Marker composition for deletion type alpha thalassemia, detection reagent and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a marker composition for deletion type alpha thalassemia, a detection reagent and application. The marker composition is a combination of any two polypeptide fragments of P1, P2, P3, P4 and P5; wherein the amino acid sequences of P1-P5 are respectively shown as SEQ ID NO: 1-5 in sequence. The invention also provides a method for separating and determining the peak area ratio of the biomarker composition based on liquid chromatography-tandem mass spectrometry. The method comprises the following steps: 1) separating polypeptide fragments in a blood sample of a patient suffering from deletion type alpha thalassemia by adopting liquid chromatography; and 2) determining by using a mass spectrometry detector, quantifying each peptide fragment by using a peak area integration method, and calculating the peak area ratio of peptide fragment markers. The marker can effectively identify various types of deletion alpha-thalassemia, and rapid screening and preliminary typing of various types of alpha-thalassemia can be realized by calculating the peak area ratio of each peptide fragment marker.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a marker composition, a detection reagent and an application of deletion type α-thalassemia. Background Art

[0002] Alpha thalassemia (abbreviated as α-thalassemia) is one of the common clinical autosomal recessive genetic diseases. Its pathogenesis is that the deletion and mutation of the α-globin gene leads to changes in its synthesis rate, causing an imbalance in the ratio of α-globin chain and β-globin chain in red blood cells, resulting in a shortened life span of red blood cells and the formation of hemolytic anemia. The disease is widely distributed around the world. In my country, it mainly occurs in provinces south of the Yangtze River, with a high incidence in Guangdong, Guangxi, Hainan and other places. It has a high prevalence and is extremely harmful.

[0003] α-thalassemia is highly heterogeneous in genetics and is mainly divided into two types: deletion type and non-deletion type. Deletion type α-thalassemia is a hereditary hemolytic anemia caused by the deletion of the α-globin gene, which leads to reduced or complete loss of α-globin chain synthesis. According to the number of α genes missing, it can be divided into the following categories: ① α-thalassemia static type (α + ): Only one α-globin gene is inactivated on both chromosomes. Common genotypes include αα / -α 3.7 (Abbreviation -α 3.7 ), αα / -α 4.2 (Abbreviation -α 4.2 ), α CS α / αα (abbreviated as α CS ), α QS α / αα (abbreviated as α QS ) and α WS α / αα (abbreviated as α WS );②α-thalassemia intermedia (α 0 ): Inactivation of two α-globin genes, common genotypes include αα / --SEA (abbreviated as-- SEA ), -α 3.7 / -α 3.7 and -α 4.2 / -α 3.7 ; ③Hemoglobinopathy (Hb H disease): 3 α-globin genes are missing, common genotypes include -α 3.7 / --SEA and -α 4.2 / --SEA; ④ Lethal hydrops fetalis syndrome (Hb Bart's hydrops fetalis syndrome): All four α-globin genes are missing. Different types of α gene defects lead to different degrees of loss of peptide chain activity, which can lead to different clinical manifestations. Therefore, genetic testing for deletion α-thalassemia will help in the diagnosis and treatment of deletion α-thalassemia.

[0004] At present, the clinical screening for α-thalassemia mainly relies on hemoglobin electrophoresis or high-performance liquid chromatography to analyze hemoglobin composition and detect the content of Hb Bart's. 3.7 , -α 4.2 , α CS , α QS , α WS ) and some mild α-thalassemia have no clinical symptoms or only mild microcytic hypochromic anemia, which is difficult to screen through quantitative detection of Hb Bart's. In addition, Hb Bart's is extremely unstable and has high requirements for sample sampling and preservation. It is often impossible to detect due to factors such as sample hemolysis, degradation, improper preservation, and age, which limits its clinical application.

[0005] Some existing technologies use PCR methods to detect α-thalassemia. For example, patent CN104212910B discloses a single-tube amplification kit for simultaneous detection of α- and β-thalassemia genes, which uses PCR to detect three deletion types of α-thalassemia (--SEA, -α3.7 and -α4.2), three mutant types of α-thalassemia (CS, QS, WS) and 19 mutant types of β-thalassemia. For another example, patent CN103602752B discloses a primer set and kit for detecting rare deletion types of thalassemia, which uses multiplex Gap-PCR technology to detect 11 rare deletion genotypes, specifically including 6 α-thalassemia deletion genotypes (-- 11.1 、-- FIL 、-- THAI , -α 27.6 , -α 2.4 , -α 2.8 ) and 5 β-thalassemia deletion genotypes. These existing methods can detect relatively few types of deletional α-thalassemia, most of which are detections of known mutation sites, and cannot perform universal screening for deletional α-thalassemia based on phenotype.

