Mass spectrometry with data-dependent acquisition

Abstract

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

Description

Field of invention

[0001] The present invention relates generally to the field of mass spectrometry and in particular to methods of mass spectrometry with data-dependent acquisition (DDA). State of the art

[0002] The analysis and investigation of proteins using mass spectrometry, known as proteomics, focuses on the identification and quantification of proteins present in a specific biological state within a cell. Proteins consist of a chain of amino acids, called the primary sequence, and this sequence is the primary criterion used for protein identification in proteomic analyses. However, proteins can also be modified by alterations such as phosphon units or glycans, known as post-translational modifications (PTMs). These modifications have been shown to affect the resulting biology of the modified protein, highlighting the need to identify these modified states and characterize the extent of the modification, e.g., location, modification structure, etc.

[0003] Unfortunately, these modifications occur substoichiometrically, resulting in a lower relative abundance compared to their unmodified counterparts, making it difficult to efficiently select and analyze these species. To circumvent the lower abundance of modified proteins, both online and offline enrichment techniques have been employed, retaining the protein-derived modified peptides while discarding a large proportion of the unmodified peptides.

[0004] In the related field of structural biology, scientists investigate the higher-order structure of proteins and their complexes. Several analytical tools can be used for such investigations. One such tool in the field of mass spectrometry is crosslinking mass spectrometry. In crosslinking, a crosslinker, a small molecule with two reactive termini at opposite ends, is introduced into the sample. These reactive termini bind to reactive amino acids along the protein backbone, forming covalent bonds with both the amino acid and the crosslinker. The other reactive end of the crosslinker is free to move within the space defined by the molecule's length.If another reactive amino acid is present within this spatial distribution, this amino acid also binds to the molecule, thereby connecting the two sites of the protein, which allows the scientist to spatially map the structure of the protein / protein complex.

[0005] While this technique provides important structural information, it also presents analytical challenges. When proteins are enzymatically digested for analysis using proteomic workflows, the crosslinked peptides are present in substoichiometric amounts and are therefore difficult to analyze, similar to PTMs. Analysis of crosslinked samples reveals that the majority of precursors selected for fragmentation are unmodified peptides, not crosslinked peptides. Recently, the chemical composition of the crosslinker was customized to allow scientists to enrich samples for crosslinked peptides, similar to the enrichment steps used for phosphorylated peptides. However, this requires additional sample handling, either online or offline with a mass spectrometer.

[0006] In both examples, an analytical challenge lies in identifying these modified precursors and selectively providing them as a target for mass spectrometric analysis.

[0007] It is assumed that there is still room for improvement in devices and methods for mass spectrometry. Brief description

[0008] One initial aspect concerns a method for mass spectrometry, including: Ionizing a sample to produce sample ions; (i) Analyzing the sample ions by performing one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions of interest in the MS1 spectrum; and (iii) Analyzing the sample ions by performing one or more MS2 mass analysis scans, each MS2 mass analysis scan targeting one of the one or more precursor ions identified from the MS1 data; the procedure further includes the following: For each of the one or more precursor ions identified from the MS1 data: Determine a value from the MS1 data that is characteristic of a collision cross section (CCS) for that precursor ion; and Select, based on the CCS indicative value(s), which precursor ion(s) the one or more MS2 bulk analysis scans should target, and / or determine, based on the CCS indicative value(s), an order in which the one or more MS2 bulk analysis scans should target the precursor ions.

[0009] Embodiments provide a data-dependent acquisition (DDA) mass spectrometry procedure in which one or more precursor ions are identified from MS1 data, and subsequently one or more MS2 mass analysis scans are triggered to target the identified precursor ion(s) of interest.

[0010] In embodiments, precursor ions of interest are identified based on a value indicative of their collision cross-section (CCS) (a “CCS-indicative value”) as determined from the MS1 data, and / or an order in which the precursor ions are analyzed by the MS2 scans is determined based on their CCS-indicative value(s). This contrasts with conventional DDA methods, such as the “Top-N” methods, where precursor ions are analyzed by MS2 scans in an order determined based on their intensity in the MS1 data.

[0011] Embodiments build upon previous work (such as that described in UK patent application GB 2,612,580, the entire contents of which are incorporated herein by reference) which demonstrated that CCS-indicative values ​​can be obtained directly from MS1 data. This contrasts with conventional methods for determining CCS values, which employ specialized ion mobility analyzers, such as drift cells, traveling wave ion guidance systems, trapped ion mobility separators (TIMS), and the like, to separate the electrodes in the CCS domain. The inventors have now recognized that the ability to determine CCS-indicative values ​​directly from MS1 data (without ion mobility separation) enables a DDA method in which precursor ions of interest are selected and / or prioritized based on their CCS-indicative value(s).Therefore, in embodiments described herein, CCS values ​​or values ​​indicative of CCS values, as determined from the MS1 mass measurement itself, are used to distinguish ions of interest and to promote an intelligent DDA workflow.

[0012] In some embodiments, for each precursor ion for which a CCS-indicative value is determined from the MS1 data, this value is compared to a value or values ​​indicative of an expected collision cross section (CCS) or range of collision cross sections (CCS) for the precursor ion, and precursor ions to be targeted by the MS2 scan(s) are selected based on this comparison. Identifying precursor ions with CCS-indicative values ​​that deviate from an expected CCS-indicative value or range makes it possible to identify and preferentially target modified sample ions, such as, in particular, modified and cross-linked peptides and / or proteins.This in turn increases the instrument analysis time used to target these ions of interest (which may be present in low frequencies and substoichiometric amounts), and reduces or minimizes the need for sample enrichment prior to MS analysis.

[0013] It is therefore recognized that embodiments provide an improved method of mass spectrometry.

[0014] In this process, the sample is ionized, i.e., by an ion source, to produce sample ions. These sample ions can be, for example, peptide and / or protein ions. The sample can be supplied to the ion source by a separation device such as a capillary electrophoresis apparatus or a chromatographic separation device (e.g., a liquid chromatography [LC] or gas chromatography [GC] apparatus). Additionally or alternatively, the ions can be separated by a separation device such as an ion mobility separator, a differential ion mobility separator, or a device configured to separate ions according to their mass-to-charge (m / z) ratio.

[0015] The method can involve performing a multitude of repeated cycles, each cycle comprising performing steps (i), (ii), and (iii) as described above. Therefore, steps (i) through (iii) can be repeated cyclically. The method can also include repeatedly performing cycles during a separation run of the separating device.

[0016] In a first step (i) of each cycle, the sample ions are analyzed by performing one or more MS1 mass analysis scans to obtain MS1 ​​data, i.e., by using a mass analyzer to mass-analyze the sample ions. The mass analyzer can be any suitable mass analyzer, such as an orbital trap mass analyzer, e.g., an Orbitrap™ mass analyzer, a time-of-flight (ToF) mass analyzer, such as a multi-reflection time-of-flight (MR-ToF) mass analyzer, an ion cyclotron resonance (ICR) mass analyzer, and so on.

[0017] In specific embodiments, the mass analyzer is an orbital trap mass analyzer, wherein the orbital trap mass analyzer is operated at a relatively high pressure, e.g., such that the timescale for ion-gas collisions within the mass analyzer is on a similar scale to the time required for a mass analysis scan. For example, an orbital trap mass analyzer is typically operated at a pressure in the range of 10 -10 mbar or 10 -11 operated at mbar (i.e., to minimize ion-gas collisions during each mass analysis scan), but in embodiments an orbital trap mass analyzer is operated at a pressure ≥ 10 -9 mbar (e.g. between 1 × 10 -9 and 9 × 10 -9The MS1 data is operated at a pressure of [mbar] (so that ion-gas collisions are significant during each mass analysis scan). As described in UK patent application GB 2,612,580, operating the orbital trap mass analyzer in this pressure regime allows the estimation of CCS values ​​of ions from the width of the ion peaks in the MS1 data.

