Method for determining positive and false positive of polypeptide data in mass spectrometry MRM scan mode

Abstract

The application mainly relates to a method for determining the positivity and false positivity of polypeptide data in a mass spectrometry MRM scanning mode. Polypeptide information of target polypeptides and RT predicted polypeptides in a sample is obtained by a high-resolution mass spectrometer. Then, the target polypeptides and the RT predicted polypeptides are subjected to MRM scanning to obtain polypeptide information and accompanying peak characteristics of the target polypeptides and the RT predicted polypeptides. A short-gradient and high-throughput polypeptide MRM scanning method is established to scan the target polypeptides and the RT predicted polypeptides to obtain polypeptide information and accompanying peak characteristics of the target polypeptides and the RT predicted polypeptides. Finally, the accompanying peak characteristics are combined to find the positive signal of the target polypeptides. The correlation between the target polypeptides and the accompanying peaks under the original LC gradient and the correlation between the predicted target polypeptides and the accompanying peaks in the new MRM method are compared and analyzed to achieve the purpose of quickly and accurately determining the positive signal of the target polypeptides, and the method is suitable for the case that the RT of the target polypeptides cannot be determined in the high-throughput MRM acquisition process.

Description

Technical Field

[0001] This invention relates primarily to the field of mass spectrometry data determination technology, and in particular to a method for determining positive and false positive results for peptide data in mass spectrometry MRM scanning mode. Background Technology

[0002] Triple quadrupole mass spectrometers possess the capability to quantify large quantities of peptides in a short time. Currently, proteomics research has progressed from a non-targeted proteomics stage to a targeted proteomics stage. To improve efficiency, there is a need to transform non-targeted, low-throughput methods into high-throughput, targeted methods. However, during this method transformation, various problems may arise in triple quadrupole mass spectrometer MRM data, such as inaccurate retention time prediction, weak signals making it difficult to correctly identify positive signals, and interference from stray peaks. Therefore, accurate identification of the target peptide is crucial when establishing high-throughput MRM methods.

[0003] The existing SCIEX method for peptide signal scanning via MRM mode using a triple quadrupole mass spectrometer (MIDAS method) is summarized below:

[0004] 1) Data is acquired using a high-resolution mass spectrometer to obtain the retention time and fragment ion information of the target peptide, as well as the retention time and ion fragment information of the RT-predicted peptide (RT-predicted peptides are generally stable, highly expressed proteins encoded by housekeeping genes), and a peptide information database is constructed.

[0005] 2) Analyze the fragment ion information obtained from the high-resolution mass spectrometer, select the 3 to 5 fragment ions with the strongest signals, and establish 3 to 5 MRM ion pairs using the mass-to-charge ratio of the precursor ion of the peptide and the mass-to-charge ratio of these fragment ions.

[0006] 3) Using the same LC gradient as when constructing the database, perform MRM scans on the target peptide and the RT-predicted peptide to obtain the fragment ion peak shape and relative intensity of the fragment ions of the target peptide under the MRM method, as well as the true retention time of the RT-predicted peptide.

[0007] 4) Establish a short-gradient, high-throughput peptide MRM scanning method to scan the target peptide and the RT-predicted peptide. The retention time of the RT-predicted peptide under the short-gradient method can be obtained. Furthermore, a retention time correction curve can be generated to predict the retention time of the target peptide under the short-gradient method.

[0008] 5) By analyzing the predicted retention time of the target peptide, the chromatographic behavior of fragment ions, and the relative strength of fragment ions, the correct target peptide signal can be found, and the true and accurate retention time of the target peptide can be confirmed.

