A method for analyzing the glycosylation ratio of apolipoprotein Apo AI based on far infrared technology

Abstract

The present invention discloses a method for analyzing the glycation ratio of apolipoprotein ApoA-I based on far-infrared technology, that is, detecting the glycation reaction of the lysine active site in apolipoprotein ApoA-I, and the frequency shift amplitude of the far-infrared characteristic absorption peak caused by it. Specifically, it includes detecting whether the far-infrared characteristic absorption peak of apolipoprotein ApoA-I in the frequency range of 0.5-2THz moves to low frequency / high frequency, and calculating the glycation ratio (unglycated / glycated) of apolipoprotein ApoA-I according to the far-infrared characteristic absorption frequency before and after glycation of the lysine active site. The method of the present invention has the advantages of being non-invasive, simple to operate, fast, and low-cost, and provides accurate measurement conditions for maintaining life and health.

Description

Technical Field

[0001] The invention relates to the technical field of glycosylated protein determination, in particular to a method for analyzing the glycosylation ratio of apolipoprotein ApoA-I based on far infrared technology. Background Art

[0002] Apolipoprotein Apo AI is one of the important carrier proteins in human blood. In life, it can provide detection conditions for the effects of diet and lifestyle on human glycation, thereby achieving a healthy life. In medical treatment, it can also assist in the detection of certain diseases.

[0003] In scientific research laboratories, mass spectrometry is used to analyze glycated apolipoprotein Apo AI. The basic process of conventional mass spectrometry is as follows: (1) Enzymatically cut the protein into peptides; (2) Measure the relative molecular mass of all peptides using a mass spectrometer; (3) Determine the molecular weight of each peptide in the absence of any modification based on the protein sequence information. If the measured molecular weight of some peptides is found to increase, and the increase corresponds exactly to the molecular weight of one or several monosaccharide residues, it can be assumed that the peptide has been glycated; (4) Detect the suspected peptides through secondary or multistage mass spectrometry to confirm their glycation sites and glycation types.

[0004] However, mass spectrometry has high requirements for technicians in sample pretreatment, instrument operation and spectral analysis, and has low accuracy in analyzing glycosylation structures with highly similar conformations, which limits its promotion and widespread use as a routine clinical testing method.

[0005] Far-infrared technology is an analytical tool that is highly sensitive to the vibration and rotation of biomolecules. Without the need for external labeling, the spectral fingerprint of a specific sample can be presented based on the response between the vibration and rotation energy levels of biomolecules and far-infrared radiation, thereby distinguishing the molecular structure of apolipoprotein Apo AI before and after glycation, and is expected to provide accurate determination of the glycation ratio of apolipoprotein Apo AI. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the glycation ratio of apolipoprotein Apo AI based on far-infrared technology, which is used to confirm the far-infrared spectral parameters that can clearly distinguish between the two states of non-glycated and glycated lysine active sites in apolipoprotein Apo AI, thereby calculating the glycated ratio of apolipoprotein Apo AI in the human body.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for analyzing the saccharification ratio of apolipoprotein Apo AI based on far infrared technology, characterized in that it comprises the following steps:

[0009] S1: Far infrared testing of apolipoprotein Apo AI, lysine active sites and its glycation products;

[0010] S2: Correct the far-infrared spectrum baseline in the frequency range of 0.5-2THz to eliminate the differences between different spectra;

[0011] S3: normalize the far-infrared spectrum in the frequency range of 0.5-2THz;

[0012] S4: Determine the average center frequency of the far-infrared absorption peak of apolipoprotein Apo AI in the range of 0.5-2 THz Determine its error range;

[0013] S5: Determine the average center frequency of the far-infrared characteristic peaks detected at the lysine active site and its saccharification products within 0.5-2THz and

[0014] S6: According to the formula Calculate the unglycosylated C Lys and glycosylated C AGEs Proportion.

[0015] Furthermore, the lysine active site in S1 includes lysine and various derivatives thereof, wherein the side chains of the lysine derivatives contain ε-amino groups.

[0016] Furthermore, the lysine glycation product in S1 is a product obtained by non-enzymatic modification of the ε-amino group of lysine by reducing sugar.

[0017] Furthermore, the spectral baseline correction methods in S2 are used to eliminate instrument noise, background signals and interference signals, including polynomial fitting method, wavelet transform method, partial least squares method and local standard deviation method.