[0018] In each cycle, one or more precursor ions are identified from the MS1 data, and the sample ions are then analyzed by performing one or more MS2 bulk analysis scans, each MS2 bulk analysis scan targeting one or more of the precursor ions identified from the MS1 data. Precursor ions can initially be identified from the MS1 data by any suitable method, such as appropriate data processing and / or peak detection.

[0019] Step (ii), identifying one or more precursor ions in the MS1 data, may involve identifying a variety of different precursor ions in the MS1 data. Step (iii), analyzing the sample ions by performing one or more MS2 bulk analysis scans, may involve performing a variety of MS2 bulk analysis scans, each MS2 bulk analysis scan targeting a different precursor ion from the variety of different precursor ions identified from the MS1 data.

[0020] For each MS2 mass analysis scan, the procedure can include: isolating the target precursor ions from the sample ions; fragmenting the isolated precursor ions to produce fragment ions; and using a mass analyzer to perform mass analysis of the fragment ions. The step of isolating the target precursor ions from the sample ions can involve using a mass filter, such as a quadrupole mass filter, to select the target precursor ions according to their mass-to-charge (m / z) ratio. Sample ions with an m / z within a narrow isolation window (e.g., ≤ 5 Da, such as 2 Da) centered on the m / z of the target precursor ion can be passed on, while sample ions with an m / z outside the isolation window can be attenuated.Each MS2 bulk analysis scan of the multitude of MS2 bulk analysis scans can target a different one from the multitude of different precursor ions by changing the m / z of the center of the isolation window for each different target precursor ion.

[0021] In some embodiments, e.g., when the mass analyzer is an orbital trap mass analyzer, the method may involve performing a single MS1 mass analysis scan in each cycle to obtain the MS1 data, followed by performing a single MS2 mass analysis scan for each cycle of the one or more target precursor ions. However, this is not necessary, and if, for example, a different type of mass analyzer is used (such as a ToF mass analyzer, which generates individual mass spectra by averaging the results of multiple scans) and / or if more complex analytical techniques are employed, more than one MS1 mass analysis scan may be used in a cycle to obtain the MS1 data, and / or more than one MS2 mass analysis scan may be performed with respect to each of the one or more target precursor ions.

[0022] In this procedure, for each of the one or more precursor ions identified from the MS1 data, a value is determined from the MS1 data that is indicative of a collision cross section (CCS) for that precursor ion (i.e., a "CCS-indicative value"). A CCS-indicative value can be determined for some, most, or all of the precursor ions identified from the MS1 data.

[0023] The CCS indicative value can be the CCS value itself or another value indicative of the CCS value of a precursor ion. Each CCS indicative value is determined (directly) from the MS1 data, e.g., from a single MS1 spectrum (and without reference to the drift time of the ion mobility).

[0024] In specific embodiments, the value indicative of a precursor ion's collision cross section (CCS) is the width of an ion peak in the MS1 data associated with that precursor ion (and subsequent steps for using the CCS indicative value are performed using this width). Alternatively, the actual value of the collision cross section (CCS), or another value indicative of a precursor ion's collision cross section (CCS), can be determined from the width of an ion peak in the MS1 data associated with that precursor ion (and subsequent steps for using the CCS indicative value can be performed using this value). In embodiments, any suitable measure for the ion peak width can be used, such as the full width at half maximum (FWHM).These embodiments are particularly, but not exclusively, suitable for embodiments in which, as described, for example, in British patent application GB 2,612,580, the mass analyzer is an orbital trap mass analyzer operated in a suitable pressure regime to enable the estimation of CCS values ​​of ions from the width of the ion peaks in the MS1 data.

[0025] However, other methods could be used to determine or estimate CCS-indicative values ​​from MS1 data (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 for determining CCS-indicative values ​​using a time-of-flight (ToF) mass analyzer. This is done by obtaining two datasets (e.g., by performing two scans or two sets of scans), whereby either (i) the ion path length or (ii) the gas pressure in the ion path, or both, are varied between the two datasets. By comparing the intensity of corresponding ion peaks in the two datasets, the CCS of the ions causing the corresponding ion peaks can be determined.

[0026] Therefore, the mass analyzer can be a time-of-flight (ToF) mass analyzer (such as a multireflection time-of-flight (MR-ToF) mass analyzer) configured to determine the mass-to-charge ratio (m / z) of ions by determining the time-of-flights of ions along an ion path, and the step (i) of analyzing the sample ions by performing one or more MS1 mass analysis scans to obtain MS1 ​​data can include the following: Operating the mass analyzer in a first operating mode and analyzing sample ions by determining flight times of the sample ions along the ion path 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

[0027] Operating the mass analyzer in a second operating mode and analyzing sample ions by determining flight times of the sample ions along the ion path 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 differs from the first path length and / or the second pressure differs from the first pressure.

[0028] 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 (ToF) mass analyzer can include one or more ion reflectors (e.g., of one of the various types described in GB 2303690.8), and in the first operating mode, ions can be caused to undergo n reflections in the one or more ion reflectors, where n is an integer ≥ 0, and in the second operating mode, ions can be caused to undergo m reflections in the one or more ion reflectors, where m is an integer > n.

[0029] In these embodiments, the step of determining a value indicative of a precursor ion's collision cross section (CCS) from the MS1 data may include: comparing the intensity of a precursor ion peak in the first data set with the intensity of a corresponding precursor ion peak in the second data set and, based on the comparison, determining a value indicative of the precursor ion's CCS. The procedure may include: determining a ratio of the intensity of the ion peak in the first data set to the intensity of the corresponding ion peak in the second data set, and determining the CCS-indicative value for the precursor ion using this ratio.For example, in some embodiments, the value indicative of a precursor ion's collision cross-section (CCS) is this ratio (and the subsequent steps for using the CCS-indicative value are performed using this ratio). Alternatively, the actual value of the collision cross-section (CCS) can be determined from the ratio, for example, by comparing the ratio with a calibration to determine the precursor ion's CCS (and the subsequent steps for using the CCS-indicative value can be performed using this ratio).

[0030] In general, the procedures described herein use the CCS indicative values ​​(e.g., as determined for several precursor ions identified in the MS1 data) to select which precursor ion(s) of the several precursor ions identified in the MS1 data the one or more MS2 bulk analysis scans should target, and / or to determine an order in which the one or more MS2 bulk analysis scans should target the precursor ions. Then, in step (iii), the one or more MS2 bulk analysis scans are performed, with each MS2 bulk analysis scan targeting one of the selected precursor ions and / or targeting the precursor ion(s) in the determined order.

[0031] The procedure may include: (a) selecting which precursor ion(s) of the identified precursor ions (i.e., selecting some, but not all, of the identified precursor ions) the MS2 bulk analysis scan(s) should target based on the CCS indicative value(s) (without specifying an order); or (b) specifying an order in which the MS2 bulk analysis scan(s) should target the precursor ions based on the CCS indicative value(s) (without selecting only some of the precursor ions of the identified precursor ions as targets); or (c) both selecting which precursor ion(s) of the identified precursor ions (i.e.,Selection of some, but not all, of the identified precursor ions) that the MS2 bulk analysis scan(s) should target based on the CCS indicative value(s), as well as determining an order in which the MS2 bulk analysis scan(s) should target the precursor ions based on the CCS indicative value(s).

