A mass spectrometry data correction method based on multiple ion matching

Abstract

The application belongs to the field of proteomics, and particularly relates to a mass spectrum data correction method based on multiple ion matching; comprising: simulating fragmentation of protein sequences of a to-be-tested protein according to ion types and calculating theoretical ion mass; performing deconvolution processing on mass spectrum data of the to-be-tested protein to obtain experimental ion mass; pairing the theoretical ion mass with the experimental ion mass to obtain all matching ion pairs; performing filtering and deduplication processing on the matching ion pairs to obtain processed matching ion pairs; calculating average mass errors of all the matching ion pairs; correcting the mass spectrum data according to the average mass errors to obtain corrected mass spectrum data; repeatedly performing the correction step until the average mass errors between all the matching ion pairs after deduplication are less than a mass error threshold, and obtaining corrected mass spectrum data; the application can use smaller ion matching errors in the ion matching process, and ensures accurate matching of ions and accurate identification of subsequent proteins.

Description

Technical Field

[0001] This invention belongs to the field of proteomics, specifically relating to a mass spectrometry data correction method based on multiple ion matching. Background Technology

[0002] Proteomics is the study of all proteins from cells to tissues in a specific organism, aiming to determine the expression, quantification, modification, and mutation states of all proteins within the organism. Mass spectrometry is the most important method in proteomics research, characterized by high sensitivity and high throughput. In a mass spectrometer, protein ion fragments are represented by the mass-to-charge ratio and ionic intensity. The accuracy of the mass-to-charge ratio measurement greatly affects the accuracy of ion mass measurement, and consequently, the accuracy of subsequent protein identification. Therefore, improving the accuracy of mass spectrometry instruments is one way to improve the accuracy of protein identification. Currently, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) can achieve a resolution of one million, enabling accurate measurement of the ion mass-to-charge ratio (mass-to-charge ratio) of protein ion fragments. However, the accumulation of ions in the ion trap of the mass spectrometer can cause a leftward or rightward shift in the measured mass-to-charge ratio data, leading to errors in subsequent protein identification. In particular, when there are multiple ions with similar masses in the mass spectrometry data, such as ions with the same sequence containing phosphorylation modification (79.9663 Da) and sulfonation modification (79.9568 Da), distinguishing between these two ions requires a lower ion matching error. When the mass spectrum shifts, using a low ion matching error may result in no matching or matching of the wrong ion, making it difficult to distinguish between these ions with similar masses. This hinders the study of protein post-translational modifications and protein function.

[0003] To prevent mass spectrometry data shifts caused by systematic errors in mass spectrometers, researchers have proposed standard instrument calibration protocols and lock-mass-based correction methods. However, the standard instrument calibration protocol method requires frequent instrument calibration during mass spectrometry experiments to maintain relative stability. In reality, it is impossible to keep a mass spectrometer consistently stable and without deviation. The lock-mass-based correction method requires adding standard samples with known molecular weights during protein sample preparation. Then, during protein identification, the mass of the known molecules is continuously determined. By calculating the deviation between the measured mass of the known molecules and the theoretical mass, the systematic error of the mass spectrometer can be estimated, thus correcting the mass spectrometry data. This method places higher demands on the algorithms for sample preparation and subsequent data processing. Furthermore, the correction relies on the continuous identification of standard molecules in the mass spectrometry data. If the standard molecules cannot be identified in the mass spectrometer due to low intensity interference from noise, this correction method will fail.

[0004] In summary, although existing methods can reduce the systematic error of mass spectrometers, they still have the following drawbacks:

[0005] (1) Methods based on standard instrument calibration protocols require frequent manual calibration of the mass spectrometer, which reduces the throughput of protein identification, and instrument calibration cannot keep the mass spectrometer from shifting indefinitely.