[0009] In establishing high-throughput MRM methods, determining the specific positive signal of the target peptide in short-gradient MRM data is the most challenging aspect. Existing techniques determine positive signals based on the consistency of fragment ion chromatographic behavior (whether they elute simultaneously, reach their peak points simultaneously, and terminate elution simultaneously), the relative strength of fragment ions, and predicted retention times. This method can correctly identify a large number of peptides. However, inaccuracies can sometimes occur. For example, 1. When the target peptide signal is weak, the MRM signal fluctuations of its fragment ions can be affected by background noise and instrument fluctuations, leading to deviations. Even positive signals may exhibit inconsistent chromatographic behavior due to background noise, making accurate identification impossible. 2. Retention time prediction algorithms are applicable to most peptides, but some peptides, due to their unique physicochemical properties or unusual retention times (e.g., actual RT is earlier or later than all RT-predicted peptides), may fail to provide accurate RT predictions. In such cases, traditional methods often require additional information.

[0010] This technical solution provides an additional dimension for identifying positive signals and eliminating false positive signals by introducing criteria for determining the accompanying peaks of the target peptide. This offers a more reliable method for confirming positive signals of the target peptide when establishing high-throughput MRM methods.

[0011] The foregoing background information is intended to help those skilled in the art understand prior art that is similar to the present invention, and to facilitate the understanding of the inventive concept and technical solution of this application. It should be clearly stated that, in the absence of clear evidence that the above content was disclosed before the filing date of this patent application, the foregoing background information should not be used to evaluate the novelty of the technical solution of this application. Summary of the Invention

[0012] To address at least one of the technical problems mentioned in the background section, the present invention aims to provide a method for determining positive and false positive results in peptide data scanned using a triple quadrupole mass spectrometer in MRM mode. By comparing and analyzing the correlation between the target peptide and the accompanying peak under the original LC gradient and the correlation between the predicted target peptide and the accompanying peak in the new MRM method, the method achieves the goal of rapidly and accurately determining the positive signal of the target peptide. This method is applicable to situations where the target peptide RT cannot be determined during high-throughput MRM acquisition.

[0013] Methods for determining positive and false positive results for peptide data in MRM mass spectrometry scanning mode include:

[0014] First, peptide information of the target peptide and RT-predicted peptide in the sample is obtained by high-resolution mass spectrometry, and a peptide information database is constructed.

[0015] Next, using the same LC gradient as when constructing the database, MRM scans were performed on the target peptide and the RT predicted peptide to obtain peptide information and associated peak characteristics of the target peptide and the RT predicted peptide.

[0016] A short-gradient, high-throughput peptide MRM scanning method was re-established to scan the target peptide and the RT-predicted peptide, obtaining peptide information and characteristics of the accompanying peaks of the target peptide and the RT-predicted peptide under short gradient conditions.

[0017] Finally, the positive signal of the target peptide was found by analyzing the predicted retention time of the target peptide, the chromatographic behavior of fragment ions, the relative strength of fragment ions, and the characteristics of accompanying peaks.

[0018] As an optimization of the technical solution of this application, the method for determining positive and false positive results in MRM mode scanning peptide data specifically includes the following steps:

[0019] S1. Obtain peptide information of target peptides and RT-predicted peptides in samples using high-resolution mass spectrometry, and construct a peptide information database.

[0020] S2. Analyze the peak information obtained in step S1, select the 3-5 fragment information with the strongest signal, and establish 3-5 MRM ion pairs using the mass-to-charge ratio of the precursor ion of the polypeptide and the mass-to-charge ratio of these fragment ions.

[0021] S3. Using the same LC gradient as in step S1, perform MRM scanning on the target peptide and the RT predicted peptide to obtain peak information of the target peptide and the RT predicted peptide, as well as the accompanying peak characteristics of the target peptide.

[0022] S4. Establish a short gradient, high-throughput peptide MRM scanning method, scan the target peptide and the RT predicted peptide to obtain the retention time of the RT predicted peptide under the short gradient method, generate a retention time correction curve, and predict the retention time of the target peptide under the short gradient method.

[0023] S5. Find the correct target peptide signal by analyzing the predicted retention time of the target peptide, fragment ion chromatographic behavior, relative strength of fragment ions, and characteristics of accompanying peaks.