[0032] Other factors (in combination with the CCS indicative value(s)) may be considered when selecting the precursor ions and / or determining the sequence. Therefore, the procedure may include: selecting, based on the CCS indicative value(s) and one or more other factors, which precursor ion(s) the MS2 bulk analysis scan(s) should target, and / or determining, based on the CCS indicative value(s) and one or more other factors, a sequence in which the one or more MS2 bulk analysis scans should target the precursor ions. The one or more other factors may include, for example, the intensity of the precursor ion in the MS1 data and / or the m / z ratio of the precursor ion in the MS1 data, etc.One or more of the other factors may also include, or instead include, the number of times an MS2 bulk scan has previously targeted the precursor ion, and / or the time elapsed since an MS2 bulk scan previously targeted the precursor ion. For example, precursor ions that have been targeted by a desired number of (one or more) MS2 scans may be added to an exclusion list, which may be time-limited. Therefore, it should be noted that this precursor ion(s), which would be selected based (purely) on its CCS indicative value(s) in the manner described above, below, and elsewhere herein, may in fact be excluded from being targeted by the MS2 bulk scan(s) (i.e., due to one or more other factors).

[0033] The selection of which precursor ion(s) of the identified precursor ions the MS2 bulk analysis scan(s) should target, based on the CCS indicative value(s), can be carried out in any suitable manner. For example, (only) the precursor ion(s) whose CCS indicative value is greater than and / or less than a threshold and / or lies within or outside a range can be selected, or a specific fixed number or proportion of identified precursor ions that have the highest or lowest CCS indicative value or that are closest to a CCS indicative target value can be selected.

[0034] Similarly, the determination of the sequence in which the MS2 bulk analysis scan(s) should target the precursor ions based on the CCS indicative value(s) can be carried out in any suitable manner. For example, the precursor ions can be ordered from the highest to the lowest CCS indicative value, from the lowest to the highest CCS indicative value, and so on.

[0035] In some embodiments, the selected precursor ions are precursor ions belonging to a specific chemical class. For example, if the sample is a heterogeneous mixture of different chemical classes (if it includes, for instance, two or more peptides, lipids, steroids, etc.), each of the different chemical classes may exhibit different trend lines in the CCS-indicative-m / z space. In this context, it has been recognized that a relationship exists between the CCS-indicative-value and m / z for ions of different chemical classes. These relationships take the form of a "trend line" for each different chemical class. In practice, there will be some variation in CCS-indicative-values ​​for ions belonging to a specific chemical class and a specific m / z, but this variation is typically small enough to distinguish ions of different chemical classes in the CCS-indicative-m / z space.Therefore, in embodiments, precursor ions from only one of the chemical classes can be selected for analysis and / or preferentially targeted by one or more MS2 mass analysis scans, e.g. by selecting (one) precursor ion(s) based on its / their CCS indicative value(s) and its / their m / z and / or determining the order based on CCS indicative value(s) and m / z.

[0036] In specific embodiments, the method comprises, for each of the one or more precursor ions for which a CCS indicative value is determined, the following: comparing the determined CCS indicative value with a value or values ​​indicative of an expected collision cross section (CCS) or range of collision cross sections for that precursor ion. Such a comparison may be performed for one or more, or most or all, of the precursor ions for which a CCS indicative value is determined. Then, the step of selecting the precursor ions to target the MS2 bulk analysis scan(s) may include: selecting, based on the comparison(s), which of the identified precursor ions the one or more MS2 bulk analysis scans will target.Similarly, the step of determining an order in which the MS2 mass analysis scan(s) should target precursor ions may include: determining, based on the comparison(s), an order in which the one or more MS2 mass analysis scans should target the precursor ions.

[0037] In some embodiments, the method may, for each of the one, several, most, or all of the precursor ions for which a CCS indicative value is determined, include: calculating a difference between the determined CCS indicative value and the expected CCS indicative value; then selecting (only) the precursor ion(s) for which the calculated difference is greater (or less) than a threshold value; or selecting a specific fixed number or proportion of identified precursor ions with the highest or lowest calculated difference. Additionally or alternatively, the order may be determined by arranging the precursor ions from highest to lowest calculated difference or from lowest to highest calculated difference.

[0038] In specific embodiments, the comparison step for each of the one, several, most, or all of the precursor ions for which a CCS indicative value is determined may include: Determining whether the determined CCS indicative value (a) is greater than a maximum expected CCS indicative value for that precursor ion. Then, the selection step based on the comparison may include: if it is determined that (a) the determined CCS indicative value is greater than the maximum expected CCS indicative value: Selecting that precursor ion to target one of the MS2 bulk analysis scans. Then, in step (iii), one of the MS2 bulk analysis scans may target that selected precursor ion.Additionally or alternatively, the step of determining a sequence based on the comparison may include the following: if it is determined that (a) the determined CCS indicative value is greater than the maximum expected CCS indicative value: assigning a relatively high priority to this precursor in the sequence. The procedure may, where appropriate, include the following if it is determined that (a) the determined CCS indicative value is less than the maximum expected CCS indicative value: not selecting this precursor (and not targeting this precursor with the MS2 bulk analysis scan(s)) and / or assigning a relatively low priority to this precursor in the sequence.

[0039] Additionally or alternatively, the comparison step for each of the one, several, most, or all of the precursor ions for which a CCS indicative value is determined may include: Determining whether the determined CCS indicative value (b) is less than a minimum expected CCS indicative value for that precursor ion. Then, the selection step based on the comparison may include: if it is determined that (b) the determined CCS indicative value is less than the minimum expected CCS indicative value: Selecting that precursor ion to target one of the MS2 bulk analysis scans. Then, in step (iii), one of the MS2 bulk analysis scans may target that selected precursor ion.Additionally or alternatively, the step of determining a sequence based on the comparison may include the following: if it is determined that (b) the determined CCS indicative value is less than the minimum expected CCS indicative value: assigning a relatively high priority to this precursor in the sequence. The procedure may, where appropriate, include the following if it is determined that (b) the determined CCS indicative value is greater than the minimum expected CCS indicative value: not selecting this precursor (and not targeting this precursor with the MS2 bulk analysis scan(s)) and / or assigning a relatively low priority to this precursor in the sequence.

[0040] Additionally or alternatively, the comparison step for each of the one, several, most, or all of the precursor ions for which a CCS indicative value is determined may include: Determining whether the determined CCS indicative value (c) is outside an expected range of CCS indicative values ​​for that precursor ion. Then, the selection step based on the comparison may include: if it is determined that (c) the determined CCS indicative value is outside the expected range of CCS indicative values: Selecting that precursor ion to target one of the MS2 bulk analysis scans. Then, in step (iii), one of the MS2 bulk analysis scans may target that selected precursor ion.Additionally or alternatively, the step of determining a sequence based on the comparison may include the following: if it is determined that (c) the determined CCS indicative value is outside the expected range of CCS indicative values: assigning a relatively high priority to this precursor in the sequence. The procedure may, where appropriate, include the following if it is determined that (c) the determined CCS indicative value is within the expected range of CCS indicative values: not selecting this precursor (and not targeting this precursor with the MS2 bulk analysis scan(s)) and / or assigning a relatively low priority to this precursor in the sequence.

[0041] In these embodiments, an MS2 scan will target precursor ions with a relatively high priority in the sequence in step (iii) before precursor ions with a relatively low priority.

