CN110658251A

CN110658251A - Use of a characteristic protein composition or mass spectral model for characterizing thalassemia

Info

Publication number: CN110658251A
Application number: CN201910945987.7A
Authority: CN
Inventors: 何昆; 吕倩; 安鹏; 牛燕燕; 马庆伟; 向华
Original assignee: Changsha Xianghua Mass Spectrometry Medical Technology Co ltd
Current assignee: Changsha Xianghua Mass Spectrometry Medical Technology Co ltd; Academy of Military Medical Sciences AMMS of PLA
Priority date: 2019-10-01
Filing date: 2019-10-01
Publication date: 2020-01-07

Abstract

The invention provides a characteristic protein fragment composition for detecting thalassemia and a mass spectrum model for evaluating the curative effect of a thalassemia drug or the curative method. Wherein the sequences of the characteristic protein fragment compositions are respectively shown as SEQ ID No. 1-3. The characteristic protein fragment composition or the mass spectrum model can be used for diagnosing and screening the thalassemia and evaluating the curative effect of the thalassemia treatment method and the curative effect of the mediterranean anemia, the method is simple and easy to operate, the accuracy is high, and a new method and a new thought are provided for the thalassemia diagnosis and screening, the mediterranean anemia treatment method and the curative effect evaluation of the mediterranean anemia.

Description

Use of a characteristic protein composition or mass spectral model for characterizing thalassemia

Technical Field

The invention belongs to the field of biotechnology, and relates to a composition or mass spectrum model for detecting characteristic hemoglobin of thalassemia, and a method for rapidly identifying hemoglobin abnormality and evaluating curative effect of a characteristic protein, especially a hemoglobin fragment-related disease treatment method or a drug by using the composition or the model. Also, the invention provides diagnostic products and uses involving the composition or model.

Background

Hemoglobin, also known as hemoglobin, is a heterotetrameric complex of 2 beta-gene globins combined with 2 alpha-gene globins, which is a major component of red blood cells and can bind oxygen, transporting oxygen and carbon dioxide. The hemoglobin content reflects the degree of anemia well. Hemoglobinopathies are a group of the largest genetic human monogenic diseases in the world and are also a collective name of thalassemia and abnormal hemoglobinopathies, wherein thalassemia has great harm to human beings and is common in southern China and is higher in the places of Guangdong, Guangxi, Hainan and the like [1 ]. The most fundamental characteristic is that one or more globin peptide chains (namely any one of the globin chains of alpha-, beta-, gamma-and delta-) in the hemoglobin caused by the globin gene defect are difficult to synthesize or can not be synthesized, and finally, the components in the hemoglobin are changed, and even abnormal hetero and pure tetramer Hb pairing polymers are generated.

The major patients are mainly chronic progressive hemolytic anemia, while the minor patients have no clinical symptoms particularly evident [2 ]. According to the inhibition of globin chain synthesis, the blood is divided into four types, namely alpha-thalassemia, beta-thalassemia, gamma-thalassemia and delta-thalassemia, wherein alpha-thalassemia and beta-thalassemia are the most common. Still at present, there is no means with obvious effect on treating thalassemia patients, so that accurate and scientific screening and diagnosis of the thalassemia are very important.

The onset of alpha-thalassemia is due to the loss of alpha chain synthesis, and the onset of beta-thalassemia is due to the loss of beta chain synthesis. Among the α thalassemias, there are the categories resting, mild, intermediate and severe. Static type: the erythrocyte has normal shape, the Hb Bart's content in the umbilical cord blood at birth is 0.01-0.02, but the erythrocyte disappears after 3 months. And (3) light: the morphology of the red blood cells is slightly changed, such as different sizes, shallow staining in the center, abnormal shape and the like; the osmotic fragility of the rhodosporium decreases; denatured globin corpuscle positive; HbA2 and HbF levels were normal or slightly low. The content of Hb Bart's in the umbilical blood of the infant patient is 0.034-0.140, and the Hb Bart's completely disappears in 6 months after the infant patient takes a baby. Intermediate type: changes in peripheral and myeloid manifestations resemble severe beta-thalassemia; decreased osmotic fragility of erythrocytes; denatured globin corpuscle positive; HbA2 and HbF levels were normal. About 0.25Hb Bart's and a minor amount of HbH in the blood at birth; with the age, HbH gradually replaces HbBart's, and the content of the HbBart's is 0.024-0.44. The inclusion body formation test was positive. Heavy: peripheral blood mature red blood cell morphology changes such as severe beta-thalassemia, significantly increased nucleated red blood cell counts and reticulocyte counts. Almost all of the hemoglobin was HbBart's, or a small amount of HbH at the same time, no HbA, HbA2 and HbF.

