5 or 6 when dependent on claim 2, the method further comprising determining the one or more values indicative of an expected collision cross section (CCS) or collision cross section range from a calibration, wherein the calibration is a calibration generated by analysing one or more samples of unmodified precursor ions.

8. The method of any preceding claim, the method further comprising, for each of one or more of the MS2 mass analysis scans: selecting a fragmentation energy to be used in performing the MS2 mass analysis scan based on the CCS indicative value of the precursor ion targeted by the MS2 mass analysis scan; and / or selecting a fragmentation method to be used in performing the MS2 mass analysis scan based on the CCS indicative value of the precursor ion targeted by the MS2 mass analysis scan.

9. A mass spectrometry method, the method comprising: ionising a sample to produce sample ions; (i) analysing the sample ions by performing one or more MS1 mass analysis scans so as to obtain MS1 data; (ii) identifying one or more precursor ions from the MS1 data; and (iii) analysing the sample ions by performing one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data; wherein the method further comprises, for each of one or more precursor ions identified from the MS1 data: determining a value indicative of a collision cross section (CCS) of the precursor ion from the MS1 data; and for each of one or more of the MS2 mass analysis scans: selecting a fragmentation energy to be used in performing the MS2 mass analysis scan based on the CCS indicative value for the precursor ion targeted by the MS2 mass analysis scan; and / or selecting a fragmentation method to be used in performing the MS2 mass analysis scan based on the CCS indicative value for the precursor ion targeted by the MS2 mass analysis scan.

10. The method of any one of the preceding claims, wherein: the value indicative of the collision cross section (CCS) of a precursor ion is a width of an ion peak in the MS1 data corresponding to the precursor ion; and / or the step of determining a value indicative of the collision cross section (CCS) of a precursor ion comprises determining the value indicative of the collision cross section (CCS) of the precursor ion from a width of an ion peak in the MS1 data corresponding to the precursor ion.

11. The method of any one of the preceding claims, wherein the step (i) of analysing the sample ions by performing one or more MS1 mass analysis scans comprises using an orbitrap mass analyser to mass analyse the sample ions.

12. The method of claim 11, wherein the orbiting trapping mass analyzer operates at a pressure of > 10 -9 mbar.

13. The method of any one of claims 1 to 10, wherein: the step (i) of analysing the sample ions by performing one or more MS1 mass analysis scans comprises using a time-of-flight (ToF) mass analyser to mass analyse the sample ions, wherein the time-of-flight (ToF) mass analyser is configured to determine a mass-to-charge ratio (m / z) of an ion by determining a time of flight of the ion along an ion path; the step of using a time-of-flight (ToF) mass analyser to mass analyse the sample ions comprises: operating the mass analyser in a first operating mode and analysing the sample ions by determining a time of flight of the sample ions along the ion path so as to obtain a first set of MS1 data, wherein in the first operating mode (i) the ion path has a first path length and (ii) the ion path is maintained at a first pressure; and operating the mass analyser in a second operating mode and analysing the sample ions by determining a time of flight of the sample ions along the ion path so as to obtain a second set of MS1 data, wherein in the second operating mode (i) the ion path has a second path length and (ii) the ion path is maintained at a second pressure, wherein the second path length is different to the first path length and / or the second pressure is different to the first pressure; and the step of determining a value indicative of the collision cross section (CCS) of a precursor ion from the MS1 data comprises: comparing an intensity of a precursor ion peak in the first set of data to an intensity of a corresponding precursor ion peak in the second set of data; and determining the value indicative of the CCS of the precursor ion based on the comparison.

14. The method of any one of the preceding claims, wherein: Step (ii) of identifying one or more precursor ions from the MS1 data comprises identifying a plurality of different precursor ions from the MS1 data; and Step (iii) of analysing the sample ions by performing one or more MS2 mass analysis scans comprises performing a plurality of MS2 mass analysis scans, wherein each MS2 mass analysis scan of the plurality of MS2 mass analysis scans targets a different precursor ion of the plurality of different precursor ions identified from the MS1 data.

15. The method of any one of the preceding claims, wherein for each MS2 mass analysis scan, the method comprises: isolating the targeted precursor ion from the sample ions; fragmenting the isolated precursor ion of interest so as to generate fragment ions; and mass analysing the fragment ions so as to obtain MS2 data.

16. The method of claim 15, wherein the step of isolating the targeted precursor ion from the sample ions comprises using a mass filter to select the targeted precursor according to its mass to charge ratio (m / z), optionally wherein the mass filter is a quadrupole mass filter.

17. The method of any one of the preceding claims, wherein the method comprises performing a plurality of repeated cycles, wherein each cycle comprises performing steps (i), (ii) and (iii).

18. The method of claim 17, wherein: the sample is provided by and / or separated by a separation device; and the method comprises repeatedly performing a cycle during a separation run of the separation device.

19. A non-transitory computer readable storage medium storing computer software code which, when executed on a processor, performs the method of any one of the preceding claims.

20. A control system for an analytical instrument, the control system being configured to cause the analytical instrument to perform the method of any one of claims 1 to 18.

21. An analytical instrument comprising the control system of claim 20.

22. An analytical instrument comprising: an ion source configured to ionise a sample to generate sample ions; a mass filter configured to filter ions using an isolation window; a fragmentation device configured to fragment sample ions so as to generate fragment ions; a mass analyser; and a control system configured to: (i) cause the instrument to perform one or more MS1 mass analysis scans so as to obtain MS1 data; (ii) identify one or more precursor ions from the MS1 data; and (iii) cause the instrument to perform one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets a precursor ion of the one or more precursor ions identified from the MS1 data; wherein the control system is further configured to: ​ For each precursor ion identified from the MS1 data: determining from the MS1 data a value indicative of the collision cross section (CCS) of the precursor ion; and selecting which precursor ion or precursor ions the one or more MS2 mass analysis scans target based on the CCS indicative value and / or determining an order in which the one or more MS2 mass analysis scans target precursor ions based on the CCS indicative value.

23. An analytical instrument comprising: an ion source configured to ionise a sample to produce sample ions; a mass filter configured to filter ions using an isolation window; at least one fragmentation device configured to fragment sample ions so as to produce fragment ions; a mass analyser; and a control system configured to: (i) cause the instrument to perform one or more MS1 mass analysis scans so as to obtain MS1 data; (ii) identify one or more precursor ions from the MS1 data; and (iii) cause the instrument to perform one or more MS2 mass analysis scans, wherein each MS2 mass analysis scan targets one of the one or more precursor ions identified from the MS1 data; wherein the control system is further configured to: For each precursor ion identified from the MS1 data: determine from the MS1 data a value indicative of the collision cross section (CCS) of the precursor ion; and For each of one or more of the MS2 mass analysis scans: select a fragmentation energy to be used when performing the MS2 mass analysis scan based on the CCS indicative value of the precursor ion that the MS2 mass analysis scan targets; and / or select a fragmentation method to be used when performing the MS2 mass analysis scan based on the CCS indicative value of the precursor ion that the MS2 mass analysis scan targets. ​

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