[0006] Therefore, finding diagnostic markers for various types of deletion α-thalassemia is of great significance for the diagnosis and treatment of deletion α-thalassemia. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a biomarker composition for deletion α-thalassemia, which can effectively identify different types of deletion α-thalassemia and provide support for early screening and preliminary typing of various types of deletion α-thalassemia.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A biomarker composition for deletion type α-thalassemia, wherein the biomarker composition for deletion type α-thalassemia is a combination of any two polypeptide fragments of P1, P2, P3, P4, and P5; the amino acid sequence of P1 is shown in SEQ ID NO: 1, the amino acid sequence of P2 is shown in SEQ ID NO: 2, the amino acid sequence of P3 is shown in SEQ ID NO: 3, the amino acid sequence of P4 is shown in SEQ ID NO: 4, and the amino acid sequence of P5 is shown in SEQ ID NO: 5.

[0010] Furthermore, the biomarker composition for deletion-type α-thalassemia is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, and P5 and P2.

[0011] Further, the deletion type α thalassemia includes α + , α 0 and / or HbH.

[0012] Furthermore, the α + The biomarker composition is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, P5 and P2; and / or the α 0 The biomarker composition of is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, P5 and P2; and / or the biomarker composition of HbH is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, P5 and P2.

[0013] Furthermore, the α + Include-α 3.7 , -α 4.2 , α CS , α QS and / or α WS ; the α 0 include-- SEA , -α 3.7 / -α 3.7 and / or -α 4.2 / -α 3.7 ; The HbH includes -α 3.7 / --SEA and / or -α 4.2 / --SEA.

[0014] Furthermore, the -α 3.7The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2, more preferably any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1; the -α 4.2 The biomarker composition is any one or more of the following combinations: P5 and P2, P4 and P2; CS The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the α QS The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P2, P4 and P1, more preferably any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P2; the α WS The biomarker composition is P3 and P1; the - SEA The biomarker composition is any one or more of the following combinations: P3 and P1, P3 and P2; 3.7 / -α 3.7 The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 4.2 / -α 3.7 The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 3.7 / --The biomarker composition of SEA is any one or more of the following combinations: P3 and P1, P3 and P2, P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 4.2 / --The biomarker combination of SEA is any one or more of the following combinations: P3 and P1, P3 and P2, P5 and P1, P5 and P2, P4 and P1, P4 and P2.

[0015] The second purpose of the present invention is to provide a detection reagent for deletion type α-thalassemia.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

[0017] A detection reagent for deletion type α-thalassemia, the detection reagent comprising any one or more of reagent A, reagent B, reagent C, and reagent D; the reagent A is a mixture of methanol and water; the reagent B is a mixture of acetonitrile, methanol, and water; the reagent C is a pancreatic enzyme solution; and the reagent D is a mixture of acetonitrile and water.

[0018] Preferably, in the reagent A, the volume ratio of methanol to water is 1:4.

[0019] Preferably, in the reagent B, the volume ratio of acetonitrile, methanol and water is 2:1:7.

[0020] Preferably, the concentration of the pancreatic enzyme solution is 5 g / L.

[0021] Preferably, in the reagent D, the volume ratio of acetonitrile to water is 3:17.

[0022] Furthermore, the detection reagent also includes normal standard samples, deletion type α-thalassemia heterozygous standard samples, and homozygous standard samples.

[0023] A third object of the present invention is to provide a method for separating and determining the peak area ratio of the aforementioned biomarker composition.

[0024] To achieve the above object, the present invention adopts the following technical solutions:

[0025] The method for separating and determining the peak area ratio of the aforementioned biomarker composition adopts a liquid chromatography-tandem mass spectrometry method for separation, qualitative, quantitative and ratio determination, and the liquid chromatography-tandem mass spectrometry method comprises the following steps:

[0026] (1) Sample pretreatment: using the aforementioned detection reagent to treat the sample to be tested to obtain a sample solution containing the polypeptide fragment; the sample to be tested is an ex vivo blood sample of a patient with deletion α-thalassemia or a patient suspected of having deletion α-thalassemia;

[0027] (2) Liquid chromatography separation: The chromatographic column uses silica gel particles modified with cyano functional groups as filler, deionized water containing formic acid as mobile phase A, and acetonitrile containing formic acid as mobile phase B, and the peptide fragments in the sample solution are separated by gradient elution;

[0028] The gradient elution procedure is as follows:

[0029] Time-min Mobile phase A - volume Mobile phase B - volume Starting 87 13 0.5 82 18 1.0 78 22 1.5 70 30 1.8 6 94 2.5 5 95 3.5 87 13 ;

[0030] (3) Mass spectrometry: Mass spectrometry is used for determination, and the peak area integration method is used to quantify each peptide segment;

[0031] (4) Ratio calculation: Based on the peak areas of each peptide segment measured in step (3), calculate the peak area ratio of the peptide marker.

[0032] Furthermore, the ex vivo blood sample is any one or more of venous blood, fingertip blood, heel blood, and whole blood specimens.

[0033] Furthermore, in step (1), the sample to be tested is eluted, denatured, and enzymatically hydrolyzed to release peptides such as P1, P2, P3, P4, and P5.