Beta-thalassemia is classified into severe, mild and intermediate types. (1) Heavy: the peripheral hemogram is microcytochrome anemia, the size of red blood cells is different, a central shallow staining area is enlarged, and abnormal shapes, target shapes, fragmented red blood cells, nucleated red blood cells, spot-colored red blood cells, polychromatic red blood cells, haugh-pericytes and the like appear; reticulocytes are normal or elevated. The myeloid cells are marked by active erythroid cell proliferation, which is dominated by middle and late erythroid cells, and the mature erythrocytes are changed as in peripheral blood. The osmotic fragility of the red blood cells is obviously reduced. HbF levels are significantly increased, mostly >0.40, which is an important basis for diagnosing severe beta-thalassemia. The skull X-ray film can show that the inner plate and the outer plate of the skull become thinner, the plate barrier is widened, and vertical short hair-like bone spurs appear between the bone cortex. (2) And (3) light: the characteristic of the model is that the mature red blood cells have slight morphological changes, the infiltration of the red blood cells is more brittle and normal or reduced, and the hemoglobin electrophoresis shows that the HbA2 content is increased (0.035-0.060). HbF content was normal. (3) Intermediate type: the change of peripheral hemogram and bone marrow picture is severe, the infiltration brittleness of red blood cells is reduced, the HbF content is 0.40-0.80, and the HbA2 content is normal or increased.

Because there is no effective treatment for thalassemia, abnormal alpha and b globin genes carried by couples can be inherited to offspring, and according to the genetic condition, if both couples are alpha poor gene carriers, 25% of couples are at risk of delivering a heavy alpha poor child and causing intrauterine edema and stillbirth of fetus. Currently, the birth of children patients can only be avoided clinically through prenatal screening, and no effective treatment measures are available for alpha-thalassemia homozygous fetuses. Therefore, the purpose of prenatal diagnosis is to prevent the birth of homozygous fetus and reduce the birth of heterozygous fetus.

There are many non-genetic detection techniques for thalassemia, such as hemoglobin electrophoresis, blood routine, erythrocyte osmotic fragility test, etc. Currently, a suitable prenatal screening method is to examine Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin (MCH) by a hematology analyzer. MCH is more reliable than MCV because red blood cells may swell when stored at room temperature. The carriers can be basically and completely screened by taking MCH <27pg or MCV <80fl as a standard. When MCH or MCV is less than this standard, hemoglobin electrophoresis should be performed, and if HbA2 is < 2.5%, it is highly suspected to be an alpha poor gene carrier. If HbH inclusion bodies are found, then intermediate form α -thalassemia can be diagnosed. Whole blood ferritin assays should also be noted to rule out iron deficiency anemia. The method has low operation requirement, low cost, but poor sensitivity and low specificity, and the hemoglobin electrophoresis method widely applied in recent years also has the defects of poor accuracy and poor repeatability. Moreover, MCV and erythrocyte osmotic fragility tests of part of patients can be normal, which is the main reason for missed diagnosis of beta-thalassemia gene carriers, and the missed diagnosis rate reaches 13.23%.

With the development of prenatal screening and imaging technologies, more and more pregnant women of middle and late pregnancy are required to perform prenatal diagnosis. Among them, ultrasonic diagnosis is one of the simple and feasible methods for detecting Bart's edematous fetuses in the middle of pregnancy. Bart's edematous fetuses can also be distinguished by ultrasonically detecting the heart-chest ratio, the placenta thickness and the amniotic fluid volume of the fetuses after 12 weeks of pregnancy. The method has the advantages that the method has reports of diagnosing different types of fetal anemia by detecting the blood flow velocity of artery in the brain of a fetus through ultrasonic Doppler at home and abroad, so that invasive operation for reducing the fetal anemia risk by more than 70 percent is considered to be the best method for diagnosing the fetal anemia. Because of its non-traumatic property, simple and quick, it is more acceptable by patients at present.

The villus biopsy is suitable for the early pregnancy, generally performed in 8-10 weeks of pregnancy, and is rarely used for pregnant women requiring continuous pregnancy at present because the natural abortion rate reaches 2% -3%.

Amniotic fluid and umbilical vessel puncture blood taking examination amniotic fluid puncture is the most common prenatal diagnosis method for fetal chromosomal diseases at present, the puncture abortion rate is only 0.15% -0.2%, amniotic cavity puncture is limited to 16-23 weeks of gestation, umbilical vessel puncture is a breakthrough prenatal diagnosis technology developed after 80 years in the 20 th century, and the prenatal diagnosis success rate, accuracy and prenatal diagnosis range are greatly improved.