[0006] (2) The lock mass-based correction method makes sample preparation and subsequent identification algorithms more complex, posing a greater challenge to protein identification algorithms after sample preparation techniques. Standard samples may fail to be identified due to noise and other issues, which poses a risk of this method failing. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention proposes a mass spectrometry data correction method based on multiple ion matching, which includes:

[0008] S1: Simulate fragmentation of the protein sequence of the protein to be tested according to the ion type and calculate the theoretical ion mass; obtain the mass spectrometry data of the protein to be tested;

[0009] S2: Deconvolution of the mass spectrometry data to obtain the experimental ion mass;

[0010] S3: Pair the theoretical ion mass with the experimental ion mass to obtain all matched ion pairs; filter the matched ion pairs to obtain filtered matched ion pairs;

[0011] S4: Perform deduplication on the filtered matched ion pairs to obtain deduplicated matched ion pairs;

[0012] S5: Calculate the average mass error of all matched ion pairs after deduplication;

[0013] S6: Correct the mass spectrometry data based on the average mass error to obtain the corrected mass spectrometry data;

[0014] S7: Repeat steps S2 to S6 until the average mass error between all matched ion pairs after deduplication is less than the mass error threshold, and obtain the corrected mass spectrometry data.

[0015] Preferably, in step S1, the process of simulating protein fragmentation of the protein sequence to be tested and calculating the theoretical ionic mass includes:

[0016] The N-terminal amino acids of the protein sequence are fixed, and the peptide bonds at the C-terminus of the protein sequence are theoretically digested to obtain the first protein subsequence. The theoretical mass of the first protein subsequence is calculated. Based on the theoretical mass of the first protein subsequence, the theoretical ion masses after N-terminal ion fragmentation in the corresponding mass spectrometry dissociation modes are calculated sequentially.

[0017] The C-terminal amino acids of the protein sequence are fixed, and the peptide bonds at the N-terminus of the protein sequence are theoretically digested to obtain the second protein subsequence. The theoretical mass of the second protein subsequence is calculated. Based on the theoretical mass of the second protein subsequence, the theoretical ion mass after C-terminal ion fragmentation in the corresponding mass spectrometry dissociation mode is calculated.

[0018] Preferably, in step S3, the process of filtering the matched ion pairs includes: calculating the mass error between the two ions in the matched ion pair and retaining matched ion pairs with an absolute mass error of less than or equal to 20 ppm.

[0019] Furthermore, the formula for calculating the mass error between the two ions is as follows:

[0020]

[0021] Here, Error represents the mass error between the two ions, A represents the experimental ion mass, and B represents the theoretical ion mass.

[0022] Preferably, in step S4, the process of deduplicating the filtered matched ion pairs includes: sorting all matched ion pairs present in an experimental ion according to the absolute value of their mass error, retaining the matched ion with the smallest absolute value of its mass error, and removing the other matched ion pairs.

[0023] Preferably, in step S6, the formula for correcting the mass spectrometry data is:

[0024]

[0025] Where mz1 represents the corrected peak mass-charge ratio, and mz2 represents the uncorrected peak mass-charge ratio. avg This represents the average quality error.

[0026] Preferably, in step S7, the quality error threshold is 2 ppm.

[0027] The beneficial effects of this invention are as follows: This invention directly and rapidly corrects the mass spectrometry data shift by estimating the system error of the mass spectrometer through multiple ion matching, making the mass spectrometry data shift less than 2 ppm; it avoids frequent manual calibration of the mass spectrometer and avoids the sample preparation process and identification of known molecules. This invention does not have the risk of failure and can prevent inaccurate ion mass measurement caused by the mass spectrometer system error and mass spectrometry data shift. In addition, it can use a smaller ion matching error in the ion matching process to perform more accurate matching of ions with similar masses, ensuring accurate ion matching and subsequent accurate identification of proteins. Attached Figure Description

[0028] Figure 1 This is a flowchart of the mass spectrometry data correction method based on multiple ion matching in this invention. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention proposes a mass spectrometry data correction method based on multiple ion matching, such as... Figure 1 As shown, the method includes the following:

[0031] S1: Simulate fragmentation of the protein sequence of the protein to be tested according to the ion type and calculate the theoretical ion mass; obtain the mass spectrometry data of the protein to be tested.