[0042] As described above and elsewhere herein, identifying precursor ions with CCS indicative values ​​that deviate in this way from an expected CCS indicative value or range makes it possible to identify and preferentially target modified sample ions, such as modified and cross-linked peptides and / or proteins. For example, precursor ions exhibiting CCS indicative values ​​higher than expected may, in specific embodiments, be cross-linked peptides or glycosylated peptides. Precursor ions with CCS indicative values ​​lower than expected may be phosphorylated peptides.

[0043] In embodiments, the value or values ​​indicative of an expected collision cross section (CCS) or range of collision cross sections can be determined from a calibration, such as a calibration curve. The method can then, for each of the one, several, most, or all of the precursor ions for which a CCS-indicative value is determined, include: determining a value or values ​​indicative of an expected collision cross section (CCS) or range of collision cross sections for that precursor ion from the calibration, and using that value or those values ​​in the comparison step.

[0044] The calibration can include an expected CCS indicative value and / or an expected range of CCS indicative values ​​as a function of the mass-to-charge ratio (m / z). For example, the calibration can include a maximum expected CCS indicative value as a function of the mass-to-charge ratio (m / z) along with a minimum expected CCS indicative value as a function of the mass-to-charge ratio (m / z), which together can form an expected range of CCS indicative values ​​as a function of the mass-to-charge ratio (m / z), i.e., an expected region in the space CCS indicative value - m / z. Then, a precursor can be selected and / or prioritized in the sequence if its specific CCS indicative value lies outside this expected region.

[0045] In embodiments, the selected precursor ions are modified precursor ions, such as modified or cross-linked peptide and / or protein ions. The calibration can then 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, if the sample ions are peptide and / or protein ions, the calibration can be generated by analyzing one or more samples of unmodified peptide and / or protein ions. The calibration can be generated by measuring a value (e.g., peak width) that is characteristic of the collision cross-section for numerous unmodified precursor ions with varying m / z ratios, and by creating the calibration from these measured values, e.g., by averaging, etc.

[0046] As mentioned above, the target precursor ions are fragmented during each MS2 mass analysis scan. In some embodiments, the fragmentation energy and / or fragmentation method used in each of the multiple MS2 mass analysis scans is / are the same. Alternatively, either the fragmentation energy and / or the fragmentation method, or both, can be varied between some or all of the multiple MS2 mass analysis scans and can be selected, for example, based on the CCS indicative value of the target precursor ion to obtain improved MS2 data.

[0047] Therefore, the procedure for each of the one, several, most, or all MS2 mass analysis scans may include: selecting a fragmentation energy for use when performing that MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by that MS2 mass analysis scan (and using the selected fragmentation energy when performing the MS2 mass analysis scan). Additionally or alternatively, the procedure for each of the one, several, most, or all MS2 mass analysis scans may include: selecting a fragmentation method for use when performing that MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by that MS2 mass analysis scan (and using the selected fragmentation method when performing the MS2 mass analysis scan).The fragmentation method can be selected from a multitude of possible fragmentation methods, which can include any combination of two or more fragmentation methods, such as collision-induced dissociation (CID), electron-induced dissociation (EID), photodissociation, and so on. Numerous other types of fragmentation are possible.

[0048] In these embodiments, the fragmentation energy and / or the fragmentation method can be selected depending, for example, on the specific chemical class as derived from the CCS indicative value and the m / z of the target precursor ion, in order to obtain, for example, improved MS2 data.

[0049] Additionally or alternatively, the fragmentation energy and / or the fragmentation method can be selected based on the comparison (e.g., in a manner similar to that described above). For example, the fragmentation energy and / or the fragmentation method can be selected depending on whether (and / or by how much) the CCS indicative value of the precursor ion deviates from an expected CCS indicative value or range. This allows for the obtaining of improved MS2 data for unmodified and modified / crosslinked peptides and / or proteins, etc.

[0050] These “decision-driven fragmentation” procedures can be performed together with or independently of the DDA procedure described above, which is based on the CCS indicative value(s).

[0051] Therefore, a second aspect provides a method of mass spectrometry, including: Ionizing a sample to produce sample ions; (i) Analyzing the sample ions by performing one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions of interest in the MS1 spectrum; and (iii) Analyzing the sample ions by performing one or more MS2 mass analysis scans, each MS2 mass analysis scan targeting one of the one or more precursor ions identified from the MS1 data; wherein the procedure for each of the one or more precursor ions identified from the MS1 data further comprises: determining a value from the MS1 data that is characteristic of a collision cross section (CCS) of that precursor ion; and for each of the one or more MS2 bulk analysis scans: Selecting a fragmentation energy for use when performing the MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 mass analysis scan; and / or Selecting a fragmentation method for use when performing the MS2 bulk analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 bulk analysis scan.

[0052] This aspect can, and in embodiments does, include any one or any number of the optional features described herein.

[0053] Another aspect is a non-volatile, computer-readable storage medium that stores computer software code which, when executed on a processor, performs the procedure(s) described above.

[0054] Another aspect is providing a control system for an analytical instrument, such as a mass spectrometer, wherein the control system is configured to cause the analytical instrument to perform the procedure(s) described above.

[0055] Another aspect is provided by an analytical instrument, such as a mass spectrometer, which includes the control system described above.

[0056] Another aspect is provided by an analytical instrument such as a mass spectrometer, comprehensively: an ion source configured to ionize a sample in order to produce sample ions; a mass filter configured to select ions using an isolation window; a fragmentation device configured to fragment sample ions in order to produce fragment ions; a mass analyzer; and a control system configured to do the following: (i) Inducing the device to perform one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions from the MS1 data; and (iii) Inducing the device to perform one or more MS2 bulk analysis scans, each MS2 scan targeting one of the one or more precursor ions identified from the MS1 data; the control system is further configured to do the following: For each of the one or more precursor ions identified from the MS1 data: Determine a value from the MS1 data that is characteristic of a collision cross section (CCS) for that precursor ion; and Select, based on the CCS indicative value(s), which precursor ion(s) the one or more MS2 bulk analysis scans should target, and / or determine, based on the CCS indicative value(s), an order in which the one or more MS2 bulk analysis scans should target the precursor ions.

[0057] Another aspect is provided by an analytical instrument such as a mass spectrometer, comprehensively: an ion source configured to ionize a sample in order to produce sample ions; a mass filter configured to select ions using an isolation window; at least one fragmentation device configured to fragment sample ions to produce fragment ions; a mass analyzer; and a control system configured to do the following: (i) Inducing the device to perform one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions from the MS1 data; and (iii) Inducing the device to perform one or more MS2 bulk analysis scans, each MS2 scan targeting one of the one or more precursor ions identified from the MS1 data; the control system is further configured to do the following: For each of the one or more precursor ions identified from the MS1 data: Determine a value from the MS1 data that is characteristic of a collision cross section (CCS) for that precursor ion; and for each of the one or more MS2 bulk analysis scans: Selecting a fragmentation energy for use when performing this MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 mass analysis scan; and / or Selecting a fragmentation method for use when performing this MS2 bulk analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 bulk analysis scan.