The gene chip diagnosis technology thalassemia diagnosis gene chip (ThalachitTM) is a new technology for identifying known thalassemia genotypes in China based on a DNA chip technology, the gene chip diagnosis technology adopts a fluorescence labeling and primer extension method on the basis of nucleic acid amplification, can improve the sensitivity and specificity of a detection result, can finish alpha and beta thalassemia gene diagnosis on one chip due to the high flux characteristic of the gene chip, and is suitable for large-area general investigation. The genetic diagnosis of the thalassemia in China starts in the 80 th century, and successively goes through 5 development stages of DNA dot hybridization, restriction enzyme zymogram analysis, Restriction Fragment Length Polymorphism (RFLP) linkage analysis, oligonucleotide (ASO) probe hybridization, PCR in vitro gene amplification and the like, the accuracy of the thalassemia detection can be greatly improved, the omission ratio of the thalassemia is reduced, and therefore the genetic detection is also called as a gold standard for diagnosing the thalassemia. And becomes the most common thalassemia gene diagnosis method in clinic at present.

However, the above methods either have the disadvantages of poor sensitivity and low specificity due to the need of biopsy sampling, or have the disadvantages of long test reaction time and expensive PCR reagent, so that a new analysis method is required to achieve a fast, accurate, cheap and convenient classification result.

A matrix-assisted laser desorption ionization ion source and a time-of-flight mass analyzer. The principle of MALDI is the process of irradiating a co-crystallized thin film formed by a sample and a matrix with laser light, the matrix absorbing energy from the laser light to be transferred to biomolecules, and the ionization process transferring protons to or from the biomolecules to ionize them. Therefore, the method is a soft ionization technology and is suitable for measuring mixtures and biomacromolecules. MALDI-TOF-MS has characteristics such as sensitivity height, degree of accuracy height and resolution ratio height, provides a powerful analysis and test means for fields such as life science, and is playing more and more important role.

Chinese patent application 201810001598.4, the title of the invention, "a method for detecting glycated hemoglobin" discloses a method for detecting glycated hemoglobin using a time-of-flight mass spectrometer, which can obtain a mass spectrogram of a sample to be detected in the range of 3000-; the ratio of glycated hemoglobin was obtained by the formula A/(A + B) × 100%. Although this method employs MALDI-TOF-MS detection, the purpose is to detect the binding product of hemoglobin and blood glucose, and accurate quantitative analysis by the difference in mass-to-charge ratios of glycated hemoglobin and non-glycated hemoglobin does not allow monitoring of globin chains of different configurations.

In conclusion, a mass spectrum model for detecting the characteristic protein of thalassemia by using MALDI-TOF-MS technology and a related diagnosis technology thereof are urgently needed in China.

Disclosure of Invention

The invention aims to overcome the defects of a technology for detecting abnormal hemoglobin of thalassemia, and provides a mass spectrum model for detecting characteristic hemoglobin of thalassemia, a treatment method and drug efficacy evaluation and a preparation method thereof. The invention is based on the principle that a MALDI-TOF-MS mass spectrometer is utilized to determine and compare hemoglobin spectrograms of blood samples of thalassemia patients and healthy people, and corresponding hemoglobin fragment markers are screened out.

Therefore, the first object of the present invention is to provide a signature protein marker composition for diagnosing thalassemia, which comprises 3 signature protein fragments, wherein the sequences of the signature protein fragments are shown in SEQ ID Nos. 1-3:

SEQ ID No.1：

MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR

SEQ ID No.2：

MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

SEQ ID No.3：

MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDAIKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH，

wherein, the characterization mass-to-charge ratios of the three characteristic protein (SEQ ID NO:1-3) peaks are respectively as follows: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment) of the three protein peaks, and m/z deviation allowed within 0.15%. Wherein the characteristic protein sequence SEQ ID NO 1 exists in blood of fetuses, children and adults; the content of the characteristic protein sequence SEQ ID NO 2 is extremely low in the fetal period and can not be detected almost, and the content is gradually increased to be detected after birth; the characteristic protein sequence SEQ ID NO 3 is mainly present in the blood of fetuses and infants up to 2 years old, and is very low in content and hardly detectable in healthy adults.