[0018] Furthermore, in S3, normalization processing is used to eliminate the differences between different spectra, including maximum and minimum value normalization, standard deviation normalization, and mean normalization.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention provides a far-infrared technology-based measurement. Compared with the mass spectrometry method which uses the molecular weight of a specific peptide segment as the measurement object of the glycation of apolipoprotein Apo AI, the far-infrared method provided by the present invention uses the lysine active site in apolipoprotein that is most susceptible to glycation reaction as the measurement object, does not require external labeling, and distinguishes between the two states of apolipoprotein Apo AI that has not undergone glycation and the one that has undergone glycation through the specific far-infrared characteristic frequency according to the response changes between the molecular vibration and rotation energy levels and far-infrared radiation before and after the glycation reaction of the lysine active site. Compared with the mass spectrometry method, the present method has lower instrument operation requirements and simpler information analysis, and is more suitable for promotion and use in clinical testing.

[0021] 2. The present invention provides an auxiliary diagnosis method for coronary heart disease based on far-infrared technology, which can confirm the far-infrared spectral parameters that can clearly distinguish the two states of unglycosylated and glycosylated lysine active sites in apolipoprotein Apo AI, thereby calculating the glycation ratio of apolipoprotein Apo AI in the human body. In terms of dietary health, it can understand the effects of different diets and lifestyles on the glycation ratio of apolipoprotein Apo AI, which is helpful to formulate more effective nutritional recommendations and lifestyle changes to maintain health. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the process of the present invention;

[0023] Figure 2 is the far infrared spectrum of the lysine active site of the present invention;

[0024] Figure 3 is the far infrared spectrum of the lysine saccharification product Nε-carboxymethyl lysine of the present invention;

[0025] Figure 4 is the far infrared spectrum of the lysine saccharification product Nε-carboxyethyl lysine of the present invention;

[0026] Figure 5 This is the far infrared spectrum of apolipoprotein Apo AI of a healthy subject of the present invention;

[0027] Figure 6 The present invention compares the detection results of the far infrared technology and mass spectrometry technology in the analysis of the saccharification ratio of apolipoprotein Apo AI. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] See also Figure 1-6 In an embodiment of the present invention, a method for analyzing the saccharification ratio of apolipoprotein Apo AI based on far infrared technology is provided, comprising the following steps:

[0030] Step 1: Purchase lysine standard and prepare a 10mmol / L lysine aqueous solution; divide the solution into three equal parts; one part is directly tested; the second part is incubated with a 20mmol / L glyoxal aqueous solution for 14 days, and after dialysis and removal of the incubation solution, the Nε-carboxymethyl lysine standard is obtained; the third part is incubated with a 20mmol / L methylglyoxal aqueous solution for 14 days, and after dialysis and removal of the incubation solution, the Nε-carboxyethyl lysine standard is obtained;

[0031] Artificially synthesized apolipoprotein Apo AI was purchased and prepared into a 10mmol / L apolipoprotein Apo AI aqueous solution; the solution was equally divided into two portions; one portion was directly tested; the second portion was incubated with a 20mmol / L methylglyoxal aqueous solution for 14 days; after dialysis and removal of the incubation solution, a glycated apolipoprotein Apo AI standard was obtained.

[0032] Step 2: Perform far infrared testing on lysine and its saccharification products. The active sites of lysine include lysine and lysine derivatives, wherein the side chains of lysine derivatives contain ε-amino groups; the lysine saccharification products are products of non-enzymatic modification of ε-amino groups by reducing sugars;

[0033] Step 3: Perform far infrared testing on apolipoprotein Apo AI and its saccharification products. The far infrared testing includes the following steps:

[0034] Drop the sample on the detection substrate and let it dry so as to collect far-infrared spectral data;

[0035] The detection substrate is a material with high far-infrared transmittance and high chemical stability;

[0036] Step 4: 0.5-2THz (17-67cm -1 ) band, each far-infrared spectrum is preprocessed, including the following steps:

[0037] a) If necessary, perform smoothing to reduce spectral noise caused by instrument noise;

[0038] b) Baseline correction to eliminate spectral differences caused by baseline frequency shift;

[0039] c) Since the far-infrared characteristic peaks of the lysine acid active site and its glycation products mostly fall within the 0.5-2 THz frequency band, the far-infrared spectrum of apolipoprotein Apo AI within the same frequency band is normalized;

[0040] Step 5: The specific data analysis method is as follows:

