阅读说明：本技术 质谱分析方法和质谱系统 (Mass spectrometry method and mass spectrometry system ) 是由 周鑫锋 邱然 孙文剑 于 2020-04-02 设计创作，主要内容包括：本发明提供了一种质谱分析方法和质谱系统,在实施该质谱分析方法的过程中,子离子的强度数据、与第一物理化学特性关联的子离子的第一参数、与第二物理化学特性关联的子离子的第二参数均被记录,从而形成谱图数据集,在解卷积步骤中,根据包含第一参数和第二参数的二维特征,对谱图数据集进行解卷积,以将来自同一母离子的子离子归类。通过以上方式,本发明提供的质谱分析方法和质谱系统能够检知部分与其他离子的谱峰严重重叠的离子,从而,提高针对数据非依赖性采集数据解析的定性和定量能力。(In the process of implementing the mass spectrometry, intensity data of daughter ions, a first parameter of the daughter ions associated with a first physicochemical characteristic, and a second parameter of the daughter ions associated with a second physicochemical characteristic are all recorded, so that a spectrogram data set is formed, and in a deconvolution step, the spectrogram data set is deconvoluted according to a two-dimensional characteristic comprising the first parameter and the second parameter, so that the daughter ions from the same parent ion are classified. Through the mode, the mass spectrometry method and the mass spectrometry system provided by the invention can detect the ions with the parts which are seriously overlapped with the spectral peaks of other ions, thereby improving the qualitative and quantitative capability of analyzing data independent acquisition data.)

1. A method of mass spectrometry comprising the steps of:

a first separation step of separating the sample based on the difference of the first physicochemical characteristic to obtain a plurality of groups of intermediates;

a second separation step of separating the intermediate or the ionized intermediate based on a difference in a second physicochemical characteristic to obtain a plurality of sets of parent ions;

a dissociation step, in which at least a part of the parent ions are dissociated, and a plurality of daughter ions are generated corresponding to the dissociated parent ions;

a detection step of detecting at least the intensity of the daughter ions generated in the dissociation step;

in carrying out the method of mass spectrometry, intensity data of said daughter ions, a first parameter of said daughter ions associated with said first physicochemical characteristic, a second parameter of said daughter ions associated with said second physicochemical characteristic are recorded, thereby forming a set of spectral data,

characterized in that the mass spectrometry method further comprises,

a deconvolution step of deconvolving the spectrogram dataset according to a two-dimensional feature including the first parameter and the second parameter to classify the daughter ions from the same parent ion.

2. The method of mass spectrometry of claim 1, wherein in the step of detecting, the intensities of the plurality of sets of parent ions are also detected.

3. The method of mass spectrometry of claim 2, wherein the detecting step comprises

Stopping or reducing the dissociation energy applied to the parent ions, thereby detecting the intensities of the plurality of groups of parent ions.

4. The method of mass spectrometry of claim 1, wherein the deconvoluting step comprises

A heat map generation step, based on the spectrogram data set, establishing a coordinate system by using the first parameter and the second parameter, and generating a plurality of heat maps, wherein each heat map is associated with a mass-to-charge ratio or a mass-to-charge ratio range of the daughter ions;

and clustering analysis, namely clustering the three-dimensional features which occupy the same first parameter range and the same second parameter range in different heat maps and are matched with each other according to a clustering analysis model.

5. The method of mass spectrometry of claim 4, further comprising:

and a step of training a clustering model, which is to train the clustering analysis model according to the score of the clustering result in the step of clustering analysis.

6. The method of mass spectrometry of claim 4, further comprising:

an MS/MS spectrum generation step, wherein an MS/MS spectrum of the clustered daughter ions is generated based on the mass-to-charge ratio or the mass-to-charge ratio range corresponding to the heat map in which the clustered feature distribution is located;

and a searching and matching step, namely searching a pre-established database according to the corresponding relation between the MS/MS spectrum generated in the MS/MS spectrum generating step and the parent-child ions, and performing substance qualification on the parent ions.

7. The method of mass spectrometry of claim 6, wherein in the MS/MS spectrum generation step, the intensity data for the clustered sub-ions in the thermal map is integrated to determine the intensity of the sub-ions of the corresponding mass to charge ratio or range of mass to charge ratios in the MS/MS spectrum.

8. The method of mass spectrometry of claim 1, wherein in the step of detecting, the daughter ion intensity data is acquired using a data-independent acquisition method.

9. A mass spectrometry system comprising:

a first separation unit for separating the sample based on the difference of the first physicochemical characteristic to obtain a plurality of sets of intermediates;

a second separation unit for separating the intermediate or the ionized intermediate based on a difference in a second physico-chemical property to obtain a plurality of sets of parent ions;

the dissociation unit is used for dissociating at least one part of the parent ions, and the dissociated parent ions correspondingly generate a plurality of daughter ions;

a detection unit at least for detecting the intensity of the daughter ions generated by the dissociation unit,

the mass spectrometry system is capable of recording intensity data of the daughter ion, a first parameter of the daughter ion associated with the first physicochemical characteristic, and a second parameter of the daughter ion associated with the second physicochemical characteristic, thereby forming a spectrogram dataset,

characterized in that the mass spectrometry system further comprises,

a processor for deconvolving the spectrogram dataset according to a two-dimensional feature comprising the first parameter and the second parameter to classify the daughter ions from the same parent ion.

10. The mass spectrometry system of claim 9, wherein the first separation unit is a chromatograph and the second separation unit is an ion mobility spectrometer.

11. The mass spectrometry system of claim 10, wherein the ion mobility spectrometer is one or a combination of a mobility tube ion mobility spectrometer, a field asymmetric waveform ion mobility spectrometer, a traveling wave ion mobility spectrometer, a breath type ion mobility spectrometer, a tandem trap ion mobility spectrometer, and a U-type ion mobility spectrometer.

12. The mass spectrometry system of claim 10, wherein the ion mobility spectrometer comprises:

the ion introducing device is used for introducing the intermediate into a post-stage device or temporarily storing the received intermediate and simultaneously releasing the intermediate to the post-stage device;

ion mobility separating means, disposed downstream of the ion introducing means, for receiving the intermediate and separating the intermediate during movement according to a difference in ion mobility.

13. The mass spectrometry system of claim 9, wherein the dissociation unit is a collision cell, a photo dissociation cell, or an electron capture dissociation cell.

14. The mass spectrometry system of claim 9, wherein the dissociation unit and the detection unit are integrated into the same ion trap mass analyzer.

15. The mass spectrometry apparatus of claim 9, wherein the detection cell comprises a first mass analyser which is a quadrupole mass analyser, an ion trap mass analyser or a time of flight mass analyser and a second mass analyser which is a quadrupole mass analyser, a time of flight mass analyser or a fourier transform type mass analyser.