[0024] As an optimization of the technical solution of this application, the accompanying peak refers to a peak signal whose retention time is close to that of the target polypeptide and can be distinguished from noise.

[0025] As an optimization of the technical solution of this application, the retention time with the target polypeptide is close to: |RT 目标多肽 -RT 伴随峰 |≤2min.

[0026] As an optimization of the technical solution of this application, the ability to distinguish from noise refers to: intensity 伴随峰 ≥ Twice the average noise level of the instrument.

[0027] As an optimization of the technical solution of this application, the short gradient, high-throughput peptide MRM scanning method uses the same LC gradient as when obtaining sample peak information by high-resolution mass spectrometry.

[0028] As an optimization of the technical solution of this application, the peak information of the target peptide and the RT predicted peptide in the sample includes: the retention time information and fragment ion information of the target peptide and the retention time information and fragment ion information of the RT predicted peptide.

[0029] As an optimization of the technical solution of this application, the short gradient, high-throughput peptide MRM scanning method includes the high-throughput triple quadrupole MRM method.

[0030] As an optimization of the technical solution of this application, the accompanying peak characteristics of the target polypeptide include the retention time and relative intensity of the accompanying peak.

[0031] As an optimization of the technical solution of this application, it also includes verifying that the peak position of the target polypeptide obtained by relabeling isotopes is a positive signal.

[0032] The aforementioned method for determining positive and false positive results of peptide data in the MRM mass spectrometry scanning mode is applied in identifying target peptides in mass spectrometry data.

[0033] As an optimization of the technical solution of this application, the application includes:

[0034] When the predicted retention time of the target peptide deviates significantly, the method described above is used to determine the target peptide in the mass spectrometry data; and / or

[0035] When the signal of the target polypeptide is weak, the method is used to determine the target polypeptide in the mass spectrometry data; and / or

[0036] The method described is used to identify the target peptide in mass spectrometry data when the target peptide signal cannot be distinguished from the noise signal.

[0037] A computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the various processes of the method for determining positive and false positive peptide data in the above-described mass spectrometry MRM scanning mode.

[0038] The beneficial effects of this application are as follows:

[0039] This invention provides an auxiliary method for determining positive signals in peptide data scanned using a high-throughput triple quadrupole mass spectrometer (MRM) method. Combining traditional determination methods, this method helps correctly identify positive signals from complex MRM signals during the establishment of a high-throughput peptide quantitative MRM method using a triple quadrupole mass spectrometer. Based on the target peptide information acquired by the high-resolution mass spectrometer, this method confirms the retention time of the target peptide in triple quadrupole MRM mode. By comparing and analyzing the correlation information between the target peptide and its accompanying peaks under the original LC gradient and the predicted correlation information between the target peptide and its accompanying peaks in the new MRM method, the true signal of the target peptide is determined. Building upon existing techniques that determine the signal based on the chromatographic behavior and relative intensity of different fragment ions, this method introduces the accompanying peak of the target peptide to determine the true signal. The introduction of this accompanying peak determination criterion provides an additional dimension for identifying positive signals and eliminating false positive signals, thus providing a more reliable determination for confirming positive signals of target peptides during the establishment of a high-throughput MRM method. Attached Figure Description

[0040] To make the above and / or other objects, features, advantages and examples of the present invention more apparent and understandable, the accompanying drawings used in the specific embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is the MRM data and MRM chromatogram of a complex sample peptide.

[0042] Figure 2 This is a flowchart of the method for determining positive and false positive results in the mass spectrometry data of this application;

[0043] Figure 3 This is a schematic diagram illustrating the determination of positive and false positive results in mass spectrometry data using the method described in this application when there is a significant deviation in the retention time prediction.

[0044] Figure 4 This is a schematic diagram illustrating the determination of positive and false positive results in mass spectrometry data using the method described in this application when the signal is weak and the traditional method is not reliable enough to determine the target peptide.

