Sugar chain molecular weight calibration method and system based on capillary electrophoresis
By combining multi-level standards and internal standards with a cubic polynomial calibration model and dynamic segmented correction, the accuracy and consistency issues of glycan molecular weight calibration in existing technologies have been resolved, achieving high-precision and wide-coverage molecular weight prediction.
Patent Information
- Application Number
- CN202511304440.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-09
AI Technical Summary
Existing technologies for calibrating the molecular weight of sugar chains suffer from problems such as a limited number of standards, a narrow calibration range, poor robustness to system fluctuations, a lack of unified calibration strategies and error control mechanisms, and inconsistent internal standard usage mechanisms, resulting in insufficient calibration accuracy and consistency.
A calibration model with broad coverage was established by using multi-level standards combined with 2-aminobenzamide-labeled maltoheptaose as an internal standard, through relative migration time correction and a cubic polynomial calibration model, combined with dynamic piecewise correction.
It achieves accurate prediction of glycan molecular weight within different migration time ranges, with prediction error controlled within 3%, improving the robustness and repeatability of the calibration method and making it suitable for various experimental platforms.
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Figure CN121090641A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of bioinformatics and glycomics detection technology, and particularly relates to a glycan molecular weight calibration method and system based on capillary electrophoresis. BACKGROUND
[0002] Currently, in the field of glycoprotein drug development, disease-related glycomics research and quality control, the separation analysis and molecular weight determination of glycan structure have become a core link. High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are the current mainstream separation and detection means. The traditional molecular weight calibration method generally uses a linear calibration model based on a small number of glycan standards, that is, a one-to-one correspondence is established between the migration time of the standard and its known GU value, so as to estimate the GU value of the glycan to be measured. However, the existing technology still has obvious deficiencies in the following aspects. For example: the number of standard is limited, the calibration range is narrow: most of the commercial or laboratory self-built linear calibration curves only use the middle interval of 1GU8, and the calibration ability for low-mobility glycan below GU1 and complex high-mobility glycan above GU10 is insufficient, resulting in a significant decrease in calibration accuracy as the GU value increases. Poor robustness to system fluctuations: capillary electrophoresis migration time is easily affected by voltage, buffer concentration, temperature changes and other factors, especially when analyzing across batches or comparing multiple instrument platforms, migration time fluctuations will accumulate errors, and the existing linear model cannot dynamically compensate or correct it. Lack of unified calibration strategy and error control mechanism: the existing technology mainly relies on empirical linear fitting, lacks segmented fine modeling means for low GU and high GU regions, and cannot achieve accurate prediction in the whole interval under a unified model. At the same time, there is a lack of quantifiable evaluation standards for prediction errors, making it difficult to optimize systematically. The use of internal standards is missing or not uniform: some methods do not introduce internal standards for migration time normalization, or do not specify a uniform internal standard variety and theoretical value, resulting in poor calibration consistency between different batches or different operators, and insufficient reproducibility of measurement results.
[0003] Therefore, there is an urgent need for a glycan molecular weight calibration method that can support multiple standard construction, support migration time normalization correction, and have dynamic segmented fitting and error self-checking capabilities, to improve the reliability and accuracy of overall glycomics research and quality control. SUMMARY
[0004] In view of the above technical deficiencies, the purpose of the present application is to provide a sugar chain molecular weight calibration method based on capillary electrophoresis, aiming to solve the technical problem that the calibration method in the prior art only uses a single standard and relies on a linear model, especially under the condition that the migration time fluctuates significantly or the GU value exceeds the linear coverage range, high-precision calibration cannot be achieved.
[0005] To solve the above technical problems, the present application adopts the following technical solutions: The present application provides a sugar chain molecular weight calibration method based on capillary electrophoresis, The sugar chain molecular weight calibration method based on capillary electrophoresis comprises: Step S10: Obtain a multi-level standard set for molecular weight calibration, the multi-level standard set comprising a first standard GU1, a second standard GU2, a third standard GU3 and a fourth standard GU4; and introduce 2-aminobenzamide-labeled maltopentaose as an internal standard; Step S20: Collect the standard migration time of the i-th standard in the multi-level standard set and the sugar chain migration time of the sugar chain to be measured; Collect the internal standard migration time , based on the internal standard migration time and the sugar chain migration time , calculate the relative migration time ; Step S30: Introduce the relative migration time correction factor , based on the relative migration time correction factor , correct the relative migration time to obtain the corrected relative migration time ; obtain a molecular weight set of the multi-level standard from the multi-level standard set, based on the molecular weight set and the corrected relative migration time establish a cubic polynomial calibration model , the cubic polynomial calibration model outputs the predicted value of the molecular weight; Step S40: Perform dynamic segment correction on the cubic polynomial calibration model to obtain an optimized calibration model ; Step S50: Based on the optimized calibration model , predict the molecular weight of the sugar chain to be measured, and output the final predicted value of the molecular weight.