Technical Field

The invention relates to the field of mass spectrometry, in particular to a mass spectrometry method and a mass spectrometry system.

Background

High resolution tandem mass spectrometry has become an important analytical instrument in omics analysis (including metabolomics, proteomics, and the like) at present. For more complex samples in omics analysis, a high-throughput, high-sensitivity, high-coverage mass spectrometry data acquisition method is required. The traditional method is Data Dependent Acquisition (DDA) proposed by Ducret et al in 1998, in which a scan is first performed on parent ions (or "precursor" ions), then the parent ions with higher abundance are selected, and sequentially enter a collision cavity for dissociation, and a spectrogram of the daughter ions (or "product" ions) is obtained. This method allows a high analyte coverage and is therefore still a widely used collection method to date.

However, since the data-dependent acquisition method can only monitor the daughter ion information of one parent ion mass-to-charge ratio channel per daughter ion scan, the utilization efficiency and flux of parent ions are low when performing tandem mass spectrometry, and when a large amount of analytes flow out of a chromatographic column at the same time, many parent ions with low abundance are still not monitored; meanwhile, because the mass-to-charge ratio channel of the parent ion corresponding to the child ion scanning event in each cycle is constantly changed, it cannot be guaranteed that the child ions of the analyte are uniformly detected for many times in the chromatographic outflow time, and then only the ion flow graph of the parent ion of the analyte can be used for quantitative analysis, not the ion flow graph of the child ion, so that the selectivity and the accuracy of the quantitative analysis in omics analysis are influenced. The method is characterized in that the method comprises the following steps of selecting parent ions to perform tandem mass spectrometry, wherein the parent ions with high abundance are preferentially selected each time the parent ions are selected to perform tandem mass spectrometry, and the relative abundance of each ion in the same batch of samples is different in different times of sample injection analysis, so that the difference can cause the randomness of parent ion monitoring, and the randomness can cause deviation or errors in the analysis of the biometric data.

In recent years, Data Independent Acquisition (DIA) has been developed rapidly, and compared with the DDA method, the DDA method has higher sensitivity, dynamic range, analysis throughput, and better quantitativity. Typical representatives of this method are the MSE method described in U.S. Pat. No. 5,671,7130 to Micromass and the SWATH (sequential-window acquisition of all the electronic mass spectra) method described in U.S. Pat. No. 5,8809770 to AB Sciex.

The SWATH method mainly aims at target analysis, so that a precursor ion pre-scan is not required, the precursor ion is generally directly segmented according to mass number, for example, each segment is 25Da, then each segment of the precursor ion selected by a quadrupole rod is introduced into a collision cavity together for dissociation, a daughter ion spectrogram is recorded and compared with a database, and the daughter ion intensity is used for quantification and is usually used for quantitative analysis of a target, but not suitable for qualitative analysis. The SWATH method has the following defects that: on one hand, the method is excessively dependent on the establishment of a high-quality daughter ion spectrum database and the matching of experimental conditions, and on the other hand, the non-targeted metabonomic analysis is difficult to implement. In recent years, alternatives to the SWATH method that do not rely on spectral libraries have also been developed in great measure, such as DIA-Umpire (Tsou et al, 2015), FT-ARM (Weisbrid et al, 2012), and PECAN (Ting et al, 2017).

In the MSE method, parent ion scanning is firstly carried out, then, a wider mass section and even all parent ions are put into a collision cavity together for dissociation, a child ion spectrogram is recorded, the parent ions and the child ions of the same analyte have the same characteristics in the aspects of retention time or peak type and the like in a chromatogram, a deconvolution algorithm is used for correlating the parent ions and the child ions, further, the child ion mass spectrogram of a single substance is obtained, and the subsequent qualitative analysis and the quantitative analysis both depend on the child ion mass spectrogram obtained by deconvolution.

In order to further improve the sensitivity and increase the dimension of orthogonal separation, more and more manufacturers are coupling ion mobility based separation devices to chromatograph-tandem mass spectrometry equipment, and since ion mobility based separation devices, such as ion mobility spectrometers (the time scale of the analysis period is, for example, 10MS), and chromatographs (the time scale of the analysis period is, for example, 1200s) and mass spectrometers (the TOF-MS is, for example, 100 μ s), operate at different orders of frequency, they can be effectively matched, connected, and subsequently separated and analyzed.

In some technical solutions in the prior art, for data analysis of a chromatography-ion mobility spectrometry-mass spectrometry system, it is necessary to deconvolute a mass spectrometry data set by using chromatography data and ion mobility spectrometry data, so as to classify daughter ions from the same parent ion.

Specifically, the data set obtained by the chromatography-ion mobility spectrometry-mass spectrometry system at least comprises data of four dimensions of mass-to-charge ratio, retention time, ion mobility and intensity of each daughter ion, and if determining which daughter ions are from the same parent ion, the judgment mode is usually that the daughter ions should have substantially the same retention time and substantially the same ion mobility.

One advantage of the chromatography-ion mobility spectrometry-mass spectrometry system is that it can provide a separation means of ion mobility spectrometry, and further separate partially or completely overlapped peaks in the chromatogram by using the difference of ion mobility properties, thereby improving the identification capability of the parent ion.

In the prior art, a method for performing deconvolution by using chromatographic data and ion mobility spectrometry data generally adopts the following mode, firstly, an intensity-retention time spectrogram of a daughter ion is established without considering the ion mobility spectrometry data, and the chromatographic spectrogram is subjected to deconvolution; secondly, establishing an ion intensity-ion mobility spectrogram aiming at the spectrum peak data of the edge of the set confidence interval by using the retention time for deconvolution operation, and then performing deconvolution on the ion mobility spectrogram; finally, the daughter ions with substantially the same or similar peak pattern and retention time (or daughter ions with substantially the same or similar peak pattern and ion mobility) in each mass-to-charge ratio value or mass-to-charge ratio window are classified into one type, and are determined to be from the same parent ion. Of course, the order of deconvolution for chromatographic data and deconvolution for ion mobility spectrometry data may also be interchanged, and will not be described herein again.

In the process of implementing the present invention, the inventors found that in the prior art, in the process of deconvoluting a mass spectrum data set by using two pieces of data with different dimensions, deconvolution tasks with two different dimensions are performed in sequence, that is, first, data with a first dimension is used to deconvolute the mass spectrum data set, after that, data with another dimension is used to deconvolute data at a critical position of a confidence interval in the deconvolution operation with the first dimension, and finally, results of two deconvolution are synthesized to determine which sub-ion peaks are classified into several types and which sub-ion pairs are classified into which sub-ion pairs respectively.

The above deconvolution method performed successively results in that if the chromatographic peak of a certain daughter ion a is substantially overlapped with the daughter ion B, and the ion mobility spectral peak of the certain daughter ion a is substantially overlapped with the daughter ion C, the daughter ion a cannot be detected in the above process.

