CROSS-REFERENCE TO RELATED PATENT APPLICATION

This application claims priority to and the benefit of Chinese Patent Application Serial No. 202010256258.3, filed Apr. 2, 2020, which is incorporated herein in its entirety by reference.

TECHNICAL FIELD

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

BACKGROUND

Currently, high resolution tandem mass spectrometry has become an important analytical instrument for omics analysis (including metabonomics, proteomics, and the like). For complex samples in the omics analysis, a mass spectral data acquisition method with high throughput, high sensitivity and high coverage is needed. A conventional method is data dependent acquisition (DDA) proposed by Ducret et al. in 1998, in which parent ions (or “precursor” ions) are scanned, and then parent ions with a relative high abundance are selected and sequentially enter a collision chamber for dissociation and obtain a daughter ion (or “product” ion) spectrogram. The method can achieve higher analyte coverage, and thus is still a widely adopted acquisition method.

However, since the DDA method can only monitor daughter ion information in one mass-to-charge ratio channel of the parent ions during each daughter ion scanning, utilization efficiency and throughput of the parent ions are low when tandem mass spectrometry analysis is performed, and many parent ions with a lower abundance are still not monitored when a large amount of analytes elute from a chromatographic column at the same time. Meanwhile, since the mass-to-charge ratio channel of the parent ions corresponding to daughter ion scanning events in each cycle is constantly changing, it cannot be guaranteed that the daughter ions of the analytes are detected more uniformly for several times in a chromatographic elution time, and thus a quantitative analysis can be performed only by using an ion current chromatogram of the parent ions of the analytes instead of an ion current chromatogram of the daughter ions, thereby affecting selectivity and accuracy of the quantitative analysis in the omics analysis. Since the parent ions with a higher abundance are preferably selected each time the parent ions are selected for the tandem mass spectrometry analysis, and relative abundances of the ions in the same batch of samples can vary among different injection analyses, and thus this kind of difference will lead to randomness of parent-ion monitoring, and the randomness may cause deviations or errors in biostatistics analysis.

In recent years, data independent acquisition (DIA) has developed rapidly due to a higher sensitivity, a higher dynamic range, a higher analysis throughput, and a better quantitative feature compared to the DDA approach. The DIA method is represented by an MSE method described in the U.S. Pat. No. 6,717,130 of Micromass and an SWATH (sequential-window acquisition of all theoretical mass spectra) method described in the U.S. Pat. No. 8,809,770 of AB Sciex.

The SWATH method is mainly directed to targeted analysis, so that instead of performing a parent-ion pre-scanning, the parent ions usually are directly segmented according to a mass number window, for example, each segment is 25 Da, then each segment of parent ion selected by a quadrupole is introduced into a collision chamber together for dissociation, a daughter-ion spectrogram is recorded and compared to a database, and a daughter-ion intensity is used for quantification. Therefore, and it is often used for targeted quantitative analysis, and is not suitable for qualitative analysis. The SWATH method has non-negligible deficiencies as follows. On the one hand, the SWATH method relies too much on the establishment of a high-quality daughter-ion spectrogram database and the matching of experimental conditions; on the other hand, it is difficult to implement untargeted metabolomics analysis. In recent years, alternatives to the SWATH method that do not rely on the spectrogram database have also gained long-term development, such as DIA-Umpire (Tsou et al., 2015), FT-ARM (Weisbrod et al., 2012), and PECAN (Ting, et al., 2017).

In the MSE method, a parent-ion scanning is performed first, then a wider mass segment or even all the parent ions are put into the collision chamber for dissociation, a daughter-ion spectrogram is recorded, and a deconvolution algorithm is used to correlate a parent ion and a daughter ion to further obtain a daughter-ion mass spectrogram of a single substance according to that the parent ion and the daughter ion of the same analyte have the same features in aspects of a retention time or a peak shape in a chromatogram. The subsequent qualitative and quantitative analyses depend on the daughter-ion mass spectrogram obtained by deconvolution.

In order to further improve the sensitivity and increase dimensions of orthogonal separation, more and more manufacturers are coupling ion mobility-based separation devices to a chromatographic-tandem mass spectrometry device. Since the ion mobility-based separation devices, such as an ion mobility spectrometry instrument (e.g., a time scale of an analysis cycle is 10 ms), and the chromatogram (e.g., a time scale of an analysis cycle is 1200 s), mass spectrum (e.g., TOF-MS is 100 μs) work at different magnitudes of frequencies, these devices can effectively cooperate and connect with each other for cascading separation and analysis.

In some technical solutions in the prior art, with respect to data analysis of a chromatogram-ion mobility spectrum-mass spectrum system, it is necessary to deconvolute a mass spectrometry data set by using chromatographic data and ion mobility spectrum data to categorize the daughter ions from the same parent ion.