[0032] Obtain the protein sequence of the protein to be tested; process the protein to be tested using a mass spectrometer to obtain the mass spectrometry data of the protein to be tested.

[0033] The theoretical ion mass is calculated based on the protein sequence, specifically:

[0034] The N-terminal amino acids of the protein sequence are fixed, and the peptide bonds at the C-terminus of the protein sequence are theoretically digested to obtain the first protein subsequence. The theoretical mass of the first protein subsequence is calculated. Based on the theoretical mass of the first protein subsequence, the theoretical ion masses after N-terminal ion fragmentation in the corresponding mass spectrometry dissociation modes are calculated sequentially.

[0035] The C-terminal amino acids of the protein sequence are fixed, and the peptide bonds at the N-terminus of the protein sequence are theoretically digested to obtain the second protein subsequence. The theoretical mass of the second protein subsequence is calculated. Based on the theoretical mass of the second protein subsequence, the theoretical ion mass after C-terminal ion fragmentation in the corresponding mass spectrometry dissociation mode is calculated.

[0036] Save all theoretical ionic masses generated from the protein sequence as a list of theoretical ionic masses.

[0037] S2: Deconvolve the mass spectrometry data to obtain the experimental ion mass.

[0038] Deconvolve the mass spectrometry data using any deconvolution software to obtain the mass of the experimental ions present in the mass spectrometry data and save it as a list of experimental ion masses.

[0039] S3: Pair the theoretical ion mass with the experimental ion mass to obtain all matched ion pairs; filter the matched ion pairs to obtain filtered matched ion pairs.

[0040] The theoretical ion mass is paired with the experimental ion mass. Specifically, the experimental ion mass list is iterated, and each experimental ion is paired with each theoretical ion in the theoretical ion mass list. After the iteration is completed, all possible pairing results are obtained, that is, all matched ion pairs are obtained.

[0041] Filtering of matched ion pairs involves: calculating the mass error between the two ions in the matched ion pair and retaining matched ion pairs with an absolute mass error value less than or equal to 20 ppm. The formula for calculating the mass error between the two ions is as follows:

[0042]

[0043] Here, Error represents the mass error between the two ions, A represents the experimental ion mass, and B represents the theoretical ion mass.

[0044] S4: Perform deduplication on the filtered matched ion pairs to obtain deduplicated matched ion pairs.

[0045] For an experimental ion, all matching ion pairs are sorted according to the absolute value of their mass error. The matching ion with the smallest absolute mass error is retained, and other matching ion pairs are discarded. All experimental ions are deduplicated to obtain all deduplicated matching ion pairs. For example, an experimental ion has a deconvolution mass of 10000 Da in the mass spectrometry data. Within a 20 ppm error range, it matches two theoretical ions with masses of 9999.9 Da and 9999.8 Da, respectively. According to the formula in step S3, the mass errors between the experimental ion and the two theoretical ions are 10 ppm and 20 ppm, respectively. The theoretical ion with the larger mass error is deleted, and the theoretical ion with the smaller error is retained as the sole matching result for the experimental ion.

[0046] S5: Calculate the average mass error of all matched ion pairs after deduplication.

[0047] Calculate the average of all quality errors to obtain the average quality error.

[0048] S6: Correct the mass spectrometry data based on the average mass error to obtain the corrected mass spectrometry data.

[0049] The mass spectrometry data are corrected based on the average mass error to compensate for the data shift caused by the mass spectrometer system error. The mass-charge ratio correction formula is as follows:

[0050]

[0051] Where mz1 represents the corrected peak mass-charge ratio, and mz2 represents the uncorrected peak mass-charge ratio. avg This represents the average quality error.

[0052] S7: Repeat steps S2 to S6 until the average mass error between all matched ion pairs after deduplication is less than the mass error threshold, and obtain the corrected mass spectrometry data.

[0053] Repeat steps S2 to S6 until the average mass error between all matched ion pairs after deduplication is less than the mass error threshold; preferably, the mass error threshold is 2 ppm.