Specifically, it is assumed that the following three types of daughter ions are obtained by dissociation in a certain analysis process of a chromatography-ion mobility spectrometry-mass spectrometry system, and are named as lona, lonb, and lonc:

	retention time	Peak pattern of chromatogram	Ion mobility	Peak type of mobility spectrum
					IonA	R1	TypeA1	M1	TypeA2
IonB	R1	TypeA1	M2	TypeB
					IonC	R2	TypeC	M1	TypeA2

From the above analysis, it can be known that lona, lonb and lonc belong to different types of daughter ions, and ideally should be assigned to different parent ions. However, when the deconvolution method in the prior art is adopted, the retention time and the chromatographic peak pattern of the IonA and the IonB are the same, the chromatographic peaks of the two are superposed to present a single peak, and the whole three ions present double peaks on the chromatographic spectrum; similarly, the mobility spectrum peaks of the ion nA and the ion nC are superposed to form a single peak, and the whole three ions also form double peaks on the ion mobility spectrum. Because the chromatogram and the ion mobility spectrum both show double peaks, no matter which dimension data is used for deconvolution of a mass spectrum data set, the existing step-by-step deconvolution method can only identify two ions from the mass spectrum data set, so that certain types of daughter ions, such as ion A, are hidden in the deconvolution process, and the ion identification capability of the system is influenced.

Disclosure of Invention

In view of the above problems, the present invention provides a mass spectrometry method capable of identifying ions having a portion that is heavily overlapped with the peaks of other ions, for example, a certain chromatographic peak is substantially overlapped with a daughter ion B, and an ion mobility spectral peak is substantially overlapped with a daughter ion C, so as to improve the qualitative and quantitative abilities of analyzing data acquired independent of data.

The mass spectrometry method comprises the following steps: a first separation step of separating the samples based on the difference in the first physicochemical characteristic to obtain a plurality of sets of intermediates; a second separation step of separating the intermediate or the ionized intermediate based on the difference in the second physicochemical characteristic to obtain a plurality of groups of parent ions; a dissociation step of dissociating at least a part of the parent ions to generate a plurality of daughter ions corresponding to the dissociated parent ions; a detection step of detecting at least the intensity of the daughter ions generated in the dissociation step; in the course of implementing the method of mass spectrometry, intensity data of the daughter ions, a first parameter of the daughter ions associated with the first physicochemical property, and a second parameter of the daughter ions associated with the second physicochemical property are all recorded, thereby forming a spectrogram dataset, wherein the method of mass spectrometry further comprises a deconvolution step of deconvolving the spectrogram dataset according to a two-dimensional feature comprising the first parameter and the second parameter, so as to classify the daughter ions from the same parent ion.

Through the above manner, because the two-dimensional feature including the first parameter and the second parameter is used in the deconvolution process, and the two-dimensional feature includes two-dimensional parameters at the same time, when deconvolution is performed on lona, lonb and lonc in the background art, it can be determined that lona, lonb and lonc should be assigned to different parent ions according to different two-dimensional features, so that ions of the type lona, which have serious ion peak overlap with other ions (lonb and lonc) and can be hidden in the analysis process of the prior art, can be accurately classified in the deconvolution process of the present invention, and qualitative capability and quantitative capability of analyzing data for data non-dependency acquisition are improved.

In an optional technical solution of the present invention, in the detecting step, the intensities of the plurality of groups of parent ions are also detected.

In an alternative embodiment of the present invention, the detecting step includes stopping or reducing the dissociation energy applied to the parent ions, thereby detecting the intensities of the plurality of groups of parent ions.

In an optional technical scheme of the invention, the deconvolution step comprises a heat map generation step, wherein a coordinate system is established by using a first parameter and a second parameter based on a spectrogram data set to generate a plurality of heat maps, and each heat map is associated with a mass-to-charge ratio or a mass-to-charge ratio range of daughter ions; and clustering analysis, namely clustering the three-dimensional features which occupy the same first parameter range and the same second parameter range in different heat maps and are matched with each other according to a clustering analysis model.

In an optional technical scheme of the invention, the method further comprises a clustering model training step, and the clustering analysis model is trained according to the scores of the clustering results in the clustering analysis step.

In an optional technical scheme of the invention, the method further comprises an MS/MS spectrum generation step, wherein an MS/MS spectrum of the clustered daughter ions is generated based on the mass-to-charge ratio or the mass-to-charge ratio range corresponding to the heat map in which the clustered feature distribution is located; and a searching and matching step, namely searching a pre-established database according to the corresponding relation between the MS/MS spectrum generated in the MS/MS spectrum generating step and the parent-child ions, and performing substance qualification on the parent ions.

In an optional technical solution of the present invention, in the MS/MS spectrum generating step, the intensity data of the clustered sub-ions in the heat map is integrated, so as to determine the intensity of the sub-ions corresponding to the mass-to-charge ratio or the mass-to-charge ratio range in the MS/MS spectrum.

In an optional technical scheme of the invention, in the detection step, a data-independent acquisition method is adopted for acquiring the intensity data of the daughter ions.

The present invention also provides a mass spectrometry system comprising: a first separation unit for separating the sample based on the difference of the first physicochemical characteristic to obtain a plurality of sets of intermediates; a second separation unit for separating the intermediate or the ionized intermediate based on the difference in the second physicochemical characteristic to obtain a plurality of groups of parent ions; the dissociation unit is used for dissociating at least one part of parent ions, and the dissociated parent ions correspondingly generate a plurality of daughter ions; the mass spectrometry system can record the intensity data of the daughter ions, a first parameter of the daughter ions associated with the first physicochemical characteristic, and a second parameter of the daughter ions associated with the second physicochemical characteristic, thereby forming a spectrogram dataset, and further comprises a processor for deconvoluting the spectrogram dataset according to a two-dimensional feature comprising the first parameter and the second parameter, so as to classify the daughter ions from the same parent ion.

In an optional technical scheme of the invention, the first separation unit is a chromatograph, and the second separation unit is an ion mobility spectrometer.

In an optional technical scheme of the invention, the ion mobility spectrometer is one or a combination of several of a migration tube ion mobility spectrometer, a field asymmetric waveform ion mobility spectrometer, a traveling wave ion mobility spectrometer, a respiratory ion mobility spectrometer, a cascade capture ion mobility spectrometer and a U-shaped ion mobility spectrometer.

In an alternative aspect of the invention, an ion mobility spectrometer comprises: the ion introducing device is used for introducing the intermediate into the post-stage device or temporarily storing the received intermediate and simultaneously releasing the intermediate to the post-stage device; and ion mobility separation means, disposed downstream of the ion introduction means, for receiving the intermediate and separating the intermediate during movement according to a difference in ion mobility.

In an alternative embodiment of the present invention, the dissociation unit is a collision chamber, a photo dissociation chamber, or an electron capture dissociation chamber.

