APOE typing and quantitative analysis method

Abstract

The invention relates to an APOE typing and quantitative analysis method, and belongs to the technical field of molecular diagnosis. The invention provides a method for detecting the subtype and expression quantity of apolipoprotein E (APOE) in a simply treated plasma or tissue sample by using mass spectrometry. According to the method, a plurality of APOE protein subtype common polypeptides are used as quality control anchor points, each subtype characteristic polypeptide is calibrated, the detection stability and reliability of the characteristic polypeptides can be effectively analyzed, and the APOE subtypes can be accurately qualitative and quantitative at the same time. The method has the advantages of high efficiency, accuracy, stability and the like, and has a wide clinical application prospect.

Description

Technical Field

[0001] The present invention relates to an APOE typing and quantitative analysis method, belonging to the technical field of molecular diagnosis. Background Art

[0002] Apolipoprotein E (APOE) is a protein closely involved in lipid metabolism and is widely distributed in the central nervous system and peripheral tissues. The human gene encoding APOE is located on chromosome 19. The gene is 3647 bp in length, consisting of four exons and three introns, encoding 317 amino acids. Two single nucleotide polymorphisms (SNPs) exist in the ApoE gene: rs429358 (c.388T>C) and rs7412 (c.526C>T). These two SNPs result in amino acid changes at positions 112 and 158 of the APOE protein, resulting in the three common APOE isoforms: E2 (388T-526T), E3 (388T-526C), and E4 (388C-526C). Among them, APOE3 is considered wild type, with amino acid 112 being cysteine ​​and amino acid 158 being arginine; mutant APOE2 has cysteine ​​at both sites, while mutant APOE4 has arginine at both sites. APOE gene polymorphism is closely associated with susceptibility to a variety of diseases, especially Alzheimer's disease and cardiovascular disease. In addition, the expression levels of different APOE subtypes are closely related to physiological health status. Therefore, developing a method that can efficiently and accurately measure changes in APOE protein subtypes and expression levels has important clinical and research value.

[0003] Traditional genotyping methods mainly rely on PCR amplification and gene sequencing. However, these methods are complex, time-consuming, and cannot effectively quantify the expression products. The expression of various proteins in the human body is closely related to the health status of the individual. Quantitative comparison between different types or subtypes of proteins can provide richer biological information and provide strong data support for research in life sciences such as human protein homeostasis. Mass spectrometry technology can efficiently and accurately perform qualitative and quantitative analysis of proteins, and has the characteristics of high sensitivity, high resolution, and a wide dynamic range, which can accurately identify different subtypes of APOE. Therefore, mass spectrometry-based detection methods have the potential to play an important role in the field of APOE protein typing and quantitative detection. Summary of the Invention

[0004] A reference peptide composition for APOE typing detection comprises one or a combination of the following three peptides:

[0005] a. A polypeptide having the sequence SELEEQLTPVAEETR (mass-to-charge ratio 865.93±5);

[0006] b. A polypeptide having the sequence GEVQAMLGQSTEELR (mass-to-charge ratio 824.40 ± 5);

[0007] c. A polypeptide having the sequence LGPLVEQGR (mass-to-charge ratio 484.78±5);

[0008] Among them, the composition is used for calibration and quantitative analysis of APOE protein subtypes in mass spectrometry detection.

[0009] A method for detecting APOE protein subtypes and expression levels comprises the following steps:

[0010] a. Collect plasma or tissue samples and extract the mixture containing APOE protein by solvent precipitation or dilution;

[0011] b. performing enzymatic digestion on the mixture to generate peptide fragments of APOE protein;

[0012] c. Separating the enzymatically hydrolyzed peptides using liquid chromatography;

[0013] d. The separated peptides are introduced into a mass spectrometer for detection to obtain mass spectra of the primary parent ion and secondary fragment ions;

[0014] e. Calibrate the characteristic polypeptide signal based on the above-mentioned reference polypeptide composition and calculate the genotype and expression level of the APOE protein subtype.

[0015] The separation conditions of liquid chromatography in step c are:

[0016] The chromatographic column specification is C18 column;

[0017] Mobile phase A was an aqueous solution containing 0.05%-0.5% formic acid, and mobile phase B was a 70%-90% acetonitrile solution containing 0.05%-0.5% formic acid;

[0018] The separation gradient duration was 30-120 minutes.