[0058] These aspects may, and in some embodiments do, include any one or more of the optional features described herein. Description of the drawings

[0059] Various embodiments are now described in more detail with reference to the attached figures, wherein: Fig. 1 schematically shows a mass spectrometer according to embodiments; Fig. Figure 2 shows simulated data of the observed mass resolution for precursor ions measured during a proteomic analysis using an Orbitrap™ mass analyzer operated with a standard ultra-high vacuum (UHV); Fig. Figure 3 shows simulated data of the observed mass resolution for unmodified and modified precursor ions measured during a proteomic analysis using an Orbitrap™ mass analyzer operated with a standard ultra-high vacuum (UHV); Fig. 4 Simulated data of the observed mass resolution for ions using a standard pressure (referred to as "precursor, Std. pressure") together with real data measured using the CCS workflow of the embodiments (referred to as "precursor, elevated pressure"), showing that at elevated UHV the various charge states present in the precursors resolve into charge-state-dependent trend lines due to the inherently higher CCS value of higher charge states; Fig. 5 Data for the ions with 2+ charge state from Fig. Figure 4 shows, together with simulated data of the observed mass resolution for precursors that exhibit modifications that lead to a decrease in the CCS value (referred to as "Mod. CCS decreased"), and for precursors that exhibit modifications that lead to an increase in the CCS value (referred to as "Mod. CCS increased"); Fig. Figure 6 shows a simulated MS1 mass spectrum containing 12 precursors with different intensities and different m / z values ​​(ion isotopes are not shown); Fig. 7 the observed mass resolution for each of the precursors of Fig. 6 as a function of m / z, together with an “expected resolution” corresponding to the 2+ trend line of an unmodified standard and an “unmodified region” that is representative of the observed dispersion in the mass resolution values ​​of the 2+ ions of the unmodified standard; Fig. 8 two selection strategies based on the in Fig. 7 data shown illustrates where the MS2 analysis either targets ions that lie below the “unmodified region” ( Fig. 8B), or the MS2 analysis targets ions that lie above the “unmodified region” ( Fig. 8A); and Fig. Figure 9 schematically shows a DDA method of a mass spectrometer according to embodiments. Detailed description

[0060] Fig. Figure 1 schematically illustrates an analytical instrument, such as a mass spectrometer, that can be used in conjunction with the procedures described herein. As in Fig. As shown in Figure 1, the device includes an ion source 10, a mass filter 20, a fragmentation device 30 and a mass analyzer 40.

[0061] 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) separator, a gas chromatography (GC) separator, or a capillary electrophoresis separator, and the like, so that the sample 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 (EI) ion source, or the like.

[0062] The analyzer may additionally or alternatively include an ion separation device located downstream of the ion source and configured to separate the ions of the samples according to a physicochemical property. For example, the device may 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).

[0063] The mass filter 20 is arranged downstream of the ion source 10 and configured to receive ions from the ion source 10 (optionally via the 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 with m / z within an m / z transmission window (or "isolation window") of the mass filter are passed through the mass filter, while received ions with m / z outside the m / z transmission window are attenuated by the mass filter, i.e., not passed through the mass filter. The width and / or m / z of the center of the transmission window can be controllable (variable), for example, by appropriately controlling the RF and / or DC voltage(s) applied to the electrodes of the mass filter 20.Therefore, the mass filter 20 can be operated, for example, in a transmission mode, in which most or all ions within a relatively wide m / z window are passed through the mass filter 20, and in a filtration mode, in which only ions within a relatively narrow m / z window (centered on a desired m / z) are passed through the mass filter 20. The mass filter 20 can be any suitable type of mass filter, such as a quadrupole mass filter.

[0064] The fragmentation device 30 is located downstream of the mass filter 20 and is configured to receive most or all of the ions passed through the mass filter 20. The fragmentation device 30 can be configured to selectively fragment some or all of the received ions, i.e., to produce fragment ions. The fragmentation device 30 can operate in a fragmentation mode, in which most or all of the received ions are fragmented to produce fragment ions (which can then be passed through the fragmentation device 30), and in a non-fragmentation mode, in which most or all of the received ions are passed through without being (intentionally) fragmented. It would also be possible to implement a non-fragmentation mode by causing ions to bypass the fragmentation device 30.The fragmentation device 30 can also be operated in one or more intermediate operating modes, e.g., where the degree of fragmentation is controllable (variable). The fragmentation device 30 can also be operated in higher-order fragmentation modes (MS). N ), e.g., wherein the fragmentions are further fragmented one or more times by the fragmentation device 30.

[0065] The fragmentation device 30 can be any suitable type of fragmentation device, such as a collision-induced dissociation (CID) fragmentation device, an electron-induced dissociation (EID) fragmentation device, a photodissociation fragmentation device, and so on. Numerous other types of fragmentation are possible.

[0066] In some embodiments, the fragmentation device 30 is capable of performing more than one type of fragmentation process, or more than one type of fragmentation device is provided, allowing the device to select between performing different fragmentation processes. Similarly, the fragmentation device(s) 30 may be capable of fragmenting ions using different fragmentation energies.

[0067] In some embodiments, the fragmentation device 30 is a collision-induced dissociation (CID) fragmentation device. The CID fragmentation device may include a collision cell, which may be filled with a collision gas, for example, maintained at a relatively high pressure. Ions can be selectively fragmented in the collision cell by controlling (varying) the kinetic energy with which ions are caused to enter the collision cell. In one fragmentation operating mode, ions can be accelerated so that they enter the collision cell with a relatively high kinetic energy, which can cause most or all of the accelerated ions to be fragmented.In a non-fragmentation operating mode, ions can be caused to enter the collision cell with a relatively low kinetic energy, which may not be sufficient to cause most or all of the ions to fragment. In intermediate modes, ions can be caused to enter the collision cell with medium kinetic energy.

[0068] The mass analyzer 40 is positioned downstream of the fragmentation device 30 and is configured to receive ions from the fragmentation device 30. Therefore, depending on the operating mode 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 to determine their mass-to-charge ratio (m / z) and / or their 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 orbital trap mass analyzer (such as a Thermo Fisher Scientific Orbitrap™ FT mass analyzer), or a time-of-flight (ToF) mass analyzer, such as a multireflection time-of-flight (MR-ToF) mass analyzer.

[0069] It should be noted that Fig. Figure 1 is merely schematic and the device may include, and in some embodiments does include, any number of one or more additional components. For example, the device may include one or more ion transfer stages arranged between each of the illustrated components, including, for example, an atmospheric pressure interface and / or one or more ion conductors, lenses, and / or other optical ion devices configured to allow some or all of the ions to pass adequately through the device. The ion transfer stage(s) may include any suitable number and configuration of ion-optical devices, for example, optionally including one or more ion conductors, lenses, and / or other ion-optical devices.

[0070] In some embodiments, the device may include more than one mass analyzer. For example, the device may be a hybrid mass spectrometer with two mass analyzers of the type described in EP 3,410,463, the contents of which are incorporated herein by reference.

[0071] As in Fig. As shown in Figure 1, the device is controlled by a control unit 50, such as a suitably programmed computer, which controls the operation of various components of the device and, for example, sets the voltages to be applied to the various components of the device. The control unit 50 can also receive and process data from various components, including the analyzer(s) according to different embodiments.

[0072] The instrument can be operated in various operating modes. In particular, the instrument can be a tandem mass spectrometer that can be operated in an MS1 ​​operating mode and an MS2 operating mode.

[0073] In the MS1 operating mode (or “Full Mass Scan”), the mass filter 20 is operated in its transmission operating mode and the fragmentation device 30 in its non-fragmentation operating mode, so that, for example, a wide m / z range (e.g., the entire mass range) of non-fragmented ions (“precursor ions” or “stem ions”) is analyzed by the analyzer 40 to generate an MS1 ​​spectrum.