In an optional aspect of the invention, the dissociation unit and the detection unit are integrated into the same ion trap mass analyzer.

In an optional aspect of the invention, the detection unit comprises a first mass analyser and a second mass analyser, the first mass analyser is a quadrupole mass analyser, an ion trap mass analyser or a time of flight mass analyser, and the second mass analyser is a quadrupole mass analyser, a time of flight mass analyser or a fourier transform type mass analyser.

Drawings

FIG. 1 is a schematic diagram of a mass spectrometry system in accordance with an embodiment of the present invention;

FIG. 2 is a block diagram of a portion of the mass spectrometry system of the embodiment of FIG. 1;

FIG. 3 is a flow chart of a method of mass spectrometry according to the embodiment of FIG. 1;

FIG. 4 is a data analysis flow of a deconvolution process in the mass spectrometry method of the embodiment of FIG. 1;

FIG. 5 is a schematic representation of a two-dimensional heat map obtained for the analysis of IonA, IonB and IonC by the mass spectrometry method of the embodiment of FIG. 1.

Reference numerals: 100-chromatograph, 102-ionization device, 104-ion mobility spectrometer, 105-mass spectrometer; 106-a first mass analyser; 108-dissociation unit, 110-second mass analyzer; 112-processor.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

[ terms and explanations ] to

It should be noted that the term "deconvolution" is used herein in a broad sense, not in a mathematical sense, as a deconvolution operation. The deconvolution process in this context is a process of defining parent-child ion pairs, i.e., a process of mining additional information hidden in existing data information from existing information.

In this context, deconvolution is performed using "two-dimensional features", in other words, two-dimensional deconvolution, that is, using original data to cluster a data set without parent-child ion pair tags in a defined domain including, but not limited to, a first physical property and a second physical property by a clustering algorithm, so as to obtain a definite parent-child ion correspondence.

The present embodiment provides a chromatography-ion mobility spectrometry-mass spectrometry system 1(LC-IMS-MS), as shown in fig. 1 and fig. 2, the chromatography-ion mobility spectrometry-mass spectrometry system 1 mainly includes three device modules, namely a chromatograph 100, an ion mobility spectrometer 104, and a mass spectrometer 105.

The chromatograph 100 is a liquid chromatograph, that is, the first separation unit in the present embodiment, and is capable of separating a sample based on a difference in a first physicochemical characteristic of the sample (retention time by a column), separating the mixture sample into a plurality of groups of intermediates, and obtaining chromatographic data by chromatographic separation as first characteristic data associated with the retention time (separation time), and recording the retention time of the passing analyte, and the first parameter (retention time parameter, RT) associated with the retention time of each ion can be further inferred by using a correspondence relationship between the chromatographic data and a subsequently obtained mass spectrum data time series or peak appearance position.

The effluent from the chromatograph, i.e., the plurality of sets of intermediates, is ionized by ionization apparatus 102 and sent to ion mobility spectrometer 104.

Ion mobility spectrometer 104, i.e., the second separation unit in the present embodiment, can secondarily separate components in the sample, which are difficult to be effectively separated according to retention time, by using the difference in ion mobility. The data obtained by ion mobility spectrometer 104 is second characteristic data associated with ion mobility, such as ion mobility spectrometry data. The ion mobility spectrum data records ion mobility data of various passing parent ions, such as ion mobility or collision cross-sectional area, and the second parameter (ion mobility, M) related to the ion mobility of each ion can be further deduced by using the corresponding relation between the ion mobility spectrum data and the subsequently obtained mass spectrum data time sequence or peak position.

Next, the parent ions obtained by the secondary separation by the ion mobility spectrometer 104 are introduced into the mass spectrometer 105, the mass spectrometer 105 can dissociate the parent ions, and mass spectrometry is performed on the dissociated parent ions, and a plurality of mass spectrograms are obtained by using a data acquisition method of data-independent acquisition, thereby forming a mass spectrometric data set.

During the analysis of the mass spectral data set, the mass spectral data set may be deconvolved from the chromatographic data of chromatograph 100 and the ion mobility spectral data of ion mobility spectrometer 104. Since the daughter ions from the same parent ion have the same retention time and ion mobility and similar or identical peak patterns, the chromatographic data, ion mobility spectrometry data, can effectively assist the sorting task of the daughter ions, which is used to classify the daughter ions from the same parent ion into one class.

Specifically, in the present embodiment, the chromatograph 100, the ion mobility spectrometer 104, and the mass spectrometer 105 are provided in this order along the transmission flow path of the sample or sample ions, the chromatograph 100 is provided upstream of the transmission flow path of the sample or sample ions, the mass spectrometer 105 is provided downstream of the transmission flow path of the sample or sample ions, and the ion mobility spectrometer 104 is provided between the chromatograph 100 and the mass spectrometer 105.

An ionization device 102 is arranged between the chromatograph 100 and the ion mobility spectrometer 104, and an effluent (intermediate) which is subjected to primary separation by the chromatograph 100 is ionized into an ion beam, transmitted by an ion optical device, and sent into the ion mobility spectrometer 104 for secondary separation. Chromatograph 100 and ion mobility spectrometer 104 have different duty cycles or operating frequencies, the typical analysis duration for separation of a liquid mixture by chromatograph 100 typically varies from minutes to hours, and the operating duration for a single cycle of ion mobility spectrometer 104 is in the millisecond to second scale, so that ion mobility spectrometer 104 can be used to add separation in a second dimension before ions are sent to mass spectrometer 105, allowing efficient separation of chromatograph 100 via ion mobility spectrometer 104 based on components that are difficult to completely separate by retention time.

It should be noted that the depiction of the positional relationship of the chromatograph 100, the ion mobility spectrometer 104, and the mass spectrometer 105 in fig. 1 is merely illustrative, and does not constitute a strict limitation on the positions of the chromatograph 100, the ion mobility spectrometer 104, and the mass spectrometer 105 in the mass spectrometry system. In other embodiments, the chromatograph 100, the ion mobility spectrometer 104, and the mass spectrometer 105 may be integrated, and the relative positional relationship between each of them may be exchanged. For example, in the present embodiment, sample ions are separated by the ion mobility spectrometer 104 and introduced into the mass spectrometer 105 to perform operations such as ion mass selection, dissociation, and detection, but this position selection and matching is merely exemplary, and in other embodiments, ions may be first mass-selected by the first mass analyzer 106 of the mass spectrometer 105 and then the mass-selected ions may be transferred to the ion mobility spectrometer 104 of the next stage.