[0041] a) calibrating and assigning the far-infrared characteristic peaks of apolipoprotein Apo AI in the 0.5-2 THz frequency band according to all the far-infrared characteristic peaks of the lysine active site and its saccharification products;

[0042] b) Calculate the average center frequency of the far-infrared characteristic peak of the lysine active site within 0.5-2THz and standard deviation;

[0043] c) Calculate the average center frequency of the far-infrared characteristic peak detected by lysine glycation products within 0.5-2THz and standard deviation;

[0044] d) Calculate the average center frequency of the far-infrared absorption peak of apolipoprotein Apo AI within 0.5-2THz and standard deviation;

[0045] According to the formula Calculate the unglycosylated lysine residues (C Lys ) and saccharified (C AGEs )Proportion.

[0046] Figure 6 This is a comparison of the detection results of the far-infrared technology and mass spectrometry technology of the present invention in the analysis of the glycation ratio of apolipoprotein Apo AI. The artificially synthesized apolipoprotein Apo AI is divided into two parts on average; one part is incubated with a 1mmol / L glyoxal aqueous solution for 14 days, and the glycation sample No. 1 is obtained after dialysis and removal of the incubation solution; the second part is incubated with a 1000mmol / L glyoxal aqueous solution for 14 days, and the glycation sample No. 2 is obtained after dialysis and removal of the incubation solution. The glycation ratio of glycated sample No. 1 detected by the above-mentioned far-infrared method is 19%, and the glycation ratio of glycated sample No. 2 is 100%; the glycated ratio of glycated sample No. 1 detected by mass spectrometry is 14%, and the glycated ratio of glycated sample No. 2 is 100%. The comparison verifies that the accuracy of the determination method of the glycated ratio of apolipoprotein Apo AI based on far-infrared technology of the present invention is close to that of mass spectrometry, and it shows higher sensitivity at a lower glycation ratio.

[0047] In order to further better explain the embodiments of the present invention, the following specific implementation cases are provided:

[0048] The specific steps are as follows:

[0049] Step 1: Collect blood samples from healthy subjects. Healthy subjects are more representative of the general population.

[0050] Enriching apolipoprotein Apo AI from blood includes the following steps:

[0051] a) Add 100 μL of 180 g / L ethylenediaminetetraacetic acid solution (EDTA) to 10 mL of blood for anticoagulation, and centrifuge at 2000 g for 10 min at room temperature to obtain plasma;

[0052] b) Using density gradient centrifugation at 35,000 g for 40 h, high-density lipoprotein with a density of 1.063 to 1.21 g / mL was separated from plasma;

[0053] c) In order to avoid the interference and masking effect of albumin and immunoglobulin in plasma, high-density lipoprotein was repeatedly frozen and thawed with liquid nitrogen, and then Apo AI antibody was added to enrich apolipoprotein Apo AI by immunoprecipitation.

[0054] Step 2: Lysine derivatives are exemplified by lysine residues, and lysine glycation products are exemplified by Nε-carboxymethyllysine and Nε-carboxyethyllysine;

[0055] Step 3: Perform far infrared testing on apolipoprotein Apo AI and lysine glycation products; for the non-independent molecular structure of lysine residues, calculate their far infrared absorption frequency through molecular simulation. The specific far infrared testing steps are as follows:

[0056] a) dropping normal Apo AI, Nε-carboxymethyl lysine and Nε-carboxyethyl lysine samples on a high-resistance silicon wafer with a resistivity of 20000Ω·cm in sequence and drying them at room temperature to collect far-infrared spectral data;

[0057] b) Tested with a commercial far-infrared time-domain spectrometer produced by Advantest Instruments Co., Ltd. Before testing, the instrument must be calibrated according to the instructions provided by the instrument manufacturer to confirm whether the working conditions of the instrument are appropriate;

[0058] c) During the test, the relative humidity in the sample chamber should be kept below 2% to eliminate the influence of water vapor in the air on the test results; each spectrum is scanned 1024 times, and the spectrum resolution is 1.9 GHz.

[0059] Step 4: Since the far-infrared characteristic peaks of lysine and its saccharification products mostly fall within the 1-2THz frequency band, pre-processing is performed within the 1-2THz frequency band of each far-infrared spectrum. The specific steps are as follows:

[0060] a) Perform smoothing to reduce spectral noise caused by instrument noise;

[0061] b) Perform baseline correction to eliminate spectral differences caused by baseline frequency shift;

[0062] c) Normalize the far-infrared spectrum in the 1-2THz frequency band.