[0045] Figure 5 This is a schematic diagram illustrating the determination of positive and false positive results in mass spectrometry data using the scheme described in this application when high-throughput sample collection is performed and the peptide signal in some samples is weak, making the judgment of the target peptide unreliable. Detailed Implementation

[0046] Those skilled in the art can refer to the content of this document and appropriately replace and / or modify the process parameters to achieve the desired results. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in this invention. The products and preparation methods described in this invention have been described through preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0047] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. This invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patent cases, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail.

[0048] Unless otherwise specified, the materials, methods, and examples described herein are exemplary and not limiting. While similar or equivalent methods and materials can be used to implement or test the invention, suitable methods and materials are described herein.

[0049] To facilitate understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention will first be explained or defined.

[0050] RT: Retention time;

[0051] MRM: Multiple reaction monitoring in mass spectrometry;

[0052] PRM: Parallel reaction monitoring;

[0053] MRMhr: High-resolution time-of-flight liquid chromatography-mass spectrometry analysis;

[0054] intensity: strength;

[0055] m / z: mass-to-charge ratio.

[0056] In this application, the definition of the accompanying peak is used in whole or in part to limit the technical solution of this application.

[0057] The present invention will now be described in detail.

[0058] Example 1:

[0059] Define the accompanying peak as a peak signal that has a retention time close to that of the target peptide and can be distinguished from noise. The retention time being close to that of the target peptide means: |RT 目标多肽 -RT 伴随峰 |≤2min, able to be distinguished from noise refers to: intensity 伴随峰 ≥ Twice the average noise level of the instrument; the average noise level of the 4500MD is approximately 300.

[0060] When acquiring MRM / PRM / MRMhr data for a target peptide, complex samples can contain millions of peptides. Therefore, peptides with very similar mass-to-charge ratios often interfere with the target peptide, resulting in accompanying peaks. If certain specific peptides have Q1 / Q3 values ​​very close to the target peptide's MRM Q1 / Q3, their information will be acquired in the target peptide's MRM channel. If the retention times of these specific peptides are close to those of the target peptide, then the peaks generated by these specific peptides are defined as accompanying peaks. Figure 1 For example, the target polypeptide (m / z=600.0) produces three fragment ions, while interfering polypeptides A, B, and C are also collected in the mass spectrometry data during mass spectrometry acquisition because their mass-to-charge ratios are very close. We can then see the stable accompanying peaks A, B, and C. These accompanying peaks come from specific polypeptides that have not been resolved, so the relationship between their retention times and those of the target polypeptide can also serve as a basis for identifying the target polypeptide.

[0061] The criteria for determining accompanying peaks include:

[0062] 1. Retention times are similar, |RT 目标多肽 -RT 伴随峰 |≤2min;

[0063] 2. Can be distinguished from noise, intensity 伴随峰 ≥ Twice the average noise level of the instrument (the average noise level of the 4500MD is approximately 300).

[0064] This embodiment provides a method for determining positive and false positive results in peptide data scanned using a triple quadrupole mass spectrometer in MRM mode. The determination process is as follows: Figure 2 As shown, the specific steps include the following:

[0065] 1) Data acquisition was performed using a high-resolution mass spectrometer to obtain the retention time and fragment ion information of the target peptide, as well as the retention time and ion fragment information of the RT-predicted peptide (RT-predicted peptides are generally stable, highly expressed proteins encoded by housekeeping genes). A peptide information database was then constructed.

[0066] 2) Analyze the fragment ion information obtained by high-resolution mass spectrometry, select 3 to 5 fragment ions with the strongest signals, and establish 3 to 5 MRM ion pairs using the mass-to-charge ratio of the precursor ion of the peptide and the mass-to-charge ratio of these fragment ions.