[0034] As a preferred solution, the sample pre-treatment steps are as follows:

[0035] 1) Elution: Place the sample to be tested in a 96-well filter plate, add reagent A, and shake at low speed for 30 minutes at room temperature to obtain hemolyzed blood;

[0036] 2) Denaturation: transfer the hemolysate obtained in step 1) to a new 96-well plate, add reagent B, and let stand at room temperature for 5 minutes to obtain a mixed solution;

[0037] 3) Enzymatic hydrolysis: Add reagent C to the mixture obtained in step 2), shake at low speed to mix, and incubate at 37°C for 1 hour;

[0038] 4) Redissolution: Take a 96-well filter plate, add the sample mixture obtained in step 3) and reagent D, gently shake to dissolve, incubate at room temperature for 2 minutes, centrifuge briefly, and keep the sample from the plate for testing.

[0039] Preferably, in step (2), the mobile phase A is deionized water containing 0.15% formic acid, and the mobile phase B is acetonitrile containing 0.15% formic acid.

[0040] Preferably, in step (2), the chromatographic column specification is 1.8μm, 2.1mm×100mm.

[0041] Preferably, in step (2), the chromatographic column is ACQUITY UPLC HSS Cyano (CN) Column Waters. 1.8μm,2.1mm×100mm.

[0042] Preferably, in step (2), the mobile phase flow rate is 0.5 ml / min and the column temperature is 45°C.

[0043] Preferably, in step (2), the injection volume is 5ul.

[0044] Preferably, in step (2), the elution time is 3.5 min.

[0045] Furthermore, the mass spectrometry detection is performed in an electrospray ionization cation detection mode using a mass spectrometry mode with multiple reflection monitoring.

[0046] Further, the ion source parameters in the mass spectrometry detection include:

[0047] Capillary voltage: 3-5KV; cone voltage: 25-35V; ion source temperature: 140-160℃; desolvation temperature: 400-500℃; cone gas flow rate: 140-160L / Hr; desolvation flow rate: 900-1100L / Hr.

[0048] Preferably, the capillary voltage is 3.5 KV, the cone voltage is 30 V, the ion source temperature is 150° C., the desolvation temperature is 450° C., the cone gas flow rate is 150 L / Hr, and the desolvation flow rate is 1000 L / Hr.

[0049] Furthermore, different fragment ion pair combinations of each characteristic peptide can be used for detection.

[0050] Furthermore, in step (3), the peak area of ​​the peptide marker is automatically integrated using TargetLynx software, the peak area score of each peptide fragment-specific ion pair is calculated, and the information intensity of the mass spectrometry detection is converted into the concentration of the analyte.

[0051] According to the reference value intervals of the marker ratios of normal people and different types of deletion α-thalassemia, a database can be further established and used for screening of various types of deletion α-thalassemia. The reference value intervals are shown in Tables 10 to 15 in the embodiments, and the reference intervals may vary due to different laboratories, different experimental personnel, and different reagent batches.

[0052] Based on the peak area ratios of the measured peptide markers and combined with the above reference value range, we can further use it to identify various types of deletional α-thalassemia.

[0053] A fourth object of the present invention is to provide a use of the aforementioned biomarker composition and / or the aforementioned detection reagent in the preparation of a product for screening various types of deletional α-thalassemia.

[0054] To achieve the above object, the present invention adopts the following technical solutions:

[0055] Use of the aforementioned biomarker composition and / or the aforementioned detection reagent in the preparation of products for screening various types of deletional α-thalassemia.

[0056] Further, the deletion type α thalassemia includes α + , α 0 and / or HbH.

[0057] Furthermore, the α + Include-α 3.7 , -α 4.2 , α CS , α QS and / or α WS ; the α 0 include-- SEA , -α 3.7 / -α 3.7 and / or -α 4.2 / -α 3.7; The HbH includes -α 3.7 / --SEA and / or -α 4.2 / --SEA.

[0058] The beneficial effects of the present invention are:

[0059] 1. The present invention provides a polypeptide marker composition for deletion α-thalassemia, which can effectively identify different types of deletion α-thalassemia and provide support for subsequent screening of various types of deletion α-thalassemia.

[0060] 2. Based on non-targeted proteomics research, the present invention found that polypeptide fragments such as P1, P2, P3, P4, and P5 in the blood are closely related to deletion type α-thalassemia. By detecting these polypeptide fragments, the sensitivity of traditional methods for detecting deletion type α-thalassemia can be improved.

[0061] 3. The present invention provides a reagent for screening for deletion type α-thalassemia based on liquid chromatography-mass spectrometry technology, and a method for separating and determining the peak area ratio of a biomarker composition for deletion type α-thalassemia. The present invention measures the peak areas of peptide chains such as P1, P2, P3, P4, and P5 by liquid chromatography-tandem mass spectrometry, and by calculating the ratio of the peak areas between the indicators, rapid screening and preliminary typing of various types of α-thalassemia can be achieved.