[0006] Preferably, in step S10, the first standard GU1 is selected ; the second standard GU2 selects Man5GlcNAc2; the third standard GU3 selects Man6GlcNAc2; and the fourth standard GU4 selects Neu5Ac2Gal2GlcNAc2Man3GlcNAc2.
[0007] Preferably, in step S20, the relative migration time .
[0008] Preferably, in step S30, the relative migration time , wherein, is a preset theoretical internal standard migration time.
[0009] Preferably, in step S30, the formula of the cubic polynomial calibration model is expressed as:
[0010] , wherein, is a fitting coefficient of the cubic polynomial calibration model, and is obtained by least square fitting based on a molecular weight set including the first standard GU1, the second standard GU2, the third standard GU3, and the fourth standard GU4.
[0011] Preferably, in step S40, the cubic polynomial calibration model is dynamically segmented and corrected to obtain an optimized calibration model , and the step specifically includes: When the cubic polynomial calibration model outputs a molecular weight prediction value in the interval [1, 3], a linear optimization mode is adopted:
[0012] , wherein, and m are a linear model slope parameter and a linear model intercept parameter, respectively, and are obtained by least square fitting based on a molecular weight set of the first standard GU1, the second standard GU2, and the third standard GU3; When the cubic polynomial calibration model outputs a molecular weight prediction value in the interval [3, 15], an exponential optimization mode is adopted:
[0013] , wherein, and are a scale coefficient of an exponential model and a growth rate coefficient of the exponential model, respectively, and are obtained by least square fitting based on a molecular weight set of the third standard GU3 and the fourth standard GU4.
[0014] Preferably, in step S10, the internal standard is used to correct electrophoretic migration time fluctuation.
[0015] The application also provides a sugar chain molecular weight calibration system based on capillary electrophoresis, comprising: a standard and internal standard acquisition module, configured to acquire a multi-level standard set for molecular weight calibration, and introduce 2-aminobenzamide-labeled maltopentaose as an internal standard; a migration time acquisition and calculation module, configured to acquire a standard migration time of an i-th standard in the multi-level standard set and a preset sugar chain migration time of a to-be-tested sugar chain ; acquire an internal standard migration time , calculate a relative migration time based on the internal standard migration time and the sugar chain migration time ; a polynomial modeling module, configured to introduce a relative migration time correction factor , correct the relative migration time based on the relative migration time correction factor , and obtain a corrected relative migration time ; acquire a molecular weight set of the multi-level standard from the multi-level standard set, and establish a cubic polynomial calibration model based on the molecular weight set and the corrected relative migration time , wherein the cubic polynomial calibration model outputs a molecular weight prediction value; a segmented optimization correction module, configured to perform dynamic segmented correction on the cubic polynomial calibration model , and obtain an optimized calibration model ; a result output module, configured to perform molecular weight prediction of the to-be-tested sugar chain based on the optimized calibration model , and output a final molecular weight prediction value.
[0016] The application also provides a sugar chain molecular weight calibration device based on capillary electrophoresis, comprising a memory, a processor, and a sugar chain molecular weight calibration program based on capillary electrophoresis stored in the memory and executable on the processor, wherein the sugar chain molecular weight calibration program based on capillary electrophoresis implements the sugar chain molecular weight calibration method based on capillary electrophoresis when executed by the processor.
[0017] The application also provides a computer program product comprising a sugar chain molecular weight calibration program based on capillary electrophoresis, wherein the sugar chain molecular weight calibration program based on capillary electrophoresis implements the sugar chain molecular weight calibration method based on capillary electrophoresis when executed by a processor.
[0018] The beneficial effects of this invention are as follows: This invention uses multi-level standards to construct a wide-coverage calibration range, and combines internal standard migration time correction and polynomial fitting algorithm to achieve accurate prediction of the molecular weight of sugar chains in different migration time ranges. The prediction error is controlled within 3%, which is significantly better than the traditional linear calibration method.