While the first separation apparatus is exemplified as a general liquid chromatograph in the present embodiment, in other embodiments, a high performance liquid chromatograph, an ultra high performance liquid chromatograph, or a gas chromatograph may be used as the first separation apparatus, and the liquid chromatograph may be based on a discharge volume chromatograph, an ion exchange chromatograph, or a pH gradient chromatograph. Furthermore, the first separation apparatus may further comprise a capillary electrophoresis separation apparatus; capillary electrophoresis chromatographic separation equipment; a hard ceramic based multilayer microfluidic separation device; a permeation gel chromatography separation device; or supercritical fluid chromatographic separation equipment.

[ ionizing device ]

The intermediate obtained via chromatographic separation is passed into an ionization device 102 for ionization, the ionization device 102 comprising an ion source selected from the group consisting of: (i) an electrospray ionization ("ESI") ion source; (ii) an atmospheric pressure photoionization ("APPI") ion source; (iii) an atmospheric pressure chemical ionization ("APCI") ion source; (iv) a matrix-assisted laser desorption ionization ("MALDI") ion source; (v) a laser desorption ionization ("LDI") ion source; (vi) an atmospheric pressure ionization ("API") ion source; (vii) a desorption ionization on silicon ("DIOS") ion source; (viii) an electron impact ("EI") ion source; (ix) a chemical ionization ("CI") ion source; (x) A field ionization ("FI") ion source; (xi) A field desorption ("FD") ion source; (xii) An inductively coupled plasma ("ICP") ion source; (xiii) A fast atom bombardment ("FAB") ion source; (xiv) A liquid secondary ion mass spectrometry ("LSIMS") ion source; (xv) An electrospray desorption ionization ("DESI") ion source; (xvi) A source of nickel-63 radioactive ions; (xvii) An atmospheric pressure matrix-assisted laser desorption ionization ion source; (xviii) A thermal spray ion source; (xix) An atmospheric sampling glow discharge ionization ("ASGDI") ion source; (xx) A glow discharge ("GD") ion source; (xxi) An impactor ion source; (xxii) A real-time direct analysis ("DART") ion source; (xxiii) A laser spray ionization ("LSI") ion source; (xxiv) A sonic spray ionization ("SSI") ion source; (xxv) A matrix-assisted inlet ionization ("MAII") ion source; (xxvi) A solvent assisted inlet ionization ("SAII") ion source; (xxvii) Penning (Penning) ionizing ion source; (xxviii) A laser ablation electrospray ionization ("LAESI") ion source; (xxix) He plasma (HePl) ion source. Preferably, an electrospray desorption ionization ("DESI") ion source, a matrix-assisted laser desorption ionization ion source ("MALDI"), a real-time direct analysis ion source ("DART"), a laser ablation electrospray ionization ("LAESI") ion source plasma chamber pressure, or a real-time ion source is used.

Although the ionization device 102 is disposed between the first separation device (chromatograph 100) and the second separation device (ion mobility spectrometer 104) before the mass spectrometer 105 in this embodiment, the arrangement position and manner are merely illustrative, and in other embodiments, the relative positions of the ionization device 102 and the arrangement of the first separation device and the second separation device may be adjusted according to actual needs, and for example, when the separation of the first separation device needs to be performed on an ion sample, the ionization device 102 may be disposed before the first separation device.

[ ion mobility Spectroscopy ]

The ion mobility spectrometer 104 may employ a mobility tube ion mobility spectrometer (DTIMS), a Field asymmetric waveform ion mobility spectrometer (FAIMS), also known as a differential ion mobility spectrometer (DMS), a traveling-wave ion mobility spectrometer (TW-IMS), a respiratory ion mobility spectrometer (AIMS), a Tandem-Trapping Ion Mobility Spectrometer (TIMS), a U-type ion mobility spectrometer (a), for example, using the device structure provided in CN109003876A, or any other suitable form of ion mobility spectrometer.

In some embodiments, ion mobility spectrometer 104 includes an ion introduction device for introducing an intermediate to a subsequent device, or for temporarily storing a received intermediate and simultaneously releasing the intermediate to a subsequent device; and ion mobility separating means, provided downstream of the ion introducing means, for receiving the intermediate and separating the intermediate in the course of movement according to a difference in ion mobility.

[ Mass Spectrometry ]

Mass spectrometer 105 in this embodiment may be any mass spectrometer 105 capable of providing accurate mass measurements for the daughter ion peaks and performing data-independent acquisitions.

In this embodiment, mass spectrometer 105, in series with ion mobility spectrometer 104 operating in ion mobility filter mode, comprises a quadrupole mass analyser, a collision cell and a time-of-flight mass analyser in series. A quadrupole mass analyzer disposed at a front stage of the collision cell as a first mass analyzer 106 of the mass spectrometer 105; a collision cell disposed between the quadrupole mass analyzer and the time-of-flight mass analyzer, and configured to dissociate the passing ions as a dissociation unit 108; and a time-of-flight mass analyzer disposed at the rear stage of the collision cell as a second mass analyzer 110 of the mass spectrometer. The first mass analyzer 106 and the second mass analyzer 110 together constitute a detection unit in the present embodiment.

In some embodiments, the collision cell and the second mass analyzer 110 may also be integrated in the same mass analyzer, such as an ion trap mass analyzer. The ion trap mass analyzer can integrate multiple functions of ion transportation, mass selection, dissociation and the like, and selects ions with a specific mass-to-charge ratio or within a mass-to-charge ratio range from received or stored ions for dissociation by using a simple device structure, so that the ions can be subjected to multi-time cascade analysis on a time dimension, and more detailed information is provided for structure analysis of compounds. For example, in some embodiments, a quadrupole-in-series configuration with an ion trap mass analyzer may also be used for tandem analysis. [ Collision tank ]

In the mass spectrometer 105 provided in the present embodiment, in order to achieve independent acquisition of data, a collision cell that can vary the dissociation energy is used to control the degree to which ions passing through the collision cell are dissociated. Specifically, under low dissociation energy, ions passing through the collision cell are not dissociated or are dissociated to a low degree, so that spectrogram information of the parent ions can be acquired at the later stage of the collision cell; when the collision cell operates under high dissociation energy, ions passing through the collision cell can be dissociated to a higher degree, and by adjusting the dissociation energy, the spectrogram information of the daughter ions can be acquired at the later stage of the collision cell. The collision cell is set to be periodically switched between high-low dissociation energy, and spectrogram information of the parent ions and spectrogram information of the daughter ions can be respectively obtained at different time intervals in one period and used as the basis of a classification task between the parent ions and the daughter ions.