[0062] 4. Based on the method for separating and determining the peak area ratio of the biomarker composition for deletion type α-thalassemia provided by the present invention, severe, moderate and mild α-thalassemia can be effectively identified. The method can effectively identify the static type (α-thalassemia) of α-thalassemia on different models. + ) detection rates were greater than 82.61%, and for α-thalassemia intermedia (α 0 ) detection rates were all greater than 92.00%, and the detection rates for HbH were all 100.00%.

[0063] 5. The samples tested by this method can be venous blood, fingertip blood, heel blood, whole blood specimens, etc., and dry filter paper prepared from the blood. The samples are easy to transport and easy to achieve centralized and large-scale testing or screening. In addition, the samples tested by this method have low requirements for storage conditions. Short-term storage at room temperature (14 days) does not reduce the accuracy of the test results; under low temperature conditions (4°C and below), they can be stored for a long time (more than 1 month).

[0064] 6. The polypeptide marker composition and method for deletion-type α-thalassemia provided by the present invention have positive impacts on patients and their families at multiple levels. First, for children who may develop severe thalassemia, early identification can prevent potential complications in advance and reduce the risk of infection caused by blood transfusion therapy, thereby improving the quality of life of children. Secondly, for children with mild thalassemia, early identification can avoid being in a low-pressure and hypoxic environment for a long time, thereby preventing the occurrence of anemia symptoms. Furthermore, for couples planning to become pregnant, they can screen in advance whether both parties are carriers of thalassemia, reduce the birth of children with severe thalassemia, and achieve the goal of eugenics. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Total ion current spectrum of peptide markers detected by liquid chromatography-mass spectrometry;

[0066] Figure 2 This is the mass spectrometry identification result of the characteristic peptide segment of P1;

[0067] Figure 3 This is the mass spectrometry identification result of the characteristic peptide segment of P2;

[0068] Figure 4 This is the mass spectrometry identification result of P3 characteristic peptide;

[0069] Figure 5 This is the mass spectrometry identification result of P4 characteristic peptide;

[0070] Figure 6 This is the mass spectrometry identification result of P5 characteristic peptide;

[0071] Figure 7 This is the result diagram of the automatic peak area integration method for quantifying the peptide marker P1, where: Figure 7 -A is the result of the quantitative ion peak area integration of the P1 characteristic peptide segment, Figure 7 -B is the peak area integration result of the qualitative ion of the characteristic peptide segment of P1;

[0072] Figure 8 This is the result diagram of the automatic peak area integration method for quantifying the peptide marker P2, where: Figure 8 -A is the result of the quantitative ion peak area integration of the P2 characteristic peptide segment, Figure 8 -B is the result of the peak area integration of the qualitative ion of the characteristic peptide segment of P2;

[0073] Fig. 9 This is the result diagram of the automatic peak area integration method for quantifying peptide marker P3, where: Fig. 9 -A is the result of the quantitative ion peak area integration of the P3 characteristic peptide segment, Fig. 9 -B is the result of the peak area integration of the qualitative ion of the P3 characteristic peptide segment;

[0074] Fig.10 This is the result diagram of the automatic peak area integration method for quantifying peptide marker P4, where: Fig.10 -A is the result of the quantitative ion peak area integration of the P4 characteristic peptide segment. Fig.10 -B is the result of the peak area integration of the qualitative ion of the P4 characteristic peptide segment;

[0075] Fig.11 This is the result diagram of the automatic peak area integration method for quantifying peptide marker P5, where: Fig.11 -A is the result of the quantitative ion peak area integration of the P5 characteristic peptide segment, Fig.11 -B is the peak area integration result of the qualitative ion of the characteristic peptide segment of P5. DETAILED DESCRIPTION

[0076] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0077] In the embodiment of the present invention, the sequence information of polypeptide fragments P1-P5 is shown in Table 1.

[0078] Table 1. Sequence information of polypeptide fragments P1-P5

[0079] name Sequence number P1 SEQ ID NO: 1 P2 SEQ ID NO: 2 P3 SEQ ID NO: 3 P4 SEQ ID NO: 4 P5 SEQ ID NO: 5

[0080] In the embodiment of the present invention, the main instruments include: liquid chromatography-tandem mass spectrometer, punching forceps, centrifuge, computer, single-channel micropipettes and multi-channel micropipettes of various ranges, and chromatographic column (ACQUITYUPLC HSS Cyano (CN) Column Waters).

[0081] In the embodiment of the present invention, the main consumables include: an ordinary 96-well reaction plate, a 96-well ELISA plate with a 0.22 μm pore size filtration function, a pipette tip, a 96-well plate special aluminum foil film, a 96-well plate sealing film, and an identification barcode.

[0082] Example 1

[0083] (1) Preparation of reagents

[0084] Reagent A: A mixture of methanol and water, wherein the volume ratio of methanol to water is 1:4, used for eluting analytes in blood samples.