[0019] This invention effectively counteracts systematic errors caused by changes in voltage, conductivity, and temperature in capillary electrophoresis systems by introducing an internal standard correction mechanism and a dynamic segmented calibration strategy. It is applicable to various experimental platforms and glycomics application scenarios, and improves the robustness and repeatability of the calibration method. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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 based on these drawings without creative effort.
[0021] Figure 1 This is a schematic flowchart of the first embodiment of a glycan molecular weight calibration method based on capillary electrophoresis according to the present invention.
[0022] Figure 2 This is a schematic diagram of the equipment for a glycan molecular weight calibration method based on capillary electrophoresis according to the present invention. Detailed Implementation
[0023] 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.
[0024] Example 1: As Figure 1 The diagram shown is a flowchart of the first embodiment of the glycan molecular weight calibration method based on capillary electrophoresis of the present invention, which presents the first embodiment of the glycan molecular weight calibration method based on capillary electrophoresis of the present invention.
[0025] In the first embodiment, the glycan molecular weight calibration method based on capillary electrophoresis includes: Step S10: Obtain a multi-level standard set for molecular weight calibration, including first standard GU1, second standard GU2, third standard GU3 and fourth standard GU4; and introduce 2-aminobenzamide-labeled maltheptaose as an internal standard; Step S20: Collecting the standard migration time of the i-th standard in the multi-level standard set, respectively and the preset sugar chain migration time of the to-be-tested sugar chain ; collecting the internal standard migration time , based on the internal standard migration time and the sugar chain migration time , calculating the relative migration time ; Step S30: introducing a relative migration time correction factor , based on the relative migration time correction factor , correcting the relative migration time to obtain a corrected relative migration time ; obtaining a molecular weight set of the multi-level standard from the multi-level standard set, based on the molecular weight set and the corrected relative migration time , establishing a cubic polynomial calibration model , the cubic polynomial calibration model outputs a molecular weight prediction value; Step S40: performing dynamic segment correction on the cubic polynomial calibration model to obtain an optimized calibration model ; Step S50: based on the optimized calibration model , performing molecular weight prediction on the to-be-tested sugar chain, and outputting a final molecular weight prediction value.
[0026] It should be noted that the "relative migration time correction factor" in this embodiment refers to a ratio between the theoretical internal standard migration time and the experimentally measured internal standard migration time, which is used to eliminate the system drift error introduced by the capillary electrophoresis system under different batches, different platforms or different experimental conditions; the "multi-level standard set" refers to representative sugar chain structures covering the GU1 to GU4 interval.
[0027] It can be understood that by introducing a correction factor to normalize the relative migration time, not only the comparability of the cross-platform migration time is significantly improved, but also the generalization ability of the molecular weight calibration model under different operating conditions is enhanced, which is helpful for realizing data standardization output in actual glycomics research.
[0028] It should be understood that, compared with the traditional linear fitting which is only applicable to a local GU value interval, the present application significantly expands the coverage ability of the calibration model to a wide GU value interval by constructing a cubic polynomial model and combining a dynamic segment optimization mechanism, especially in the high migration time period, the molecular weight prediction error is still less than 3%, solving the prediction misalignment problem of the traditional method under extreme sugar chain structures.
[0029] Embodiment two: In addition, the present application provides a sugar chain molecular weight calibration system based on capillary electrophoresis, which adopts the sugar chain molecular weight calibration method based on capillary electrophoresis in the above embodiment, and can solve the technical problem of sugar chain molecular weight calibration based on capillary electrophoresis. Compared with the prior art, the beneficial effects of the sugar chain molecular weight calibration system based on capillary electrophoresis provided by the present application are the same as those of the sugar chain molecular weight calibration method based on capillary electrophoresis provided by the above embodiment, and other technical features of the sugar chain molecular weight calibration system based on capillary electrophoresis are the same as those disclosed in the above embodiment method, which will not be repeated here.