The dissociation unit 108 may be selected from the group consisting of: (i) a collision induced dissociation ("CID") fragmentation device; (ii) a surface induced dissociation ("SID") fragmentation device; (iii) an electron transfer dissociation ("ETD") fragmentation device; (iv) an electron capture dissociation ("ECD") fragmentation device; (v) an electron impact or shock dissociation fragmentation device; (vi) a light-induced dissociation ("PID") fragmentation device; (vii) a laser-induced dissociation fragmentation device; (viii) an infrared radiation induced dissociation device; (ix) an ultraviolet radiation induced dissociation device; (x) A nozzle-skimmer cone (skimmer) interfacial fragmentation device; (xi) An in-source fragmentation device; (xii) An in-source collision induced dissociation fragmentation device; (xiii) A thermal or temperature source fragmentation device; (xiv) An electric field induced fragmentation device; (xv) A magnetic field induced fragmentation device; (xvi) An enzymatic digestion or degradation fragmentation device; (xvii) An ion-ion reactive fragmentation device; (xviii) An ion-molecule reaction fragmentation device; (xix) An ion-atom reaction fragmentation device; (xx) An ion-metastable ion reactive fragmentation device; (xxi) An ion-metastable molecule reaction fragmentation device; (xxii) An ion-metastable atom reaction fragmentation device; (xxiii) An ion-ion reaction device for reacting ions to form an adduct or product ions; (xxiv) An ion-molecule reaction device for reacting ions to form an adduct or product ions; (xxv) An ion-atom reaction device for reacting ions to form an adduct or product ion; (xxvi) Ion-metastable ion reaction means for reacting ions to form an adduct or product ion; (xxvii) Ion-metastable molecule reaction means for reacting ions to form an adduct or product ion; (xxviii) Ion-metastable atom reaction means for reacting ions to form an adduct or product ion; and (xxix) electron ionization dissociation ("EID") fragmentation devices.

In the low dissociation mode, the voltage applied to the collision cell is reduced to adjust the dissociation energy to a relatively low value, and in some embodiments, the magnitude of the voltage applied to the collision cell is selected from the group consisting of: less than or equal to 5V; less than or equal to 4.5V; less than or equal to 4V; less than or equal to 3.5V; less than or equal to 3V; less than or equal to 2.5V; less than or equal to 2V; less than or equal to 1.5V; less than or equal to 1V; 0.5V or less or substantially 0V.

In the high dissociation mode, the voltage applied to the collision cell is increased to adjust the dissociation energy to a relatively high value, and in some embodiments, the magnitude of the applied voltage on the collision cell is selected from the group consisting of: more than or equal to 10V; more than or equal to 15V; more than or equal to 20V; more than or equal to 30V; not less than 40V; more than or equal to 50V; more than or equal to 100V; not less than 150V; more than or equal to 200V.

[ quadrupole mass analyzer ]

As a first mass analyser 106 of the mass spectrometer, a quadrupole mass analyser is used to select ions of a suitable mass to charge ratio value or range of mass to charge ratios to enter the collision cell for dissociation.

In this embodiment, when a mass spectrum with low dissociation energy needs to be obtained, for example, a mass spectrum of a parent ion, the collision cell may be set to operate in a low dissociation energy state, and the quadrupole mass analyzer may be used to perform scanning within the full mass-to-charge ratio range, or all ions may be guided to pass through the second mass analyzer to select the mass-to-charge ratio, so as to obtain the mass spectrum of the parent ion. When a mass spectrum with high dissociation energy needs to be obtained, for example, a sub-ion spectrum, the collision cell may be set to operate in a high dissociation energy state, and the full mass-to-charge ratio range of the ions may be scanned in a manner of selecting a plurality of mass-to-charge ratio windows step by step (stepwise), so as to obtain a plurality of sub-ion mass spectra. In this embodiment, one parent ion spectrogram and a plurality of child ion spectrograms can be obtained in each working cycle of the mass spectrometer, and in other embodiments, the number of spectrograms acquired in each working cycle can also be flexibly selected according to actual needs, and is not limited to the above manner. The parent ion spectrum and the child ion spectrum are combined to form a mass spectrum data set for operation processing by the processor 112.

It should be noted that, although the present embodiment is performed on both the parent ion spectrum and the daughter ion spectrum, in some embodiments, only the daughter ion spectrum may be acquired, and in the subsequent data analysis process, the mass spectrum data set may be clustered, daughter ions having the same or similar characteristics may be clustered, and determined to be from the same parent ion, and the identification of the species of the parent ion and the establishment of correspondence between the parent ion and the daughter ion may be selectively performed in the data analysis process.

In the data-independent acquisition mode, in most cases, each mass-to-charge ratio window contains a plurality of parent ion peaks, and if the parent ion peaks and the child ion peaks in the child ion spectrogram can be definitely assigned and paired, mass spectrum data containing dissociation information corresponding to an analyte can be obtained through analysis, and information such as a chemical formula, relative molecular mass, conformation, configuration and the like of the analyte can be identified by searching and comparing the mass spectrum data from the existing mass spectrum database.

In the present embodiment, the quadrupole mass analyzer can adjust the mass-to-charge ratio in a stepwise (stepwise) manner. Specifically, different mass-to-charge ratio windows may be sequentially selected from a plurality of mass-to-charge ratio windows formed by dividing the full mass-to-charge ratio range until the full mass-to-charge ratio range is covered. The order of selecting the mass to charge ratio windows may be from large to small or from small to large.

For analytical tasks where the analyte is predominantly a small mass ion, such as metabolomic analysis, the full mass to charge ratio range typically corresponds to a mass to charge ratio in the range of about 100-600; for analytical tasks where the analyte is primarily a polypeptide, such as proteomic analysis, the full mass to charge ratio range typically corresponds to a mass to charge ratio in the range of about 400-1400.

[ Mass-to-Charge ratio Window ]

The selected or isolated ions have mass-to-charge ratio values distributed within a specified range, i.e., the corresponding mass-to-charge ratio window. In some embodiments, each mass-to-charge ratio window may contain 5 consecutive mass-to-charge ratios, i.e., the maximum and minimum values of the range differ by 5 mass-to-charge ratios, in other words, a mass-to-charge ratio window of "+/-2.5 Dalton". The widths of the multiple mass-to-charge ratio windows are variable within the full mass-to-charge ratio range and can also be kept consistent. There may be partial overlap between multiple mass-to-charge ratio windows or no overlap at all.

In this embodiment, the ion release of the ion mobility spectrometer is synchronized with the mass analysis of the mass spectrometer. Specifically, in the present embodiment, the ion mobility spectrometer performs ion release within a certain ion mobility or ion gas phase collision cross-sectional area range, and the mass analyzer performs one or more scans of mass-to-charge ratio range synchronously, such as a scan including a full mass-to-charge ratio range of parent ions and a scan of daughter ions for a plurality of mass-to-charge ratio windows within the full mass-to-charge ratio range.

[ time-of-flight mass analyser ]

The time-of-flight mass analyzer, which is the second mass analyzer 110 of the mass spectrometer in the present embodiment, is mainly used for analyzing parent/daughter ions. The accelerated ions are separated in a time-of-flight mass analyzer according to mass-to-charge ratio and arrive at a detector in sequence, and the detector records the mass spectrum of the ions. Wherein the ion mass spectrum recorded in low dissociation mode is a low dissociation spectrum, e.g. as a parent ion spectrum; the ion mass spectrum recorded in high dissociation mode is taken as the high dissociation spectrum, for example as the daughter ion spectrum.