[0054] After the iteration is completed, the corrected mass spectrometry data is obtained, and the corrected mass spectrometry data is saved for subsequent data processing.

[0055] In summary, this invention provides a rapid and stable correction of mass spectrometry data shifts by estimating mass spectrometry system errors through multiple ion matching. This reduces the need for manual mass spectrometry correction, eliminates the need for additional sample preparation, and prevents inaccurate ion mass measurements caused by mass spectrometry data shifts due to mass spectrometry system errors. Consequently, it allows for the use of smaller ion matching errors during the ion matching process, enabling more accurate matching of ions with similar masses, thus ensuring accurate ion matching and subsequent accurate protein identification.

[0056] The above-described embodiments further illustrate the purpose, technical solution, and advantages of the present invention. It should be understood that the above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made to the present invention within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mass spectrometry data correction method based on multiple ion matching, characterized in that, include: S1: Simulate fragmentation of the protein sequence of the protein to be tested according to the ion type and calculate the theoretical ion mass; obtain the mass spectrometry data of the protein to be tested; S2: Deconvolution of the mass spectrometry data to obtain the experimental ion mass; S3: Pair the theoretical ion mass with the experimental ion mass to obtain all matched ion pairs; filter the matched ion pairs to obtain filtered matched ion pairs; S4: Perform deduplication on the filtered matched ion pairs to obtain deduplicated matched ion pairs; S5: Calculate the average mass error of all matched ion pairs after deduplication; S6: Correct the mass spectrometry data based on the average mass error to obtain the corrected mass spectrometry data; S7: Repeat steps S2 to S6 until the average mass error between all matched ion pairs after deduplication is less than the mass error threshold, and obtain the corrected mass spectrometry data.

2. The mass spectrometry data correction method based on multiple ion matching according to claim 1, characterized in that, In step S1, the process of simulating protein fragmentation of the protein sequence to be tested and calculating the theoretical ionic mass includes: The N-terminal amino acids of the protein sequence are fixed, and the peptide bonds at the C-terminus of the protein sequence are theoretically digested to obtain the first protein subsequence. The theoretical mass of the first protein subsequence is calculated. Based on the theoretical mass of the first protein subsequence, the theoretical ion masses after N-terminal ion fragmentation in the corresponding mass spectrometry dissociation modes are calculated sequentially. The C-terminal amino acids of the protein sequence are fixed, and the peptide bonds at the N-terminus of the protein sequence are theoretically digested to obtain the second protein subsequence. The theoretical mass of the second protein subsequence is calculated. Based on the theoretical mass of the second protein subsequence, the theoretical ion mass after C-terminal ion fragmentation in the corresponding mass spectrometry dissociation mode is calculated.

3. The mass spectrometry data correction method based on multiple ion matching according to claim 1, characterized in that, In step S3, the process of filtering the matched ion pairs includes: calculating the mass error between the two ions in the matched ion pair and retaining matched ion pairs with an absolute mass error value of less than or equal to 20 ppm.

4. The mass spectrometry data correction method based on multiple ion matching according to claim 3, characterized in that, The formula for calculating the mass error between two ions is: Here, Error represents the mass error between the two ions, A represents the experimental ion mass, and B represents the theoretical ion mass.

5. The mass spectrometry data correction method based on multiple ion matching according to claim 1, characterized in that, In step S4, the process of deduplicating the filtered matched ion pairs includes: sorting all matched ion pairs present in an experimental ion according to the absolute value of their mass error, retaining the matched ion with the smallest absolute value of its mass error, and removing the other matched ion pairs.

6. The mass spectrometry data correction method based on multiple ion matching according to claim 1, characterized in that, In step S6, the formula for correcting the mass spectrometry data is: Where mz1 represents the corrected peak mass-charge ratio, and mz2 represents the uncorrected peak mass-charge ratio. avg This represents the average quality error.

7. The mass spectrometry data correction method based on multiple ion matching according to claim 1, characterized in that, In step S7, the quality error threshold is 2 ppm.

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