[0074] In the MS2 operating mode, the mass filter 20 is operated in its filter operating mode and the fragmentation device 30 in its fragmentation operating mode, so that, for example, a selected narrow m / z range of precursor ions is fragmented and the resulting fragment ions (“productions” or “daughter ions”) are analyzed by the analyzer 40 to generate an MS2 spectrum.

[0075] The instrument can also be operated in one or more higher-order fragmentation modes, such as an MS3 operating mode, in which precursor ions are fragmented, at least some of the resulting fragment ions are themselves fragmented, and the second-generation fragment ions (“grandchild ions”) are analyzed by analyzer 40 to generate an MS3 spectrum. In general, the instrument can be operated in any order of fragmentation mode, i.e., in an MS N -Operating mode with N≥ 2.

[0076] As described above, an analytical challenge in proteomics is to identify and select modified peptide ions, such as PTMs and cross-linked peptides, within a proteomic workflow while minimizing additional sample handling requirements. One solution, according to embodiments, exploits the structural changes at the peptide level that result from protein modification. It has been shown in the literature that modifications to a peptide ion can affect its gas-phase structure, either through compaction (i.e., a reduction in the area / volume of the structure compared to an unmodified peptide) or through elongation (i.e., an increase in the area / volume of the structure compared to an unmodified peptide ion). These structural differences can be addressed using structure-sensitive techniques.One such technique, which is frequently used, is ion mobility, in which ions are separated with respect to their collision cross-sectional area (CCS) values, which are linked to the rotationally averaged structure that the ion assumes in the gas phase.

[0077] Recently, the inventors published work, such as that described in UK patent application GB 2,612,580, in which these CCS values ​​were measured based on the mass measurement step in an Orbitrap™ mass analyzer. This work demonstrated that at elevated UHV pressure values ​​(e.g., approximately 10 -9In the Orbitrap™ mass analyzer, the decay of the ion in the transient domain (mbar) is linked to its CCS value. In ensemble measurements, the decay rate of a single ion species—that is, an ion with a specific m / z and charge state—results in a change in the observed mass resolution (i.e., peak width) of that ion in the mass spectrum. Here, this work is extended to develop a data-driven, targeted, real-time workflow where precursors exhibiting deviations from the expected mass resolution indicate that they are modified ions. The MS2 analysis then spontaneously targets these ions, thereby increasing the utilization of the instrument operating cycle for the analysis of modified peptides and reducing or minimizing the need for pre-analysis sample enrichment.

[0078] Fig. Figure 2 shows simulated data of the observed mass resolution for precursor ions measured during a proteomic analysis using an Orbitrap™ mass analyzer operated under standard ultra-high vacuum (UHV). As shown in Fig. As shown in Figure 2, the mass resolution of an ion scales with the inverse square root of the m / z value. The UHV used by the mass analyzer is held at a certain value (e.g., approximately 10). -10 mbar or 10 -11 mbar), where the mean free path of a peptide ion is such that the ion population does not decrease noticeably during the measurement and therefore exhibits minimal losses due to collisions with the background gas. For this reason, ions with different structures and inherently different CCS values ​​show similar observed mass resolution values ​​as a function of m / z.

[0079] Fig. Figure 3 shows the corresponding simulated data for precursors with structurally influencing modifications such as glycans, cross-links, or phospho groups, which correspond to the data from Fig. 2 are superimposed. Due to the lack of ion decay over the measurement timeframe, structural differences do not lead to separation in the mass resolution domain. Therefore, as in Fig. 3 shown, due to the absence of collisions during the Orbitrap™ mass analyzer measurement, unmodified precursors and modified precursors in mass resolution versus m / z, although the two ion groups are structurally unique.

[0080] To measure CCS values ​​in the Orbitrap™ mass analyzer, the UHV is increased to a value at which the ions show significant decay on the transient detection timescale (e.g., approximately 10 -9 mbar). As in Fig. Figure 4 shows the result of this decay in the observed mass resolution.

[0081] As from Fig. As shown in Figure 4, the individual charge states begin to cluster in the mass resolution-m / z space, since ions with higher charge states generally exhibit a larger CCS value. This causes ions with higher charge states to decay more rapidly, resulting in lower observed mass resolution and separation of the different charge states in this dimension. By controlling the UHV pressure and identifying unmodified precursor ions for a known standard (e.g., Hela), these charge-state-dependent trend lines can be mapped and calibrated for later use in a targeted workflow for analyzing an unknown compound.

[0082] Furthermore, if it only refers to a single charge state in Fig. 4 is concentrated, such as the 2+ charge state, as in Fig. As shown in Figure 5, it can be demonstrated that modified ions can separate from their unmodified counterparts, either due to a higher CCS value and a lower observed mass resolution (i.e., ions that are in Fig. 5 below the grouping “No Mod.”) or due to a lower CCS value and a higher observed mass resolution (i.e., ions that are in Fig. 5 above the grouping “No Mod.”). In embodiments, this separation is used for a targeted real-time workflow.

[0083] As explained above, protein modifications occur in substoichiometric amounts and would therefore result in a small portion of the instrument's analysis time being spent on their analysis using conventional stochastic detection. Fig. Figure 6 shows an example of a simulated MS1 mass spectrum, as it would be acquired during a proteomic workflow. In this example, the MS1 scan shows 12 precursor signals with varying m / z and intensity values. In standard proteomic workflows such as DDA, the “top N” most intense of these precursors (isotopes not shown) would be selected for fragmentation, ignoring their modification status.

[0084] However, according to embodiments, the increased UHV pressure can be used to map the observed mass resolutions in real time.

[0085] Fig. Figure 7 shows the observed mass resolution for each of the precursors of Fig. 6 as a function of m / z. Furthermore, the in Fig. Figure 5 shows the 2+ trend line of the unmodified standard through the “expected resolution” and the “unmodified region”, which is representative of the dispersion observed in the mass resolution values ​​of the 2+ ions of the unmodified standard.

[0086] Using the previously established line of "expected resolution" for unmodified peptide ions (from a known compound such as Hela), it can be seen that six precursor ions exhibit a mass resolution outside the expected "unmodified region." These data suggest that these six precursors are structurally unique, and this could be due to the presence of modifications such as phosphorylation or crosslinking.

[0087] When this knowledge is incorporated into a data-driven workflow, it allows a user to selectively target precursors with higher CCS values, for example, for crosslinking or glycan analysis, or precursors with lower CCS values ​​for modifications that lead to compaction, such as phosphorylation. In such a workflow, the target precursors are those located outside the "unmodified region," and therefore a significantly higher percentage of the instrument's analysis time is devoted to precursors likely to exhibit the modification of interest.

[0088] In Fig. 8. Two selection strategies based on the in Fig. The data shown in Figure 7 illustrates this. If the user is interested in investigating modifications known to increase the CCS value of the ion, ions below the "unmodified region" can be defined as the target ( Fig. 8B), while all other ions can be excluded. If, on the other hand, the user is interested in modifications that typically show CCS compaction, such as phosphorylation, the procedure can define target values ​​for the subsequent MS2 analysis that lie above the "unmodified region" ( Fig. 8A).

[0089] Fig. Figure 9 illustrates a procedure for operating the analyzer. Fig. 1 according to embodiments. A sample is provided to a separation device (e.g., LC) for sample separation, and the eluent from the separation device is ionized in the ion source 10 (step 100). The resulting ions are analyzed by the instrument, which operates in a data-dependent acquisition (DDA) mode. Additionally or alternatively, sample ions can be separated by a separation device, such as an ion mobility separator.