[0067] 3) Using the same LC gradient as when constructing the database, perform MRM scans on the target peptide and the RT-predicted peptide to obtain the fragment ion peak shape and relative intensity of the fragment ions of the target peptide under the MRM method, as well as the true retention time of the RT-predicted peptide, and obtain the accompanying peak characteristics of the target peptide.

[0068] 4) Establish a short gradient, high-throughput peptide MRM scanning method to scan the target peptide and the RT predicted peptide. The retention time of the RT predicted peptide under the short gradient method can be obtained. Furthermore, a retention time correction curve can be generated to predict the retention time of the target peptide under the short gradient method.

[0069] 5) By analyzing the predicted retention time of the target peptide, the chromatographic behavior of fragment ions, the relative strength of fragment ions, and the characteristics of accompanying peaks, the correct target peptide signal can be found, and the true and accurate retention time of the target peptide can be confirmed.

[0070] Example 2:

[0071] Based on the foregoing embodiments, specific examples are provided to verify that when there is a significant deviation in the retention time prediction, the solution of this application can assist in identifying the correct target peptide, such as... Figure 3 As shown.

[0072] Figure 3 The A subplot shows that the target peptide was observed at RT=7.0 under the original LC gradient; and there was an accompanying peak of a certain fragment ion at 7.0+0.5 min.

[0073] Figure 3 As shown in Figure B, in the new MRM method, the predicted RT for the target peptide is 3.4, but no target peptide was found at 3.4 ± 1 min. A cluster of peaks appeared at RT = 2.1 min, but the reliability was reduced due to the large deviation in RT. However, a companion peak with the same fragment ions appeared at 2.1 ± 0.5 min, thus significantly improving the reliability of the cluster of peaks at 2.1 min as the target peptide.

[0074] Figure 3 The C value in the figure shows that, after verification with relabeled isotopes, the target peptide RT in the new MRM method is 2.1 min. This verifies the feasibility of the method.

[0075] In this embodiment, the predicted RT of the target peptide was 3.4, but in reality, there was no obvious peak shape at 3.4 ± 1 min. The peak position at RT = 2.1 min was much less reliable because it deviated too much from the predicted retention time of the target peptide. However, similar to the accompanying peak at 0.5 min from the target peptide under the original LC gradient, the same fragment ion accompanying peak appeared at RT = 2.1 min. Therefore, it was speculated that this cluster of peaks at RT = 2.1 min might be the target peptide. The correctness of the speculation was verified by relabeling isotopes.

[0076] Example 3:

[0077] Based on the foregoing embodiments, specific examples are provided to verify that when the signal is weak and traditional methods are not reliable enough in identifying the target peptide, the solution of this application can improve the reliability of the target peptide identification, such as... Figure 4 As shown.

[0078] Figure 4 Subplot A shows that the target peptide was observed at RT=13.5 under the original LC gradient; and there was an accompanying peak of a certain fragment ion at 13.5+0.4 min.

[0079] Figure 4 Subfigure B shows that in the new MRM method, the predicted RT for the target peptide is 6.9, with a cluster of peaks at RT=7.0. Due to the weak signal, the peak shapes of this cluster are not uniform, and the relative intensities of the fragment ions differ from those reproduced by the data method (the purple fragment ion signal is unstable). Therefore, the accuracy of this cluster of peaks cannot be completely determined. However, considering the appearance of a companion peak with the same fragment ions as in the database method at 7.0 ± 0.3 min, the probability of the target peptide at RT=7.0 is greatly increased.

[0080] Figure 4 Subgraph C shows that, after relabeling with isotopes, the target peptide in the new method has an RT of 7.0 min. This verifies the feasibility of the method.

[0081] Depend on Figure 4 It can be seen that there is no obvious peak at RT=6.9 min for the predicted target peptide. However, there is a cluster of peaks at RT=7.0 min, but the signal is weak and the relative strength of the fragment ions is different from that of the data method. However, at +0.3 min, there is an accompanying peak of the same fragment ions as the database method. Therefore, it is speculated that the peak at RT=7.0 min may be the target peptide. The correctness of the speculation was verified by re-indication of isotopes.