[0085] Reagent B: A mixture of acetonitrile, methanol and water, wherein the volume ratio of acetonitrile, methanol and water is 2:1:7, used for denaturation of analytes in blood samples.

[0086] Reagent C: Pancreatin solution with a concentration of 5 g / L.

[0087] Reagent D: A mixture of acetonitrile and water, in which the volume ratio of acetonitrile to water is 3:17, used for reconstitution of analytes in blood samples.

[0088] Quality control products: normal standard products, deletion type α-thalassemia heterozygous standard products, and homozygous standard products.

[0089] (2) Specimen collection

[0090] Take a drop of venous blood, fingertip blood or heel blood sample and drip it into Whatman The blood is placed on the filter paper and dried to make a dry blood filter paper specimen, which is then sealed in a sealed bag and stored at 4°C for a long time until tested. Or a whole blood specimen can be directly taken for analysis.

[0091] (3) Pretreatment and analyte extraction

[0092] 1) Use a standard puncher to punch out a 3.2mm diameter circular filter paper blood piece (or a sample equivalent to 3.2μl of whole blood) from the dried blood filter paper specimen and place it in a 96-well filter plate. Add 200μl of reagent A and shake at low speed at room temperature for 30 minutes to fully dissolve.

[0093] 2) Take 40 μl of the hemolysate, transfer it to a new 96-well plate, add 50 μl of reagent B, and let it stand at room temperature for 5 minutes.

[0094] 3) Add 10 μl of reagent C to the above mixture, shake at low speed to mix, seal with sealing film, and incubate in a 37°C incubator for 1 hour.

[0095] 4) Take a 96-well filter plate, add 10 μl of the above sample mixture and 90 μl of reagent D, gently shake to dissolve, incubate at room temperature for 2 minutes, centrifuge briefly, and keep the sample from the plate for testing.

[0096] (4) Liquid chromatography-tandem mass spectrometry detection

[0097] Using the established detection scheme, rapid injection through liquid chromatography and rapid detection through tandem mass spectrometry can achieve high-throughput injection and analysis of samples. The detection time for each sample is less than 5 minutes. In one experiment, at least 192 samples can be tested in the same batch. Normal standards, deletion α-thalassemia heterozygous standards, and homozygous standards are analyzed simultaneously for each batch of samples for quality control of the detection method.

[0098] 1) Liquid phase conditions

[0099] The chromatographic column uses silica gel particles modified with cyano functional groups as filler (ACQUITY UPLC HSS Cyano (CN) Column Waters, 1.8μm, 2.1mm×100mm), deionized water containing 0.15% formic acid as mobile phase A, acetonitrile containing 0.15% formic acid as mobile phase B, gradient elution according to Table 1; set the mobile phase flow rate to 0.5ml / min, column temperature to 45℃, injection volume to 5ul.

[0100] Table 1. Mobile phase gradient elution program

[0101] Time-min Mobile phase A - volume Mobile phase B - volume Starting 87 13 0.5 82 18 1.0 78 22 1.5 70 30 1.8 6 94 2.5 5 95 3.5 87 13

[0102] 2) Mass spectrometry conditions

[0103] The data were collected in the electrospray ionization (ESI) positive ion (+) detection mode using the multiple reaction monitoring (MRM) mass spectrometry mode. The ion source parameters are shown in Table 2.

[0104] Table 2. Mass spectrometer ion source parameters

[0105] Parameter Type Parameter Value Capillary voltage 3.5KV Cone voltage 30V Ion source temperature 150℃ Desolvation temperature 450℃ Cone hole gas flow rate 150(L / Hr) Desolventization flow rate 1000(L / Hr)

[0106] 3) Peptide marker mass spectrometry MRM mode detection parameters

[0107] According to the different mass-to-charge ratios of the target peptide markers, mass spectrometry detection parameters were set up, as shown in Table 3.

[0108] Table 3. MRM mode parameters for target peptide detection by liquid chromatography-tandem mass spectrometry

[0109] Peptides Mass-to-charge ratio (m / z) Charge type Charge number [z] Start[min] End[min] (N)CE P1 508.8030 +H 2 1.49 1.76 16 P2 658.8257 +H 2 0.75 1.05 26 P3 524.7966 +H 2 0.76 1.04 15 P4 417.7105 +H 2 0.50 0.76 16 P5 528.2922 +H 2 1.50 1.77 18

[0110] 4) Mass spectrometry identification of each characteristic peptide segment

[0111] The mass spectrometry fragment ion compositions of characteristic peptides P1 to P5 are shown in Tables 4 to 8, respectively. Figure 1 As shown, the mass spectrometry identification spectra of characteristic peptide segments P1 to P5 are as follows: Figure 2 to Figure 6 shown.