[0019] The parameters of mass spectrometry detection in step d include:

[0020] The MS1 scan range was 300–1500 m / z, the resolution was 100,000–150,000, the AGC was 200%–500%, and the ion injection time was 10–50 ms;

[0021] The MS2 scan range was 100–2500 m / z, the precursor mass range was 300–1200 m / z, the isolation window was 10–20 m / z, the resolution was 25,000–50,000, the AGC was 2000%–3000%, and the HCD collision energy was 25%–35%.

[0022] The calibration formula in step e is:

[0023]

[0024] Wherein, Peptideεn is the detected target peptide response, Peptideεn' is the corrected target peptide relative response, Peptide S1, S2, and S3 are the three reference peptides in claim 1, A1, A2, and A3 are correction coefficients, and the default value is 0.5-1.5.

[0025] The enzymatic digestion adopts a combined enzymatic digestion of trypsin and / or Lys-C enzyme, and the mass ratio of enzyme to substrate is 1:20 to 1:1000. The mass spectrometer is a high-resolution mass spectrometer, including but not limited to Orbitrap series, Q-TOF series or TripleTOF series mass spectrometers.

[0026] In step e, the results are verified by comparing samples with known NGS test results to ensure the accuracy of the test.

[0027] The beneficial effects of the present invention are:

[0028] The present invention provides an innovative method for genotyping and quantifying APOE protein using mass spectrometry. This method has the advantages of high efficiency, accuracy, and sensitivity, meets clinical needs, and has broad application prospects. It is hoped that through the promotion of this method, the ability to screen and prevent related diseases in the early stages will be improved. The method of the present invention is applicable to various fields such as clinical medicine, drug development, and genetic disease research, and has the following advantages:

[0029] 1. The sample sources are extensive and the collection and testing process is simple

[0030] 2. Suitable for qualitative and quantitative analysis of APOE protein isoforms in complex samples such as plasma containing complex matrices.

[0031] 3. Eliminate systematic errors: The calibration peptide is based on the peptide calibration within the APOE protein. The signal intensity is stable and is not affected by the mutation site. It can be used as a reference standard to improve quantitative stability and reduce errors caused by sample preparation, injection volume or instrument drift.

[0032] 4. Improve the accuracy of characteristic peptide detection: It can eliminate the non-specific effects of experimental conditions on the target APOE protein subtype characteristic peptide, thereby improving the specificity of the characteristic peptide signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of LC-MS detection of characteristic peptides and reference peptides and corresponding peptide sequences and mass-to-charge ratio information DETAILED DESCRIPTION

[0034] The present invention provides a method for detecting apolipoprotein E (APOE) subtypes and expression levels in plasma or tissue samples after simple treatment using mass spectrometry. The method comprises the following steps:

[0035] 1. Sample collection:

[0036] Blood or tissue samples are collected and a protein mixture including APOE protein is obtained by solvent precipitation or direct dilution.

[0037] 2. Enzymatic digestion of protein:

[0038] The protein mixture is enzymatically digested, using trypsin. This step breaks down the APOE protein into multiple peptide fragments.

[0039] 3. Peptide separation:

[0040] Liquid chromatography (LC) was used to separate the peptides after enzymatic digestion to improve the coverage of mass spectrometry analysis.

[0041] 4. Mass spectrometry analysis:

[0042] The separated peptides were introduced into a mass spectrometer for detection and analysis, and the mass spectra of the peptides of different subtypes of APOE proteins were obtained by detecting the mass-to-charge ratio (m / z) of the primary parent ion and secondary fragment ion of the peptides.

[0043] 5. Data processing:

[0044] The mass spectrometry results were used to analyze the characteristic peptides of the APOE protein, and the APOE genotype (ε2, ε3, ε4) and expression level were determined based on the primary parent ion spectrum and the m / z value and abundance of the resolved secondary fragment peaks.

[0045] 6. Result verification:

[0046] Verify and confirm accuracy by comparing samples with known NGS test results.

[0047] In the following examples, APOE protein subtype detection and analysis were performed using the following steps:

[0048] 1. Sample collection:

[0049] Collect plasma samples and use solvent precipitation or direct dilution to obtain a protein mixture including APOE protein. This embodiment uses the solvent precipitation method, which specifically involves the following steps: take a certain volume of plasma and add it to a 50-200mM ammonium bicarbonate solution at a plasma:ammonium bicarbonate solution dilution ratio of 1:10 for dilution and mix thoroughly. Then, add pre-cooled methanol to the plasma diluent at a plasma diluent:methanol ratio of 2:1. Vortex to mix thoroughly, centrifuge, discard the supernatant, vacuum dry the precipitate, and store in a -80°C refrigerator until use.