[0063] Step 5: Extract effective information from the above far infrared data. The data analysis steps are as follows:

[0064] a) In the 1-2 THz frequency band, the far-infrared characteristic peak of lysine residues is located at 1.55 THz, the far-infrared characteristic peak of Nε-carboxymethyl lysine is located at 1.17 THz, 1.42 THz (main peak), 1.59 THz and 1.98 THz, the far-infrared characteristic peak of Nε-carboxyethyl lysine is located at 1.43 THz (main peak) and 1.66 THz, and the far-infrared characteristic peak of normal Apo AI is located at 1.54 THz and 1.85 THz;

[0065] b) Calculate the average center frequency of the far-infrared characteristic peak of the lysine active site within 0.5-2THz Conclusion

[0066] c) Calculate the average center frequency of the far-infrared characteristic peak detected by lysine glycation products within 0.5-2THz and standard deviation. Taking the main peaks of Nε-carboxymethyllysine and Nε-carboxyethyllysine as representatives, we can conclude

[0067] d) Calculate the average mean value of the far-infrared absorption peak of normal apolipoprotein Apo AI within 0.5-2THz The average central frequency of the far-infrared absorption peak of glycosylated apolipoprotein Apo AI incubated in a high-glucose environment

[0068] e) According to the formula Calculate the unglycosylated lysine residues in normal apolipoprotein ApoA-I (C Lys ) and saccharified (C AGEs ) ratio, that is

[0069]

[0070] Result: C Lys≈1, C AGEs ≈0.

[0071] In summary: Compared with the mass spectrometry method which uses the molecular weight of a specific peptide as the measurement object of the glycation of apolipoprotein Apo AI, the far-infrared method provided by the present invention uses the lysine active site in apolipoprotein that is most susceptible to glycation reaction as the measurement object, and does not require external labeling. According to the response changes between the molecular vibration and rotation energy levels and far-infrared radiation before and after the glycation reaction of the lysine active site, the apolipoprotein Apo AI in the two states of non-glycation and glycation can be distinguished through the specific far-infrared characteristic frequency. Compared with the mass spectrometry method, the present method has lower instrument operation requirements, simpler information analysis, more accurate measurement, and is more suitable for promotion and use in clinical testing.

[0072] The types of lysine active sites and their saccharification products, testing instruments, and spectral preprocessing steps described here are intended to simplify the description of the present invention. The application, modification, and variation of the present invention are obvious to those skilled in the art.

[0073] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for analyzing the saccharification ratio of apolipoprotein Apo AI based on far infrared technology, characterized in that: The following steps are involved: S1: Far infrared testing of apolipoprotein Apo AI, lysine active sites and its glycation products; S2: Correct the far-infrared spectrum baseline in the frequency range of 0.5-2THz to eliminate the differences between different spectra; S3: normalize the far-infrared spectrum in the frequency range of 0.5-2THz; S4: Determine the average center frequency of the far-infrared absorption peak of apolipoprotein Apo AI in the range of 0.5-2 THz Determine its error range; S5: Determine the average center frequency of the far-infrared characteristic peaks detected at the lysine active site and its saccharification products within 0.5-2THz and S6: According to the formula Calculate the unglycosylated C Lys and glycosylated C AGEs Proportion.

2. The method for analyzing the saccharification ratio of apolipoprotein Apo AI based on far infrared technology according to claim 1, characterized in that: The lysine active sites in S1 include lysine and its various derivatives, among which the side chains of the lysine derivatives contain ε-amino groups.

3. The method for analyzing the saccharification ratio of apolipoprotein Apo AI based on far infrared technology according to claim 1, characterized in that: The lysine glycation product in S1 is the product of non-enzymatic modification of the ε-amino group of lysine by reducing sugar.

4. The method for analyzing the saccharification ratio of apolipoprotein Apo AI based on far infrared technology according to claim 1, characterized in that: The spectral baseline correction methods in S2 are used to eliminate instrument noise, background signals and interference signals, including polynomial fitting method, wavelet transform method, partial least squares method and local standard deviation method.

5. The method for determining the saccharification ratio of apolipoprotein Apo AI according to claim 1, characterized in that: The normalization process in S3 is used to eliminate the differences between different spectra, including maximum and minimum value normalization, standard deviation normalization and mean normalization.

Images