[0090] In data-driven detection (DDA) mode, the instrument repeatedly performs the following steps during a separation run of the separator: (i) obtaining an MS1 ​​spectrum over an m / z range of interest (step 102); (ii) identifying one or more precursor ions of interest in the MS1 spectrum; and (iii) obtaining an MS2 spectrum (or an MS N-spectrum) for each identified precursor of interest (Step 104).

[0091] Step (i) involves the instrument performing one or more MS1 mass analysis scans to generate the MS1 spectrum. Step (iii) involves, for each of the identified precursor ions of interest: isolating the precursor ion using the mass filter 20, fragmenting the isolated precursor ions in the fragmentation device 30, and mass analyzing the fragmented ions with the mass analyzer 40. Therefore, during each cycle, several MS2 spectra (or more generally, several MS) are acquired. N-Spectra) are acquired by sequentially changing the center of the (narrow) m / z window of the mass filter between each of a multitude of different target m / z values, e.g., to sequentially select (and fragment) each of a multitude of precursor ions, each with a different m / z. The multitude of different m / z values ​​corresponds to the multitude of different target precursor ions.

[0092] As in Fig. As shown in Figure 9, according to embodiments, a list of target precursor ions is generated based on CCS indicative values ​​determined for each ion peak in the MS1 data. Therefore, the device identifies peaks in the MS1 spectrum (step 110) and calculates the CCS indicative value of each identified peak in the MS1 spectrum (step 111). The calculated CCS indicative value is then compared for each peak with an expected CCS indicative value or range of expected CCS indicative values ​​for that peak (step 112). As described above, the expected CCS indicative value can be derived from a calibration curve (e.g., as shown in Figure 9). Fig. 7 shown) can be determined, which may have been created by analyzing one or more samples of unmodified precursor ions.

[0093] In step 113, if the peak has a CCS indicative value sufficiently similar to the expected value, it can be ignored or assigned a relatively low priority. If the peak has a CCS indicative value sufficiently different from the expected value, it can be added to a list of targets (in step 114) and / or assigned a relatively high priority. After all peaks in the MS1 spectrum have been considered, a complete list of MS2 targets, optionally along with a priority ranking, can be compiled and used to control the MS2 scans in step 104. This process can then be repeated for the next MS1 spectrum, and so on.

[0094] It should be noted that Fig. Section 9 shows a simplified procedure and that additional steps can be included in the workflow in practice. In particular, additional logical steps can be included between steps 102 and 104, e.g., in the manner of standard DDA workflows, such as a state-of-charge / inclusion check, a step to establish an intensity ranking (“Top-N”), and / or the use of an exclusion list or lists to exclude MS2 targets that have already been analyzed or have been analyzed recently.

[0095] It is therefore assumed that in embodiments, the differences in the CCS-guided observed mass resolutions are used to target precursors located above or below an expected unmodified region. CCS-guided decision-making regarding which ions to target can increase the instrument analysis time spent targeting ions of interest that may be present at low abundances and substoichiometric levels. This can advantageously reduce or minimize the need for sample enrichment prior to MS analysis.

[0096] Although specific embodiments have been described in detail above, several alternatives are possible. For example, CCS-indicative values ​​could instead be determined from the MS1 data using the method described in UK patent application GB 2303690.8. In this method, CCS-indicative values ​​are determined using a time-of-flight (ToF) mass analyzer, with which two datasets are obtained (i.e., by performing two scans or two sets of scans), whereby either (i) the ion path length or (ii) the gas pressure in the ion path, or both, are varied between the two datasets. By comparing the intensity of corresponding ion peaks in the two datasets, the CCS of the ions causing the corresponding ion peaks can be determined.

[0097] In some embodiments, the selected precursor ions can be precursor ions belonging to a specific chemical class. For example, if the sample is a heterogeneous mixture of different chemical classes (e.g., if it includes two or more peptides, lipids, steroids, etc.), each of the different chemical classes may exhibit distinct trend lines in the CCS-indicative value-m / z space. Precursor ions from only one of the chemical classes can then be selected for analysis and / or preferentially targeted by one or more MS2 mass analysis scans by selecting precursor ion(s) based on their CCS-indicative value(s) and their m / z, and / or by determining the order based on their CCS-indicative value(s) and m / z.

[0098] The methods described herein can also be used to trigger different MS2 fragmentation energies or processes. That is, different fragmentation strategies can be selected depending on the CCS indicative value. With further reference to Fig.7. For example, if both peptides located in the unmodified region and peptides with a lower CCS indicative value, which may therefore be phosphorylated, are present, the phosphorylated peptides can be selected and fragmented using a fragmentation method and / or fragmentation energy (e.g., EID with a specific electron energy), and the unmodified peptides can be selected later and fragmented using a different fragmentation method and / or energy (e.g., HCD or EID with a different electron energy) than the phosphorylated ones. Similarly, in the case of a heterogeneous mixture of different chemical classes (e.g., peptides, lipids, steroids, as mentioned 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 for steroids, etc.

[0099] Although the present invention has been described with reference to various embodiments, it is understood that various modifications can be made without deviating from the scope of protection of the invention as set out in the attached claims. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] GB 2,612,580 [0011, 0017, 0024, 0077] GB 2303690 [0025, 0028, 0096] EP 3,410,463

[0070]