[0082] Example 4:

[0083] Based on the foregoing embodiments, specific examples are provided to verify that in high-throughput sample collection, when the peptide signal in some samples is weak and traditional methods are not reliable enough in identifying the target peptide, the solution of this application can improve the reliability of target peptide identification. Figure 5 As shown.

[0084] Figure 5 Subplot A shows that during high-throughput sample acquisition, the target peptide was detected at RT=5.2 min; and there was an accompanying peak of a fragment ion at 5.2+0.8 min.

[0085] Figure 5 Subplot B shows that in another sample, at RT=5.2 min, there was only a very weak peak with a low signal-to-noise ratio (S / N) of approximately 2. At this point, it was impossible to confirm whether the peak was a genuine target peptide signal or noise. However, an accompanying peak also appeared at 6.0 min, therefore, the peak at RT=5.2 min was not noise, and the probability of a positive identification was greatly increased.

[0086] Figure 5 Subplot C shows that, after verification with relabeled isotopes, the suspected peak at RT=5.2 min is the target polypeptide, proving the feasibility of this verification method.

[0087] Depend on Figure 5 It is known that during high-throughput sampling, there are many signals with varying strengths. Therefore, traditional methods lack confidence in identifying the target peptide. The peak signal intensity at RT=5.2 min, where the target peptide is predicted, is very weak with an extremely low signal-to-noise ratio, making it difficult to determine whether the peak signal is the target peptide or noise. However, a comparison shows that a strong accompanying peak signal also appears at RT=5.2+0.8 min, greatly increasing the probability that it is the target peptide. The conjecture was verified by re-labeling isotopes.

[0088] In summary, the above-mentioned methods demonstrate that this technique can further improve the reliability of peptide identification, providing a more reliable basis for judgment when there are deviations in retention time prediction or when the target peptide signal is weak and traditional methods are insufficient.

[0089] Example 5:

[0090] A computer-readable storage medium is also provided, on which a computer program is stored. When the computer program is executed by a processor, it implements the various processes of the method for determining positive and false positive results in the mass spectrometry data of the above embodiments, and can achieve the same technical effect. To avoid repetition, it will not be described again here.

[0091] Computer-readable storage media include both permanent and non-permanent, removable and non-removable media, which can store information by any method or technology. Information can be computer-readable instructions, data structures, modules of programs, or other data. Examples of computer storage media include, but are not limited to, phase-change memory (PRAM), static random access memory (SRAM), dynamic random access memory (DRAM), other types of random access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM), flash memory or other memory technologies, CD-ROM, digital versatile optical disc (DVD) or other optical storage, magnetic tape, magnetic magnetic disk storage or other magnetic storage devices, or any other non-transferable medium that can be used to store information accessible by a computing device. As defined herein, computer-readable media does not include transient computer-readable media, such as modulated data signals and carrier waves.

[0092] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be described in detail here.

[0093] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0094] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

[0095] While the foregoing detailed descriptions have shown, described, and pointed out novel features applicable to various embodiments, it should be understood that various omissions, substitutions, and changes may be made to the form and details of the described apparatus or methods without departing from the spirit of this disclosure. Furthermore, the various features and methods described above may be used independently of each other or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of this disclosure. Many of the foregoing embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or applications, as well as their obvious modifications and equivalents. Therefore, the invention is not intended to be limited to the specific disclosure of the preferred embodiments herein.

[0096] All matters not covered in this invention are common knowledge.