[0112] Table 4. Mass spectrometry identification of fragment ions of P1 characteristic peptides

[0113] serial number Singly charged m / z sequence sequence Singly charged m / z serial number B1 118.0863 V LTSGDAIK 917.5302 Y9 B2 231.1703 V L TSLGDAIK 804.4462 Y8 B3 332.2180 VLT SLGDAIK 703.3985 Y7 B4 419.2500 VLTS LGDAIK 616.3665 Y6 B5 532.3341 VLTSL GDAIK 503.2824 Y5 B6 589.3556 VLTSLG DAIK 446.2609 Y4 B7 704.3825 VLTSLGD AIK 331.2340 Y3 B8 775.4196 VLTSLGDA IK 260.1969 Y2 B9 888.5037 VLTSLGDAI K 147.1128 Y1

[0114] Table 5. Mass spectrometry identification of fragment ions of P2 characteristic peptides

[0115] serial number Singly charged m / z sequence sequence Singly charged m / z serial number B1 118.0863 V NVEDAGGETLGR 1217.5757 Y12 B2 232.1292 VN VEDAGGETLGR 1103.5327 Y11 B3 331.1976 VNV EDAGGETLGR 1004.4643 Y10 B4 460.2402 VNVE DAGGETLGR 875.4217 Y9 B5 575.2671 VNVED AGGETLGR 760.3948 Y8 B6 646.3043 VNVEDA GGETLGR 689.3577 Y7 B7 703.3257 VNVEDAG GETLGR 632.3362 Y6 B8 760.3472 VNVEDAGG ETLGR 575.3148 Y5 B9 889.3898 VNVEDAGGE TLGR 446.2722 Y4 B10 990.4374 VNVEDAGGET LGR 345.2245 Y3 B11 1103.5215 VNVEDAGGETL GR 232.1404 Y2 B12 1160.5430 VNVEDAGGETLG R 175.1190 Y1

[0116] Table 6. Mass spectrometry identification of fragment ions of P3 characteristic peptides

[0117] serial number Singly charged m / z sequence sequence Singly charged m / z serial number B1 120.0655 T IIVSMWAK 947.5383 Y8 B2 233.1496 TI IVSMWAK 834.4542 Y7 B3 346.2337 TII VSMWAK 721.3702 Y6 B4 445.3021 TIIV SMWAK 622.3017 Y5 B5 532.3341 TIIVS MWAK 535.2697 Y4 B6 663.3746 TIIVSM WAK 404.2292 Y3 B7 849.4539 TIIVSMW AK 218.1499 Y2 B8 920.4910 TIIVSMWA K 147.1128 Y1

[0118] Table 7. Mass spectrometry identification of fragment ions of P4 characteristic peptides

[0119]

[0120]

[0121] Table 8. Mass spectrometry identification of fragment ions of P5 characteristic peptides

[0122] serial number Singly charged m / z sequence sequence Singly charged m / z serial number B1 132.1019 L FTVYPS 942.4931 Y8 B2 279.1703 LF TVYPSTK 795.4247 Y7 B3 380.2180 LFT VYPST 694.3770 Y6 B4 479.2864 LFTV YPSTK 595.3086 Y5 B5 642.3497 LFTVY PSTK 432.2453 Y4 B6 739.4025 LFTVYP STK 335.1925 Y3 B7 826.4345 LFTVYPS TK 248.1605 Y2 B8 927.4822 LFTVYPST K 147.1128 Y1

[0123] (5) Data analysis

[0124] The raw data generated by the tandem mass spectrometer is the instrument's response intensity to peptide fragments of different concentrations. Using TargetLynx software, the characteristic peptides are automatically integrated by the peak area integration method, the peak area score of each peptide fragment specific ion pair is calculated, and the information intensity of the mass spectrometer detection is converted into the concentration of the analyte. The results are as follows: Figure 7 to Figure 11 shown.

[0125] (6) Identification of deletion α-thalassemia

[0126] Through a large number of experiments, this study found that the ratios of the peak areas of peptides such as P1, P2, P3, P4, and P5 are associated with different types of deletional α-thalassemia, as shown in Table 9 for details.

[0127] Table 9. Correlation indicators of deletion type α-thalassemia

[0128] type Indicator 1 Indicator 2 Indicator 3 α P5 / P2 P5 / P1 P4 / P2 <![CDATA[α + ]]> P5 / P2 P5 / P1 P4 / P2 <![CDATA[αα / -α 3.7 ]]> P5 / P2 P5 / P1 P4 / P1 <![CDATA[αα / -α 4.2 ]]> P5 / P2 P4 / P2 P4 / P1 <![CDATA[α CS a / a]]> P5 / P2 P5 / P1 P4 / P2 <![CDATA[α QS a / a]]> P5 / P2 P5 / P1 P4 / P2 <![CDATA[α WS a / a]]> P3 / P1 P3 / P2 P4 / P1 <![CDATA[α 0 ]]> P3 / P1 P3 / P2 P5 / P2 αα / --SEA P3 / P1 P3 / P2 P5 / P2 <![CDATA[-α 3.7 / -a 3.7 ]]> P5 / P1 P5 / P2 P4 / P1 <![CDATA[-α 4.2 / -a 3.7 ]]> P5 / P1 P5 / P2 P4 / P1 HkDJ P3 / P1 P3 / P2 P5 / P1 <![CDATA[-α 3.7 / --SEA]]> P3 / P1 P3 / P2 P5 / P1 <![CDATA[-α 4.2 / --SEA]]> P3 / P1 P3 / P2 P5 / P1