Claims

[1] Methods of mass spectrometry, comprising: Ionizing a sample to produce sample ions; (i) Analyzing the sample ions by performing one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions of interest in the MS1 spectrum; and (iii) Analyzing the sample ions by performing one or more MS2 mass analysis scans, each MS2 mass analysis scan targeting one of the one or more precursor ions identified from the MS1 data; the procedure further includes the following: for each of one or more precursor ions identified from the MS1 data: Determine a value from the MS1 data that is characteristic of a collision cross section (CCS) of that precursor ion; and Select, based on the CCS indicative value(s), which precursor ion(s) the one or more MS2 bulk analysis scans should target, and / or determine, based on the CCS indicative value(s), an order in which the one or more MS2 bulk analysis scans should target the precursor ions. [2] The method of claim 1, wherein the method comprises: for each of the one or more precursor ions for which a CCS indicative value is determined: comparing the determined CCS indicative value with a value or values ​​indicative of an expected collision cross-section (CCS) or range of collision cross-sections for that precursor ion; and Select, based on the comparison(s) of which precursor ion(s) the one or more MS2 bulk analysis scans should target, and / or the order in which the one or more MS2 bulk analysis scans should target the precursor ions. [3] Method according to claim 2, wherein: The comparison step for each of the one or more precursor ions for which a CCS indicative value is determined includes: determining whether the determined CCS indicative value is (a) greater than a maximum expected CCS indicative value for that precursor ion; and / or (b) less than a minimum expected CCS indicative value for that precursor ion; and / or (c) outside an expected range of CCS indicative values ​​for that precursor ion; and The step of selection based on the comparison(s) includes the following: if it is determined that (a) the determined CCS indicative value is greater than the expected maximum CCS indicative value; and / or (b) the determined CCS indicative value is less than the expected minimum CCS indicative value; and / or (c) the determined CCS indicative value is outside the expected range of CCS indicative values: Selecting this precursor; and / or The step of determining the order based on the comparison(s) includes the following: if it is determined that (a) the determined CCS indicative value is greater than the expected maximum CCS indicative value; and / or (b) the determined CCS indicative value is less than the expected minimum CCS indicative value; and / or (c) the determined CCS indicative value is outside an expected range of CCS indicative values: assigning a relatively high priority to this precursor in the order. [4] Method according to claim 3, wherein: The step of selection based on the comparison(s) includes the following: if it is determined that (a) the determined CCS indicative value is less than the expected maximum CCS indicative value; and / or (b) the determined CCS indicative value is greater than the expected minimum CCS indicative value; and / or (c) the determined CCS indicative value is within the expected range of CCS indicative values: Not selecting this precursor; and / or The step of determining the order based on the comparison(s) includes the following: if it is determined that (a) the determined CCS indicative value is less than the expected maximum CCS indicative value; and / or (b) the determined CCS indicative value is greater than the expected minimum CCS indicative value; and / or (c) the determined CCS indicative value is within the expected range of CCS indicative values: assigning a relatively low priority to this precursor in the order. [5] Method according to any of the preceding claims, wherein the precursor ion(s) selected and / or given a relatively high priority in the sequence are modified precursor ions. [6] Method according to claim 5, wherein the sample ions are peptide and / or protein ions, and wherein the precursor ion(s) selected and / or given a relatively high priority in the sequence are modified or cross-linked peptide and / or protein ions. [7] The method of claim 5 or 6, if dependent on claim 2, further comprising determining the value or values ​​characteristic of an expected collision cross section (CCS) or range of collision cross sections from a calibration, wherein the calibration is a calibration produced by analyzing one or more samples of unmodified precursor ions. [8] Method according to any one of the preceding claims, further comprising for each of the one or more MS2 mass analysis scans: Selecting a fragmentation energy for use when performing the MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 mass analysis scan; and / or Selecting a fragmentation method for use when performing the MS2 bulk analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 bulk analysis scan. [9] Methods of mass spectrometry, including: Ionizing a sample to produce sample ions; (i) Analyzing the sample ions by performing one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions of interest in the MS1 spectrum; and (iii) Analyzing the sample ions by performing one or more MS2 mass analysis scans, each MS2 mass analysis scan targeting one of the one or more precursor ions identified from the MS1 data; wherein the procedure further comprises, for each of one or more precursor ions identified from the MS1 data: determining a value from the MS1 data that is characteristic of a collision cross section (CCS) of that precursor ion; and for each of the one or more MS2 bulk analysis scans: Selecting a fragmentation energy for use when performing the MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 mass analysis scan; and / or Selecting a fragmentation method for use when performing the MS2 bulk analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 bulk analysis scan. [10] Method according to any one of the preceding claims, wherein: the value that is characteristic of a 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 a collision cross section (CCS) of a precursor ion includes: determining a value indicative of a collision cross section (CCS) of a precursor ion from a width of an ion peak in the MS1 data corresponding to the precursor ion. [11] Method according to any of the preceding claims, wherein the step of (i) analyzing the sample ions by performing one or more MS1 mass analysis scans comprises the use of an orbital trap mass analyzer for mass analysis of the sample ions. [12] Method according to claim 11, wherein the orbital trap mass analyzer operates at a pressure ≥ 10 -9 is operated in mbar. [13] Method according to any one of claims 1 to 10, wherein: The step of (i) analyzing the sample ions by performing one or more MS1 mass analysis scans includes: using a time-of-flight (ToF) mass analyzer to perform mass analysis of the sample ions, wherein the time-of-flight (ToF) mass analyzer is configured to determine the mass-to-charge ratio (m / z) of ions by determining the time-of-flights of ions along an ion path; The step of using a time-of-flight (ToF) mass analyzer for mass analysis of the sample ions includes the following: Operating the mass analyzer in a first operating mode and analyzing sample ions by determining flight times of the sample ions along the ion path 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 analyzer in a second operating mode and analyzing sample ions by determining flight times of the sample ions along the ion path 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 differs from the first path length and / or the second pressure differs from the first pressure; and The step of determining a value indicative of a collision cross section (CCS) of a precursor ion from the MS1 data includes the following: Comparing the intensity of a precursor ion peak in the first data set with the intensity of a corresponding precursor ion peak in the second data set; and Determine, based on comparison, a value that is characteristic of the CCS of the precursor ion. [14] Method according to any one of the preceding claims, wherein: the step of (ii) identifying one or more precursor ions from the MS1 data includes identifying a multitude of different precursor ions from the MS1 data; and the step of (iii) analyzing the sample ions by performing one or more MS2 mass analysis scans includes performing a plurality of MS2 mass analysis scans, each MS2 mass analysis scan of the plurality of MS2 mass analysis scans targeting a different one from the plurality of different precursor ions identified from the MS1 data. [15] Method according to any of the preceding claims, wherein the method comprises the following for each MS2 bulk analysis scan: Isolating the target precursor ions from the sample ions; Fragmenting the isolated precursor ions of interest to produce fragment ions; and Mass analysis of the fragments to obtain MS2 data. [16] Method according to claim 15, wherein the step of isolating the target precursor ions from the sample ions comprises the use of a mass filter to select the target precursors according to their mass-to-charge ratio (m / z), wherein the mass filter is optionally a quadrupole mass filter. [17] Method according to any of the preceding claims, wherein the method comprises performing a plurality of repeated cycles, each cycle comprising performing steps (i), (ii) and (iii). [18] Method according to claim 17, wherein: the sample is provided from a separation device, and / or the sample ions are separated by a separation device; and The method involves repeatedly performing cycles during a separation run of the separation device. [19] Non-volatile, computer-readable storage medium that stores computer software code which, when executed on a processor, performs the method according to any of the preceding claims. [20] Control system for an analyzer, wherein the control system is configured to cause the analyzer to perform the method according to any one of claims 1 to 18. [21] Analysis device comprising the control system according to claim 20. [22] Analyzer, comprising: an ion source configured to ionize a sample in order to produce sample ions; a mass filter configured to select ions using an isolation window; a fragmentation device configured to fragment sample ions in order to produce fragment ions; a mass analyzer; and a control system configured to do the following: (i) Inducing the device to perform one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions from the MS1 data; and (iii) Inducing the device to perform one or more MS2 bulk analysis scans, each MS2 scan targeting one of the one or more precursor ions identified from the MS1 data; the control system is further configured to do the following: For each of the one or more precursor ions identified from the MS1 data: Determine a value from the MS1 data that is characteristic of a collision cross section (CCS) for that precursor ion; and Select, based on the CCS indicative value(s), which precursor ion(s) the one or more MS2 bulk analysis scans should target, and / or determine, based on the CCS indicative value(s), an order in which the one or more MS2 bulk analysis scans should target the precursor ions. [23] Analyzer, comprising: an ion source configured to ionize a sample in order to produce sample ions; a mass filter configured to select ions using an isolation window; at least one fragmentation device configured to fragment sample ions to produce fragment ions; a mass analyzer; and a control system configured to do the following: (i) Inducing the device to perform one or more MS1 bulk analysis scans to obtain MS1 ​​data; (ii) Identifying one or more precursor ions from the MS1 data; and (iii) Inducing the device to perform one or more MS2 bulk analysis scans, each MS2 scan targeting one of the one or more precursor ions identified from the MS1 data; the control system is further configured to do the following: for each of one or more precursor ions identified from the MS1 data: Determine a value from the MS1 data that is characteristic of a collision cross section (CCS) of that precursor ion; and for each of the one or more MS2 bulk analysis scans: Selecting a fragmentation energy for use when performing this MS2 mass analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 mass analysis scan; and / or Selecting a fragmentation method for use when performing this MS2 bulk analysis scan, based on the CCS indicative value of the precursor ion targeted by this MS2 bulk analysis scan.

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