In some embodiments of the present invention, the first and second mass analyzers 106, 110 may be selected from the group consisting of: (i) a quadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) paul or 3D quadrupole mass analyser; (iv) a penning trap mass analyzer; (v) an ion trap mass analyser; (vi) a sector magnetic field mass analyzer; (vii) an ion cyclotron resonance ("ICR") mass analyzer; (viii) a fourier transform ion cyclotron resonance ("FTICR") mass analyzer; (ix) an electrostatic mass analyser arranged to generate an electrostatic field having a quadrupole-logarithmic potential distribution; (x) A Fourier transform electrostatic mass analyzer; (xi) A Fourier transform mass analyzer; (xii) A time-of-flight mass analyzer; (xiii) An orthogonal acceleration time-of-flight mass analyser; and (xiv) a linear acceleration time-of-flight mass analyser.

In this embodiment, the first mass analyser 106 is a quadrupole mass analyser, and in other embodiments of the invention, the first mass analyser 106 is preferably configured as a time-of-flight mass analyser, a magnetic mass analyser or another suitable type of mass analyser, provided that it is capable of continuously selecting ions of different mass to charge ratio windows to pass through, thereby completing a scan of a selected mass to charge ratio range or a full mass to charge ratio range.

In the present embodiment, the second mass analyzer 110 is a time-of-flight mass analyzer, and in other embodiments, the form of the second mass analyzer 110 is not limited thereto, and for example, a quadrupole mass analyzer, a magnetic mass analyzer, a fourier transform type mass analyzer, or any other suitable type of mass analyzer may be preferably used.

In some embodiments, at least a portion of the first mass analyzer 106, the dissociation unit 108, and the second mass analyzer 110 may also be integrated in the same ion trap mass analyzer, for example, using the same ion trap mass analyzer to achieve dissociation and analysis of ions to reduce device size while providing detailed structural information of more compounds.

[ control procedure ]

The mass spectrometry system 1 provided in this embodiment can be applied to qualitative or quantitative analysis of metabonomics, proteomics, and the like, and specifically, when analyzing analytes in a mixture form, it can provide separation in three orthogonal dimensions for mixed analytes, including separation in retention time dimension of a chromatogram, separation in ion mobility dimension of an ion mobility spectrum, and separation in mass-to-charge ratio dimension of a mass spectrum, so that the characteristics that parent ions and daughter ions of the same analyte have the same efflux peak pattern, retention time, or ion mobility in the chromatogram and the ion mobility spectrum are utilized to perform mathematical deconvolution, and the parent ions and the daughter ions are corresponded to identify the type and content of each component in the mixed analytes.

The mass spectrometry method shown in fig. 3 is adopted for controlling different modules, and specifically, the mass spectrometry method provided by the present embodiment includes the following steps:

s1 a first separation step of separating the samples based on the difference in the first physicochemical characteristic to obtain a plurality of sets of intermediates.

In this embodiment, the samples are separated based on the difference in retention time of each component in the sample passing through the chromatographic column, and a plurality of sets of intermediates are obtained as the effluent of the chromatography.

S2 second separation step, separating the intermediate or ionized intermediate based on the difference in the second physico-chemical property, to obtain a plurality of sets of parent ions.

In the present embodiment, a sample is separated by the chromatograph 100 and then introduced into the ion mobility spectrometer 104 to be separated for the second time, and the ion mobility spectrometer 104 performs separation based on a difference in ion mobility (or physicochemical characteristics such as ion gas phase collision cross-sectional area), and obtains characteristic data related to ion mobility, for example, spectrum data of an ion mobility spectrum.

It should be noted that the process of ionizing the sample is not limited to occur after the first separation step of S1, but may occur before the first separation step of S1, in other words, the technical solution of ionizing the sample before the first separation step of S1, and still should be included in the protection scope of the present invention as long as the technical solution does not depart from the gist of the present invention.

In this embodiment, in the second separation step S2, the second separation means is operated in the filter mode, and the second separation means is operated in the filter mode to screen out and release ions having an ion mobility within a predetermined range from the received second separated matter.

S3 dissociation step, dissociating at least a portion of the parent ions, and generating a plurality of daughter ions corresponding to the dissociated parent ions.

S4 detecting step of detecting at least the intensity of the product ions generated in the dissociation step.

Dissociation is performed for the sample ions flowing out after the processing of step S2, and mass spectrometric detection is performed for daughter ions obtained after the dissociation. In step S4, mass spectrometer 105 performs mass spectrometry on the dissociated daughter ions, but mass spectrometry is not limited to mass spectrometry on only the daughter ions, and mass spectrometry on parent ions that have not been dissociated or have a low dissociation degree may be performed in some embodiments, if necessary.

In this embodiment, in the mass spectrometry step, the parent ions are analyzed by cooperation between a first mass analyzer 106 and a second mass analyzer 110 disposed in the mass spectrometer 105, and a dissociation unit 108 is disposed between the first mass analyzer 106 and the second mass analyzer 110, wherein the first mass analyzer 106 is configured to select ions in the entire mass-to-charge ratio range or ions in a partial mass-to-charge ratio range to be introduced into the dissociation unit 108, and the second mass analyzer 110 acquires mass spectrometry data of the daughter ions generated by the dissociation unit 108.

In other embodiments of the present invention, an ion trap mass analyzer may also be employed to collect parent ion mass spectrometry data and daughter ion mass spectrometry data obtained from tandem mass spectrometry, wherein tandem mass spectrometry is repeated a plurality of times in succession.

The steps of S1, S2, S3 are performed for a sample mixed with a plurality of components according to a transport path of the sample, thereby completing the separation of the sample and the data recording process in synchronization during the analysis. In some embodiments, since the execution frequency of the S1, S2 and S3 steps repeated periodically is not the same, within the execution period of one separation step, another separation step may be executed multiple times. For example, in the execution cycle of one S1 step, the S2 step may be executed a plurality of times, in other words, in the process of performing chromatographic separation and recording one chromatographic spectrum, a plurality of ion mobility spectrum separations may be performed and a plurality of ion mobility spectrum spectra may be recorded; in the process of performing ion mobility spectrometry separation and recording one ion mobility spectrometry spectrogram, multiple times of mass spectrometry separation can be performed, and multiple mass spectrometry spectrograms can be recorded.

In some embodiments, before the dissociation step of S3, a charge state determining step of determining a charge state of the parent ions according to a relationship between a mass-to-charge ratio of the parent ions and an ion mobility; and a parent ion screening step of selecting a parent ion having a predetermined charge state to pass and dissociate based on the charge state determined in the charge state determining step. The selection mode can be a functional relation established according to the first parameter, the second parameter and/or the mass-to-charge ratio parameter, and through the selection mode, the target type parent ions can be further screened out, the dissociation and mass spectrometry of the parent ions can be performed more specifically, and the qualitative and quantitative performance of the mass spectrometry is improved.