[0129] (7) Reference value range of related indicators

[0130] According to the above technical scheme of the present invention, 200 normal controls and 81 patients with α-thalassemia were analyzed, and the reference value interval (bilateral 95th) was established as follows. The reference interval may vary due to different laboratories, different experimental personnel, different instruments, different reagent batches, and different internal standard concentrations. Laboratories can establish their own reference value intervals accordingly.

[0131] Table 10. Reference intervals of P3 / P1 ratio in normal subjects and patients with α-thalassemia

[0132]

[0133] Table 11. Reference intervals of P3 / P2 ratio in normal subjects and patients with α-thalassemia

[0134]

[0135] Table 12. Reference intervals of P5 / P1 ratios in normal controls and patients with α-thalassemia

[0136]

[0137]

[0138] Table 13. Reference intervals of P5 / P2 ratio in normal subjects and patients with α-thalassemia

[0139]

[0140] Table 14. Reference intervals of P4 / P1 ratios in normal subjects and patients with α-thalassemia

[0141]

[0142]

[0143] Table 15. Reference intervals of P4 / P2 ratio in normal subjects and patients with α-thalassemia

[0144]

[0145] Example 2. Evaluation of reagent efficacy

[0146] (1) Sensitivity analysis of α-thalassemia-related indicators

[0147] The method described in the present invention is for the treatment of α-thalassemia quiescent type (α + ), α-thalassemia intermedia (α 0 ) and α-thalassemia HbH genotypes had an average detection rate of 83.70%, 82.00% and 100.00% respectively.

[0148] On a Waters Xevo TQ-S tandem mass spectrometer, the ratio of P3 to P1 is WS α / αα, αα / --SEA, -α 3.7 / --SEA and -α 4.2 The detection rates of genotypes such as / --SEA were all 100.00%. 3.7 / --SEA and -α 4.2 The sensitivity of genotype detection of / --SEA was also 100.00%. 3.7 (85.00%), α CS α / αα(100.00%), α QS α / αα(100.00%), -α 3.7 / -α3.7 (100.00%), -α 4.2 / -α 3.7 (100.00%), -α 3.7 / --SEA(100.00%) and -α 4.2 / --SEA (100.00%) detection sensitivity is also very high. P5 and P2 can efficiently detect αα / -α 3.7 (90.00%), αα / -α 4.2 (87.50%), α CS α / αα(100.00%), α QS α / αα(100.00%), -α 3.7 / -α 3.7 (100.00%), -α 4.2 / -α 3.7 (100.00%), -α 3.7 / --SEA(100.00%) and -α 4.2 / --SEA (100.00%) and other α-thalassemia genotypes. The ratio of P4 to P1 is αα / -α 3.7 (85.00%), α CS α / αα(91.67%), -α 3.7 / -α 3.7 (100.00%), -α 4.2 / -α 3.7 (100.00%), -α 3.7 / --SEA(100.00%) and -α 4.2 / --SEA (100.00%) and other genotypes. P4 / P2 can efficiently detect αα / -α 4.2 (87.50%), α CS α / αα(91.67%), α QS α / αα(100.00%), -α 3.7 / -α 3.7 (100.00%), -α 4.2 / -α 3.7 (100.00%), -α 3.7 / --SEA(100.00%) and -α 4.2 / --SEA (100.00%) and other α-thalassemia genotypes.

[0149] Table 16. Sensitivity analysis of α-thalassemia related indicators

[0150]

[0151]

[0152] (2) Specificity analysis of α-thalassemia-related indicators

[0153] The method described in the present invention is for the treatment of α-thalassemia quiescent type (α + ), α-thalassemia intermedia (α 0 ), the specificity of α-thalassemia HbH is greater than 88.50%. The ratio of P3 to P1, P3 to P2 is positive for αα / --SEA, -α 3.7 / --SEA and -α 4.2 The specificity of the detection of genotypes such as / --SEA was 100.00%. The ratio of P5 to P1 and P5 to P2 was -α 3.7 / -α 3.7 , -α 4.2 / -α 3.7 and -α 4.2 / --SEA (100.00%) had a specificity of 100.00% for αα / -α 3.7 , α CS α / αα、α QS α / αα、-α 3.7 / -α 3.7 , -α 3.7 / --The specificity of SEA detection was greater than 80.50%. The ratio of P4 to P1 was QS α / αα(100.00%), -α 3.7 / -α 3.7 (100.00%), -α 4.2 / -α 3.7 (100.00%), -α 3.7 / --SEA(96.50%) and -α 4.2 / --SEA (96.50%) and other genotypes have high specificity. P4 / P2 can efficiently detect αα / -α 3.7 (87.00%), α CS α / αα(99.00%), α QS α / αα(99.50%), -α 3.7 / -α 3.7 (100.00%), -α 4.2 / -α 3.7 (100.00%), -α 3.7 / --SEA(99.50%) and -α 4.2 / --SEA (99.50%) and other α-thalassemia genotypes.