Claims

1. A method for determining positive and false positive peptide data in mass spectrometry MRM scanning mode, characterized in that... include: First, high-resolution mass spectrometry was used to obtain peptide information of the target peptide and RT-predicted peptide in the sample, and a peptide information database was constructed. The RT-predicted peptide is a protein that is stably and highly expressed by a housekeeping gene. Next, using the same LC gradient as when constructing the database, MRM scans were performed on the target peptide and the RT predicted peptide to obtain peptide information and accompanying peak characteristics of the target peptide and the RT predicted peptide; the accompanying peak refers to the peak signal that is close to the retention time of the target peptide and can be distinguished from noise. A short-gradient, high-throughput peptide MRM scanning method was re-established to scan the target peptide and the RT-predicted peptide, obtaining peptide information and characteristics of the accompanying peaks of the target peptide and the RT-predicted peptide under short gradient conditions. Finally, the positive signal of the target peptide was found by analyzing the predicted retention time of the target peptide, the chromatographic behavior of fragment ions, the relative strength of fragment ions, and the characteristics of accompanying peaks.

2. The method for determining positive and false positive peptide data in the mass spectrometry MRM scanning mode according to claim 1, characterized in that: Specifically, the following steps are included: S1. Obtain peptide information of target peptides and RT-predicted peptides in samples using high-resolution mass spectrometry, and construct a peptide information database. S2. Analyze the peak information obtained in step S1, select the 3-5 fragment information with the strongest signal, and establish 3-5 MRM ion pairs using the mass-to-charge ratio of the precursor ion of the polypeptide and the mass-to-charge ratio of these fragment ions. S3. Using the same LC gradient as in step S1, perform MRM scanning on the target peptide and the RT predicted peptide to obtain peak information of the target peptide and the RT predicted peptide, as well as the accompanying peak characteristics of the target peptide. S4. Establish a short gradient, high-throughput peptide MRM scanning method, scan the target peptide and the RT predicted peptide to obtain the retention time of the RT predicted peptide under the short gradient method, generate a retention time correction curve, and predict the retention time of the target peptide under the short gradient method. S5. Find the correct target peptide signal by analyzing the predicted retention time of the target peptide, fragment ion chromatographic behavior, relative strength of fragment ions, and characteristics of accompanying peaks.

3. The method for determining positive and false positive peptide data in the mass spectrometry MRM scanning mode according to claim 1, characterized in that: The retention time close to that of the target peptide refers to: |RT 目标多肽 -RT 伴随峰 ≤5min.

4. The method for determining positive and false positive peptide data in the mass spectrometry MRM scanning mode according to claim 1, characterized in that: The ability to be distinguished from noise refers to: intensity 伴随峰 ≥1 times the average noise level of the instrument.

5. The method for determining positive and false positive peptide data in the mass spectrometry MRM scanning mode according to claim 1 or 2, characterized in that: The short gradient, high-throughput peptide MRM scanning method uses an LC gradient time that is shorter than the LC gradient time used when obtaining sample peak information via high-resolution mass spectrometry.

6. The method for determining positive and false positive peptide data in the mass spectrometry MRM scanning mode according to claim 1 or 2, characterized in that: The peak information of the target peptide and the RT-predicted peptide in the sample includes: retention time information and fragment ion information of the target peptide and retention time information and fragment ion information of the RT-predicted peptide.

7. The method for determining positive and false positive results of peptide data in the mass spectrometry MRM scanning mode according to any one of claims 1-6 is used in determining the target peptide in the mass spectrometry data.

8. The application according to claim 7, characterized in that: The applications include: When the predicted retention time of the target peptide deviates significantly, the method described above is used to determine the target peptide in the mass spectrometry data; and / or When the signal of the target polypeptide is weak, the method is used to determine the target polypeptide in the mass spectrometry data; and / or The method described is used to identify the target peptide in mass spectrometry data when the target peptide signal cannot be distinguished from the noise signal.

9. A computer-readable storage medium having a computer program stored thereon, the computer program being executed by a processor to implement various processes of the method for determining positive and false positive peptide data in the mass spectrometry MRM scanning mode according to any one of claims 1-6.

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