[0050] 2. Enzymatic digestion of protein:

[0051] The protein mixture is enzymatically digested, and the commonly used digestive enzyme is trypsin. This step degrades the APOE protein into multiple peptide fragments. The specific steps are as follows: dissolve the dried precipitated protein in 8M urea, add the reducing agent 50mM TCEP solution to a final concentration of 5mM, heat denaturation and reduction, add the alkylating agent 100mM IAA solution to a final concentration of 10mM, and react at room temperature in the dark for 30 minutes. After diluting the final concentration of urea to less than 1M with 50-200mM ammonium bicarbonate solution, add Lys-C enzyme and Trypsin enzyme in a ratio of 1:50-1:500 by mass of enzyme / substrate, mix well, react overnight, and then add 10% TFA to a final concentration of 0.1% to terminate the reaction.

[0052] 3. Peptide separation:

[0053] Liquid chromatography (LC) was used to separate the enzymatically digested peptides to improve the coverage of mass spectrometry analysis and identification. The LC was performed on a Vanquish Neo UHPLC system and the mass spectrometer was an Orbitrap Exploris 480 (ThermoFisher Scientific). The column specifications were 2 μm × 75 μm × 25 cm C18 column. Mobile phase A was water (containing 0.1% formic acid) and mobile phase B was 80% acetonitrile (containing 0.1% formic acid). The separation gradient was 65 min.

[0054] 4. Mass spectrometry analysis:

[0055] The separated peptides are introduced into a mass spectrometer for detection and analysis, and the mass spectra of the polypeptides of different subtypes of APOE proteins are obtained by detecting the mass-to-charge ratio (m / z) of the primary parent ion and the secondary fragment ion of the peptide. This embodiment takes the non-targeted detection method as an example, but is not limited to non-targeted detection analysis, and is also applicable to targeted detection methods. The mass spectrometer model is Orbitrap Exploris 480 (Thermo Fisher Scientific), the mass spectrometer MS1 scanning range is 400-1200m / z, the resolution is 120,000, the AGC is 300%, and the IT is 25ms; the MS2 scanning range is 150-2000m / z, the precursor mass range is 400-1000m / z, the isolation window is 15m / z, the number of single scan cycle windows is 40, the resolution is 30,000, the AGC is 2500%, and the HCD collision energy is 32%.

[0056] 5. Data processing:

[0057] The characteristic peptides of APOE protein were analyzed by mass spectrometry, based on the m / z values ​​and relative abundances of the primary parent ion and the secondary fragment peaks after fragmentation, such as Figure 1 As shown, the APOE gene phenotype (ε2, ε3, ε4) and relative or absolute expression levels are determined. When calculating the expression level of each characteristic polypeptide, the reference polypeptide detected simultaneously in the sample is used for correction calculation. The reference polypeptides are shown in Table 1. They are obtained based on the mutation site in the APOE protein sequence, the response value of the identification, and the stability screening. The three polypeptides SELEEQLTPVAEETR, GEVQAMLGQSTEELR, and LGPLVEQGR, which are close to the mutation site, stably detectable, and have a high response, are further preferred. The correction formula is:

[0058]

[0059] Where Peptide εn is the detected target peptide response, and Peptide εn' is the corrected relative target peptide response. The calibration peptides are Peptide S1: SELEEQLTPVAEETR, Peptide S2: GEVQAMLGQSTEELR, and Peptide S3: LGPLVEQGR. A1, A2, and A3 are calibration coefficients, with a default reference value of 1. This method was used to calibrate the detection results of the relatively low-response E4 peptide LGADMEDVR (SEQ ID NO. 4). Compared to the single, preferred peptide LGPLVEQGR, the corrected coefficient of variation (CV) (coefficient of variation = standard deviation / mean) was reduced from 39.9% to 9.78%. Calibration with three preferred peptides further reduced the CV to 7.28%. CV calculations were based on the signal intensity of each peptide measured in three replicate batches to assess quantitative stability. The improved stability after calibration using the preferred peptides is shown in Table 2.

[0060] Table 1 Reference peptide list

[0061]

[0062]

[0063] Table 2 Stability improvement results after calibration of preferred peptides

[0064]

[0065] 6. Verification of method reliability:

[0066] The accuracy and reliability of this method were verified by mass spectrometry analysis in 10 subjects with known APOE genotypes. The calibrated results accurately determined the APOE protein subtypes of the subjects, with a 100% concordance rate between the mass spectrometry results and the genotypes. The results are shown in Table 3.