[0154] Table 17. Specificity analysis of α-thalassemia related indicators

[0155]

[0156]

[0157] The above results show that the detection method provided by the present invention has good test efficiency, which further proves that the method can be applied in clinical practice on a large scale.

Claims

1. A biomarker composition for deletion α-thalassemia, characterized in that: The biomarker composition for deletion-type α-thalassemia is a combination of any two polypeptide fragments of P1, P2, P3, P4, and P5; the amino acid sequence of P1 is shown in SEQ ID NO: 1, the amino acid sequence of P2 is shown in SEQ ID NO: 2, the amino acid sequence of P3 is shown in SEQ ID NO: 3, the amino acid sequence of P4 is shown in SEQ ID NO: 4, and the amino acid sequence of P5 is shown in SEQ ID NO:

5.

2. The biomarker composition according to claim 1, characterized in that The biomarker composition for deletion-type α-thalassemia is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, and P5 and P2.

3. The biomarker composition according to claim 1, characterized in that The deletion type α-thalassemia includes α + , α 0 and / or HbH.

4. The biomarker composition according to claim 3, characterized in that: The α + The biomarker composition is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, P5 and P2; and / or the α 0 The biomarker composition of is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, P5 and P2; and / or the biomarker composition of HbH is any one or more of P3 and P1, P3 and P2, P4 and P1, P4 and P2, P5 and P1, P5 and P2.

5. The biomarker composition according to claim 3, characterized in that: The α + Include-α 3.7 , -α 4.2 , α CS , α QS and / or α WS ; the α 0 include-- SEA , -α 3.7 / -α 3.7 and / or -α 4.2 / -α 3.7 ; The HbH includes -α 3.7 / --SEA and / or -α 4.2 / --SEA.

6. The biomarker composition according to claim 5, characterized in that: The-α 3.7 The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 4.2 The biomarker composition is any one or more of the following combinations: P5 and P2, P4 and P2; CS The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the α QS The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P2, P4 and P1; the α WS The biomarker composition is P3 and P1; the - SEA The biomarker composition is any one or more of the following combinations: P3 and P1, P3 and P2; 3.7 / -α 3.7 The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 4.2 / -α 3.7 The biomarker composition is any one or more of the following combinations: P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 3.7 / --The biomarker composition of SEA is any one or more of the following combinations: P3 and P1, P3 and P2, P5 and P1, P5 and P2, P4 and P1, P4 and P2; the -α 4.2 / --The biomarker combination of SEA is any one or more of the following combinations: P3 and P1, P3 and P2, P5 and P1, P5 and P2, P4 and P1, P4 and P2.

7. A detection reagent for deletion type α-thalassemia, characterized in that: The detection reagent includes any one or more of reagent A, reagent B, reagent C, and reagent D; the reagent A is a mixture of methanol and water; the reagent B is a mixture of acetonitrile, methanol, and water; the reagent C is a pancreatic enzyme solution; and the reagent D is a mixture of acetonitrile and water.

8. A method for separating and determining the peak area ratio of the biomarker composition according to claim 1, characterized in that: Liquid chromatography-tandem mass spectrometry is used for separation, qualitative, quantitative and ratio determination, and the liquid chromatography-tandem mass spectrometry method comprises the following steps: (1) Sample pretreatment: treating the sample to be tested with the detection reagent described in claim 6 to obtain a sample solution containing the polypeptide fragment; the sample to be tested is an ex vivo blood sample of a patient with deletion α-thalassemia or a patient suspected of having deletion α-thalassemia; (2) Liquid chromatography separation: The chromatographic column uses silica gel particles modified with cyano functional groups as filler, deionized water containing formic acid as mobile phase A, and acetonitrile containing formic acid as mobile phase B, and the peptide fragments in the sample solution are separated by gradient elution; The gradient elution procedure is as follows: ； (3) Mass spectrometry: Mass spectrometry is used for determination, and the peak area integration method is used to quantify each peptide segment; (4) Ratio calculation: Based on the peak areas of each peptide segment measured in step (3), calculate the peak area ratio of the peptide marker.

9. The method according to claim 8, characterized in that The mass spectrometry detection is performed in an electrospray ionization cation detection mode using a mass spectrometry mode with multiple reaction monitoring.

10. Use of the biomarker composition according to any one of claims 1 to 6 and / or the detection reagent according to claim 7 in the preparation of a product for screening various types of deletion α-thalassemia.

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