Specifically, a data set obtained by the chromatogram-ion mobility spectrum-mass spectrum system contains at least data of four dimensions including mass-to-charge ratio, retention time, ion mobility, intensity for each of the daughter ions. To determine which of the daughter ions origin from the same parent ion, a determination method is generally that these daughter ions should have basically the same retention time and ion mobility.

The chromatogram-ion mobility spectrum-mass spectrum system has one advantage that it is possible to provide a separation means of ion mobility spectrum and further separate peaks that are partially or completely overlapped in the chromatogram by using different ion mobility properties, thereby improving the identification capability of the parent ions.

In the prior art, the method for deconvolution using the chromatographic data and the ion mobility spectrum data is generally adopted in the following manner. Firstly, an intensity-retention time spectrum of the daughter ions is established and a chromatographic spectrum is deconvoluted without considering the ion mobility spectrum data; then, as to spectral peak data at the edge of a set confidence interval using the retention time for deconvolution, an intensity-ion mobility spectrum of the daughter ions is established, and the ion mobility spectrum is deconvoluted; finally, the daughter ions with basically the same or similar peak shape and retention time (or the daughter ions with basically the same or similar peak shape and ion mobility) in each mass-to-charge ratio value or mass-to-charge ratio window are clustered into one category and determined to come from the same parent ion. Of course, a sequential order of deconvolution for the chromatographic data and deconvolution for the ion mobility spectrum data may also be interchanged, and the details will not be repeated herein.

In the process of implementing the present invention, the inventor found that the prior art has at least the following problems. In the prior art, in the process of deconvoluting a mass spectrometry data set by using two data of different dimensions, two deconvolution tasks of different dimensions are performed sequentially, that is, the data of the first dimension is used to deconvolute the mass spectrometry data set until completion; then the data of the other dimension is used to deconvolute the data at a critical position of a confidence interval in the first dimension deconvolution operation; finally, results of the two deconvolutions are merged to determine which category these daughter ion peaks should be clustered to and which parent-daughter ion pairs these daughter-ion peaks should be assigned to respectively.

The above sequentially performed deconvolution methods result in that if a chromatographic peak of some daughter ion A basically overlaps with a daughter ion B while an ion mobility spectrum peak thereof (daughter ion A) in turn basically overlaps with a daughter ion C, then the daughter ion A basically cannot be detected in the process described above.

Specifically, assume that the following three types of daughter ions are obtained by dissociation in a certain analysis process of the chromatogram-ion mobility spectrum-mass spectrometry system, which are IonA, IonB, and IonC respectively:

RetentionChromatographicIonMobility spectrumTimepeak shapemobilitypeak shapeIonAR1TypeA1M1TypeA2IonBR1TypeA1M2TypeBIonCR2TypeCM1TypeA2

According to the analysis of the above situation, IonA, IonB and IonC belong to different types of daughter ions, and should be assigned to different parent ions under ideal conditions. However, when adopting the deconvolution method in the prior art, since IonA and IonB have the same retention time and chromatographic peak shape, a single peak will appear after overlapping the chromatographic peaks of the two ions, and the three ions will present a double peak on the chromatographic spectrum. Similarly, a single peak will appear after overlapping mobility spectrum peaks of IonA and IonC, and the three ions will also present a double peak on the ion mobility spectrum. Since both the chromatogram and the ion mobility spectrum show double peaks, no matter any data of the above dimensions is used to deconvolute the mass spectrometry data set firstly, the existing stepwise deconvolution methods can only identify two types of ions therefrom. As a result, certain types of daughter ions, such as IonA, are hidden during the deconvolution process, which affects the ion discrimination ability of the system.

SUMMARY

In view of the above problems, the present invention provides a mass spectrometry analysis method capable of identifying ions that partially overlap spectral peaks of other ions significantly, take a certain daughter ion A as an example, in which a chromatographic peak of the daughter ion A overlaps with a daughter ion B, and an ion mobility spectrum peak of the daughter ion A overlaps with a daughter ion C; and improving the qualitative and quantitative ability of data analysis for data independent acquisition.

The mass spectrometry analysis method includes: a first separation step, separating a sample to obtain multiple groups of intermediates based on differences of a first physicochemical property; a second separation step, separating intermediates or ionized intermediates to obtain multiple groups of parent ions based on differences of a second physicochemical property; a dissociation step, in which at least part of the parent ions are dissociated, and the dissociated parent ions correspondingly generate multiple daughter ions; a detection step, in which at least an intensity of a daughter ion generated in the dissociation step is detected, during the implementation of the mass spectrometry analysis method, intensity data of 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 to form a spectrogram data set; and a deconvolution step, in which the spectrum data set is deconvoluted to categorize the daughter ions from the same parent ion according to the two-dimensional features including the first parameter and the second parameter.