[0030] Embodiment three: The present application provides a sugar chain molecular weight calibration device based on capillary electrophoresis, please refer to Figure 2A capillary electrophoresis-based sugar chain molecular weight calibration device includes at least one processor; and a memory connected to the at least one processor in communication; wherein the memory stores instructions executable by the at least one processor, the instructions being executed by the at least one processor to enable the at least one processor to perform a capillary electrophoresis-based sugar chain molecular weight calibration method of Embodiment 1 described above. The capillary electrophoresis-based sugar chain molecular weight calibration device according to the embodiments of the present application can include, but is not limited to, a mobile terminal such as a mobile phone, a notebook, a digital broadcasting receiver, a PDA (Personal Digital Assistant), a PAD (Portable Application Description), a PMP (Portable Media Player), a car terminal (e.g., a car navigation terminal), and the like, and a stationary terminal such as a digital TV, a desktop computer, and the like. The capillary electrophoresis-based sugar chain molecular weight calibration device is only one example, and should not impose any limitation on the function and the range of use of the embodiments of the present application. The capillary electrophoresis-based sugar chain molecular weight calibration device can include a processing device 1001 (e.g., a central processing unit, a graphic processing unit, or the like) that can perform various appropriate actions and processes according to programs stored in a read-only memory 1002 or loaded from a storage device 1003 to a random access memory 1004. In the random access memory 1004, various programs and data required for the operation of the capillary electrophoresis-based sugar chain molecular weight calibration device are also stored. The processing device 1001, the read-only memory 1002, and the random access memory 1004 are connected to each other through a bus 1005. An I / O interface 1006 is also connected to the bus. In general, the following systems can be connected to the I / O interface 1006: an input device 1007 including, for example, a touch screen, a touch pad, a keyboard, a mouse, an image sensor, a microphone, an accelerometer, a gyroscope, and the like; an output device 1008 including, for example, a liquid crystal display (LCD), a speaker, a vibrator, and the like; the storage device 1003 including, for example, a magnetic tape, a hard disk, and the like; and a communication device 1009. The communication device 1009 can allow the capillary electrophoresis-based sugar chain molecular weight calibration device to communicate with other devices wirelessly or by wire to exchange data. Although the capillary electrophoresis-based sugar chain molecular weight calibration device having various systems is illustrated in the drawing, it is understood that all of the illustrated systems are not required to be implemented or provided. More or less systems can be alternatively implemented or provided.
[0031] Embodiment Four: The present application also provides a computer program product comprising a computer program which, when executed by a processor, implements the steps of a method for calibrating the molecular weight of a sugar chain based on capillary electrophoresis as described above. The computer program product provided by the present application can solve the technical problem of calibrating the molecular weight of a sugar chain based on capillary electrophoresis. Compared with the prior art, the computer program product provided by the present application has the same beneficial effects as the method for calibrating the molecular weight of a sugar chain based on capillary electrophoresis provided by the above-described embodiments, and thus will not be described here.
[0032] In particular, the processes described above with reference to the flowcharts can be implemented as a computer software program according to embodiments of the present application. For example, embodiments of the present application include a computer program product comprising a computer program carried on a computer readable medium, the computer program containing program code for executing the method shown in the flowcharts. In such embodiments, the computer program can be downloaded and installed from a network via a communication device, or installed from a storage device 1003, or installed from a read-only memory 1002. When the computer program is executed by a processing device 1001, the above-described functions defined in the method of the embodiments of the present application are performed.
[0033] It should be understood that various parts of the present application can be realized in hardware, software, firmware or a combination thereof. In the description of the above-described embodiments, specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner.
[0034] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.
Claims
1. A method for calibrating the molecular weight of glycans based on capillary electrophoresis, characterized in that, The methods include: Step S10: Obtain a multi-level standard set for molecular weight calibration, including first standard GU1, second standard GU2, third standard GU3 and fourth standard GU4; and introduce 2-aminobenzamide-labeled maltheptaose as an internal standard; Step S20: Collect the standard migration time of the i-th standard in the multi-level standard set respectively. and the preset glycan migration time of the glycan to be tested ; Collection internal standard migration time Based on internal standard migration time and sugar chain migration time Calculate relative migration time ; Step S30: Introduce a relative migration time correction factor Based on relative migration time correction factor relative migration time Make corrections to obtain the corrected relative migration time. Obtain the molecular weight set of multi-level standards from the multi-level standard set, and based on the molecular weight set and corrected relative migration time... Establish a cubic polynomial calibration model cubic polynomial calibration model Output the predicted molecular weight; Step S40: Calibrate the cubic polynomial model Perform dynamic piecewise correction to obtain the optimized calibration model. ; Step S50: Based on the optimized calibration model Predict the molecular weight of the sugar chain to be tested and output the final predicted molecular weight value.