S5 deconvolving, according to the two-dimensional features including the first parameter and the second parameter, the spectrogram dataset to classify the daughter ions from the same parent ion.

In this embodiment, the step of deconvoluting S in S5 specifically includes the following steps:

s51 heat map generation step, based on the mass spectrum data set, establishing a coordinate system by the first parameter and the second parameter, and generating a plurality of heat maps, wherein each heat map is associated with the intensity distribution of the daughter ions with one mass-to-charge ratio or one mass-to-charge ratio range;

in the step of S51 heat map generation, mass spectral data of a kind of collected mass-to-charge ratio or a range of mass-to-charge ratios of daughter ions is summarized in a heat map established by using ion mobility M and retention time RT as coordinate systems. In this embodiment, the mass to charge ratio range is three mass to charge ratios in size, and therefore, each thermal map will integrate the mass spectral data of the daughter ions over three mass to charge ratio ranges.

Referring to fig. 3, because the daughter ions from the same parent ion should have substantially the same retention time and substantially the same ion mobility, these daughter ions having the same or similar retention time and ion mobility will be adjacent to each other in the thermal map, forming a "signature spot" occupying a range of retention times and a range of ion mobilities upon integration. The color gradation of a "feature spot" characterizes the ion intensity for the corresponding mass-to-charge ratio or the ion intensity integral for the corresponding mass-to-charge ratio range for the corresponding location in the retention time and ion mobility coordinate system.

In S51 heat map generation step, ions within a certain mass-to-charge ratio range may be selected and combined to generate a heat map, and the size of the mass-to-charge ratio range may be preset or may be customized by the user.

In the present embodiment, the process of classifying the daughter ions is performed by a feature matching (for example, pattern recognition) process for the "feature patch", and specifically, in order to classify the daughter ions from the same parent ion into one, the following steps are performed:

and S52, clustering analysis, namely clustering the characteristics which occupy the same first parameter range and the same second parameter range in different heat maps and are matched with each other according to the clustering model.

Although the daughter ions from the same parent ion are dissociated to different degrees, they should correspond to the parent ion having the same retention time and ion mobility in time sequence, and the chromatographic peak pattern and the ion mobility spectrum peak pattern corresponding to these daughter ions should be similar, in other words, the three-dimensional morphology of the "feature spot" obtained by combining the chromatographic peak pattern and the ion mobility spectrum peak pattern (the peak pattern in three dimensions of ion mobility-retention time-intensity) should also be similar. In the embodiment, the clustering analysis is carried out by utilizing the similarity of the three-dimensional form of the characteristic spots, the clustering accuracy can be effectively improved, and the clustering analysis can be adapted to the relatively mature characteristic matching technology at present, so that the method has good popularization and application prospects.

In the cluster analysis step of S52, the conditions set for the similarity of the parameter ranges and the matching of the "feature patches" in the cluster conditions may be set to be substantially the same or substantially the same, that is, the determination of the matching may be set to allow a certain amount of error, for example, the similarity may be higher than a threshold.

In some embodiments of the present invention, the clustering algorithm may further be implemented based on the following steps:

s521, information area division is carried out on a two-dimensional plane formed by a first physical property (such as retention time) and a second physical property (such as ion mobility), and information area blocks required by the subsequent steps are selected;

s522, characterizing the data in the selected communicated region blocks, and constructing characteristic parameters for bearing region block information;

s523, boundary characteristic division is carried out according to the obtained characteristic parameter set;

and S524, classifying the characteristic parameter sets in the same dividing unit into the same type of parent-child ion relation.

And S53MS/MS spectrum generation step, based on the mass-to-charge ratio or mass-to-charge ratio range corresponding to the heat map where the clustered 'feature spots' are located, generating the MS/MS spectrum of the clustered sub-ions, for example, the MS/MS spectrum can be constructed according to the clustering condition of the region block information in the S521 step.

Specifically, if it is found in the search process for different heat maps that the same or substantially the same "feature spot" appears at similar positions in some heat maps, for example, in fig. 4, the "feature spot" appears in m/z 164-.

In order to further identify the type of the parent ion, one way is to compare the mass spectrum generated by dissociation of the parent ion with mass spectrum data stored in the existing database to distinguish the type of the parent ion.

The comparison of mass spectrum spectrogram data needs to be based on the coincidence of peak position and peak intensity data, wherein the preliminary resolution mode of the peak position is described above and is not described herein again (if a specific mass-to-charge ratio is to be determined, the daughter ion from which mass-to-charge ratio the "feature spot" originates may be further searched).

In the mass spectrum, each mass-to-charge ratio is stronger than the peak of the daughter ion, in this embodiment, the "volume under the surface" may be used to solve the "characteristic spot" in the thermal map, that is, the intensity data of the daughter ion covered in the characteristic spot is integrated to obtain the peak strength data corresponding to the "characteristic spot", and the peak strength data may be used as the peak strength data corresponding to the daughter ion in the mass spectrum after homogenization processing.

Through the mode, the peak position and the peak intensity data can be obtained through analysis, and a mass spectrum spectrogram corresponding to the characteristic spot can be generated according to the peak position and the corresponding peak intensity data.

After the generation of the mass spectrum spectrogram of each characteristic spot is finished, executing the following steps:

and S54 searching and matching, namely searching a pre-established database according to the corresponding relation between the MS/MS spectrum generated in the S53MS/MS spectrum generation step and the parent-child ions, and performing substance qualification on the parent ions.

By searching a pre-established database, the type of the parent ions can be determined so as to meet qualitative requirements; in addition, because the peak intensity data of each group of daughter ions can be solved through integration, the concentration ratio of each group of daughter ions determined through searching can also be determined through calculating the ratio of the peak intensity data, and the quantitative requirement of omics analysis can also be met.

In some embodiments, the clustering model may be further trained according to the score of the clustering result, and specifically, the mass spectrometry method further includes: and a step of training a clustering model, namely training the clustering analysis model according to the scores of the clustering results in the step of S52 clustering analysis. The clustering model is continuously optimized through a machine learning algorithm, so that the ion identification capability can be effectively improved.

In the above manner, for the lona, lonb and lonc described in the background art, three different ions will present a distribution state as shown in fig. 5 on the heat map, and when deconvoluting for three feature spots, because of the three feature spots, different ranges are occupied on the two-dimensional heat map, the daughter ions are classified according to the feature spots, the lona can be clearly distinguished from the lonb and lonc ions, and different parent ions are searched from the database to correspond to the ion, so that the qualitative and quantitative capability of data analysis is improved.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.