Through the above methods, the deconvolution process uses two-dimensional features that include the first parameter and the second parameter at the same time. Therefore, when performing deconvolution for IonA, IonB, and IonC in the background art, it can be determined that IonA, IonB, and IonC should be assigned to different parent ions according to the difference in two-dimensional features, so that ions like IonA that significantly overlaps with ion peaks of other ions (IonB, IonC) and may be hidden in the resolution process of the prior art can also be accurately clarified in the deconvolution process of the present invention, thereby improving the qualitative and quantitative ability of data analysis for data independent acquisition.

In an alternative technical solution of the present invention, intensities of multiple groups of parent ions are also detected in the detection step.

In an alternative technical solution of the present invention, the detection step includes stopping or reducing dissociation energy applied to the parent ions, so as to detect the intensities of multiple groups of parent ions.

In an alternative technical solution of the present invention, the deconvolution step includes a heatmap generation step, in which a coordinate system is established with the first parameter and the second parameter to generate multiple heatmaps based on the spectrogram data set, and each heatmap is associated with daughter ions of one type of mass-to-charge ratio or within a mass-to-charge ratio range; and a cluster analysis step, in which three-dimensional features that occupy the same first parameter range and the same second parameter range and match each other in different heatmaps are clustered according to a cluster analysis model.

In an alternative technical solution of the present invention, the mass spectrometry analysis method further includes a cluster model training step, in which the cluster analysis model is trained according to scores of clustering results in the cluster analysis step.

In an alternative technical solution of the present invention, the mass spectrometry analysis method further includes an MS/MS spectrum generation step, in which an MS/MS spectrum of clustered daughter ions is generated based on a mass-to-charge ratio or a mass-to-charge ratio range corresponding to the heatmap where clustered feature distribution is located; and a search matching step, in which a pre-established database is searched for substance identification of the parent ion according to an MS/MS spectrum generated in the MS/MS spectrum generation step and the corresponding relationship between parent ions and daughter ions.

In an alternative technical solution of the present invention, in the MS/MS spectrum generation step, integral is performed with respect to intensity data of the daughter ions that are clustered in the heatmap so as to determine the intensity of the daughter ions corresponding to the mass-to-charge ratio or the mass-to-charge ratio range in the MS/MS spectrum.

In an alternative technical solution of the present invention, in the detection step, the intensity data of the daughter ions are acquired using a data independent acquisition method.

The invention further provides a mass spectrometry system, including: a first separation unit used for separating a sample to obtain multiple groups of intermediates based on differences of the first physicochemical property; a second separation unit used for separating intermediates or ionized intermediates to obtain multiple groups of parent ions based on differences of the second physicochemical property; a dissociation unit used for dissociating at least part of the parent ions, in which the dissociated parent ions correspond to generation of multiple daughter ions; a detection unit at least used for detecting an intensity of a daughter ion generated in the dissociation unit, and the mass spectrometry system can record the intensity data of the daughter ions, the first parameters of the daughter ions associated with the first physicochemical properties, and the second parameters of the daughter ions associated with the second physicochemical properties so as to form a spectrogram data set; and a processor used for deconvoluting the spectrogram data set according to the two-dimensional features including the first parameter and the second parameter, so as to categorize the daughter ions from the same parent ion.

In an alternative technical solution of the present invention, the first separation unit is a chromatographic instrument and the second separation unit is an ion mobility spectrometer.

In an alternative technical solution of the present invention, the ion mobility spectrometer is one or a combination among a drift tube ion mobility spectrometer, a field asymmetric waveform ion mobility spectrometer, a traveling wave ion mobility spectrometer, an aspiration ion mobility spectrometer, a tandem-trapped ion mobility spectrometer, and a U-shape ion mobility spectrometer.

In an alternative technical solution of the present invention, the ion mobility spectrometer includes: an ion introduction device used for introducing the intermediates into a post-stage device, or temporarily storing the received intermediates while releasing the intermediates to the post-stage device at the same time; and an ion mobility separation device that is provided downstream of the ion introduction device to receive the intermediates and separate the intermediates during a moving process according to differences in ion mobility.

In an alternative technical solution of the present invention, the dissociation unit is a collision chamber, a photodissociation chamber, or an electron capture dissociation chamber.

In an alternative technical solution of the present invention, the dissociation unit and the detection unit are integrated into the same ion trap mass analyzer.

In an alternative technical solution of the present invention, the detection unit includes a first mass analyzer and a second mass analyzer, in which the first mass analyzer is a quadrupole mass analyzer, an ion trap mass analyzer, or a time-of-flight mass analyzer, and the second mass analyzer is a quadrupole mass analyzer, a time-of-flight mass analyzer or a Fourier transform mass analyzer.