2. The method for calibrating the molecular weight of glycans based on capillary electrophoresis as described in claim 1, characterized in that, In step S10, the first standard product GU1 is selected. The second standard GU2 was selected from Man5GlcNAc2; the third standard GU3 was selected from Man6GlcNAc2; and the fourth standard GU4 was selected from Neu5Ac2Gal2GlcNAc2Man3GlcNAc2.
3. The method for calibrating the molecular weight of glycans based on capillary electrophoresis as described in claim 1, characterized in that, In step S20, the relative migration time .
4. The method for calibrating the molecular weight of glycans based on capillary electrophoresis as described in claim 1, characterized in that, In step S30, the relative migration time is corrected. ,in, This is the preset theoretical internal standard migration time.
5. The method for calibrating the molecular weight of glycans based on capillary electrophoresis as described in claim 1, characterized in that, In step S30, the formula for the cubic polynomial calibration model is expressed as follows: in, The fitting coefficients of the cubic polynomial calibration model are obtained by least squares fitting based on the molecular weight set of the multi-level standard set, including the first standard GU1, the second standard GU2, the third standard GU3, and the fourth standard GU4.
6. The method for calibrating the molecular weight of glycans based on capillary electrophoresis as described in claim 1, characterized in that, In step S40, the cubic polynomial calibration model is performed. Perform dynamic piecewise correction to obtain the optimized calibration model. The steps specifically include: When the cubic polynomial calibration model When the predicted molecular weight is in the interval [1, 3], a linear optimization method is used: in, and m are the slope parameter and intercept parameter of the linear model, respectively, which are obtained by fitting the molecular weight set of the first standard GU1, the second standard GU2, and the third standard GU3 using the least squares method. When the cubic polynomial calibration model When the predicted molecular weight is in the range [3, 15], an exponential optimization method is used: in, and These are the scaling coefficient and growth rate coefficient of the exponential model, respectively, obtained by fitting the molecular weight sets of the third standard GU3 and the fourth standard GU4 using the least squares method.
7. The method for calibrating the molecular weight of glycans based on capillary electrophoresis as described in claim 1, characterized in that, In step S10, the internal standard is used to correct for fluctuations in electrophoretic migration time.
8. A glycan molecular weight calibration system based on capillary electrophoresis, applied to the glycan molecular weight calibration method based on capillary electrophoresis according to any one of claims 1 to 7, characterized in that, The capillary electrophoresis-based glycan molecular weight calibration system includes: The standard and internal standard acquisition module is used to acquire a multi-level standard set for molecular weight calibration. The multi-level standard set includes first standard GU1, second standard GU2, third standard GU3 and fourth standard GU4; and introduces 2-aminobenzamide-labeled maltheptaose as an internal standard. The migration time acquisition and calculation module is used to acquire the standard migration time of the i-th standard in a multi-level standard set. and the preset glycan migration time of the glycan to be tested ; Collection internal standard migration time Based on internal standard migration time and sugar chain migration time Calculate relative migration time ; The polynomial modeling module is used to introduce a relative migration time correction factor. Based on relative migration time correction factor relative migration time Make corrections to obtain the corrected relative migration time. Obtain the molecular weight set of multi-level standards from the multi-level standard set, and based on the molecular weight set and corrected relative migration time... Establish a cubic polynomial calibration model cubic polynomial calibration model Output the predicted molecular weight; The piecewise optimization correction module is used for calibration models of cubic polynomials. Perform dynamic piecewise correction to obtain the optimized calibration model. ; The results output module is used for optimization calibration model. Predict the molecular weight of the sugar chain to be tested and output the final predicted molecular weight value.
9. A glycan molecular weight calibration device based on capillary electrophoresis, characterized in that, The capillary electrophoresis-based glycan molecular weight calibration device includes: a memory, a processor, and a capillary electrophoresis-based glycan molecular weight calibration program stored in the memory and executable on the processor. When the capillary electrophoresis-based glycan molecular weight calibration program is executed by the processor, it implements a capillary electrophoresis-based glycan molecular weight calibration method according to any one of claims 1 to 7.
10. A computer program product, characterized in that, The computer program product includes a glycan molecular weight calibration program based on capillary electrophoresis, which, when executed by a processor, implements a glycan molecular weight calibration method based on capillary electrophoresis according to any one of claims 1 to 7.