[0067] Table 3: Mass spectrometry results for subjects with known NGS results

[0068] Sample ID NGS MS Protein type SH25001 E2 / E3 E2 / E3 SH25002 E3 / E3 E3 / E3 SH25003 E3 / E3 E3 / E3 SH25004 E3 / E3 E3 / E3 SH25005 E3 / E3 E3 / E3 SH25006 E3 / E4 E3 / E4 SH25007 E3 / E4 E3 / E4 SH25008 E3 / E4 E3 / E4 SH25009 E3 / E3 E3 / E3 SH25010 E3 / E3 E3 / E3

[0069] Potential uses

[0070] 1. Alzheimer's disease screening: By detecting the APOE gene phenotype, the individual's risk of developing Alzheimer's disease is assessed and clinical preventive measures are guided.

[0071] 2. Cardiovascular disease risk assessment: Provide a basis for individualized treatment plans for cardiovascular diseases.

[0072] 3. Genetic risk assessment: Provide a scientific basis for family genetic risk assessment and enhance public awareness of related diseases.

Claims

1. A reference polypeptide composition for APOE typing detection, characterized in that: Contains the following three peptides: a. A polypeptide having a sequence of SELEEQLTPVAEETR (SEQ ID NO.1); b. A polypeptide having a sequence of GEVQAMLGQSTEELR (SEQ ID NO.2); c. a polypeptide having a sequence of LGPLVEQGR (SEQ ID NO.3); Among them, the composition is used for calibration and quantitative analysis of APOE protein subtypes in mass spectrometry detection.

2. A method for detecting APOE protein subtypes and expression levels, characterized in that: The following steps are involved: a. Collect plasma or tissue samples and extract the mixture containing APOE protein by solvent precipitation or dilution; b. performing enzymatic digestion on the mixture to generate peptide fragments of APOE protein; c. Separating the enzymatically hydrolyzed peptides using liquid chromatography; d. The separated peptides are introduced into a mass spectrometer for detection to obtain mass spectra of the primary parent ion and secondary fragment ions; e. Calibrate the characteristic polypeptide signal based on the reference polypeptide composition described in claim 1 to calculate the genotype and expression level of the APOE protein subtype.

3. The detection method according to claim 2, characterized in that The separation conditions of the liquid chromatography in step c are as follows: the chromatographic column specification is a C18 column; Mobile phase A was an aqueous solution containing 0.05%-0.5% formic acid, and mobile phase B was a 70%-90% acetonitrile solution containing 0.05%-0.5% formic acid; The separation gradient duration was 30-120 minutes.

4. The detection method according to claim 1, wherein Mass-to-charge ratio of the polypeptide shown in SEQ ID NO.1 865.93±5, the mass-to-charge ratio of the polypeptide shown in SEQ ID NO.2 is 824.40±5, and the mass-to-charge ratio of the polypeptide shown in SEQ ID NO.3 is 484.78±5.

5. The detection method according to claim 2, characterized in that The parameters of mass spectrometry detection in step d include: The MS1 scan range was 300–1500 m / z, the resolution was 100,000–150,000, the AGC was 200%–500%, and the ion injection time was 10–50 ms; The MS2 scan range was 100–2500 m / z, the precursor mass range was 300–1200 m / z, the isolation window was 10–20 m / z, the resolution was 25,000–50,000, the AGC was 2000%–3000%, and the HCD collision energy was 25%–35%.

6. The detection method according to claim 2, characterized in that The calibration formula in step e is: Wherein, Peptideεn is the detected target peptide, Peptideεn' is the corrected target peptide, Peptide S1, S2, and S3 are the three reference peptides in claim 1, respectively, and A1, A2, and A3 are correction coefficients, with a default value of 0.5-1.

5.

7. The detection method according to claim 2, characterized in that The enzymatic digestion adopts a combined enzymatic digestion of trypsin and / or Lys-C enzyme, and the mass ratio of the enzyme to the substrate is 1:20 to 1:1000.

8. The detection method according to claim 2, characterized in that The mass spectrometer is a high-resolution mass spectrometer, including but not limited to Orbitrap series, Q-TOF series or TripleTOF series mass spectrometers.

9. The detection method according to claim 2, characterized in that In step e, the results are verified by comparing samples with known NGS test results to ensure the accuracy of the test.

10. The detection method according to claim 2, characterized in that: The detection method is used to quantitatively detect the content of E4 polypeptide.

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