Due to differences in the mass-to-mass ratio and relative content of the three proteins in whole blood samples of thalassemia patients (including fetuses, children and adults) and healthy people (including fetuses, children and adults), the m/z deviation is allowed to be within +/-0.15%. In which the physiological role of the gamma protein after birth of the fetus is gradually replaced by the beta protein. The gamma protein peak intensity is therefore very low in healthy adults. But gamma compensation peaks appear in partially beta-poor patients. Thus abnormal upregulation of the gamma peak and the presence or change in m/z of the above characteristic protein can be used as an indication of the detection of thalassemia. In one embodiment, the peaks of the healthy population characteristic proteins (SEQ ID NOS: 1-3) are characterized by mass-to-charge ratios of: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment), wherein the deviation tolerance range of m/z related to the invention is +/-0.15%. Wherein the 15120m/z peak is used to determine the intensity of change in up-or down-regulation of 15859m/z or 15989 m/z. See normal human map (figure 1).

In a preferred embodiment, the peaks of the healthy population characteristic proteins (SEQ ID NOS: 1-2) are characterized by mass-to-charge ratios of: 15119m/z (. alpha. -globin fragment), 15867m/z (. beta. -globin fragment).

In another embodiment, the thalassemia patient characteristic protein (SEQ ID NOS: 1-3) peaks are characterized by mass-to-charge ratios of: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment) of the three protein peaks, and m/z deviation allowed within 0.15%. . Wherein, in the detected sample, when 15859m/z of the peak of the characterization beta globin fragment is down-regulated to express or 15989m/z of the peak of the characterization v globin fragment is up-regulated to express in the population over 2 years old, the detected sample is indicated to be a thalassemia patient or a potential patient; after the thalassemia patient is treated, when 15989m/z representing the nuzhu protein fragment peak is up-regulated relative to the original expression quantity, the curative effect of the medicine of the detected person is indicated to be good, and the medicine can be continuously used for treatment. Wherein the 15120m/z peak is used to determine the intensity of change in up-or down-regulation of 15859m/z or 15989 m/z. See also map of thalassemia patients (fig. 2).

In a preferred embodiment, the peaks of the thalassemia patient characteristic proteins (SEQ ID NOS: 1-2) are characterized by mass-to-charge ratios of: 15112m/z (. alpha. -globin fragment), 15853m/z (. beta. -globin fragment).

In any of the above embodiments, the signature protein marker composition further comprises 1 to 3 internal standard protein standards for relative quantification, preferably Apomyoglobin (Apomyoglobin), which are close to the signature protein detection range, in addition to the 3 signature protein fragments, and the peak absolute intensity of the signature protein and the variation of up-regulation or down-regulation thereof can be directly converted by comparing the peak intensities of the internal standard proteins. In a preferred embodiment, wherein the standard is Apomyoglobin (Apomyoglobin), m/z is 169952.

The second object of the present invention is to provide a mass spectrometric model of characteristic proteins for diagnosing thalassemia, which includes but is not limited to the above-mentioned polypeptide marker composition, wherein the marker composition is composed of 3 characteristic protein fragments, wherein the sequences of the characteristic protein fragments are shown in SEQ ID Nos. 1-3, respectively, and the m/z deviation is within the allowable range of + -0.15%.

In one embodiment, when the expression of 15859m/z is down-regulated or the expression of 15989m/z is up-regulated in the population over 2 years old in the sample to be detected, the human to be detected is a thalassemia patient or a potential patient; after the local thalassemia patient is treated, when 15989m/z (gamma globin fragment) is relatively up-regulated and expressed, the curative effect of the medicine of the detected person is indicated to be good, and the medicine can be continuously used for treatment.

In another preferred embodiment, the peaks of the thalassemia patient characteristic proteins (SEQ ID NOS: 1-2) are characterized by mass-to-charge ratios of: 15112m/z (. alpha. -globin fragment), 15853m/z (. beta. -globin fragment).

In any of the above embodiments, the 15120m/z peak intensity is taken as the standard intensity and is defined as 1. The cutoff value of peak intensity 15859m/z was set to 0.5. When the expression of 15859m/z is lower than 0.5, the result is indicated that the patient is a thalassemia patient or a potential patient. In the course of tracking therapeutic effect, when 15989m/z is up-regulated to over 0.1 relative to the original expression level, it indicates that the tested patient is the thalassemia patient or potential patient with the currently adopted therapeutic method or medicine with better therapeutic effect.

It is a third object of the present invention to provide a diagnostic product for diagnosing thalassemia comprising the above-described marker composition, or comprising the above-described mass spectrometry model.

In one embodiment, the diagnostic product is selected from the group consisting of a diagnostic kit consisting of whole blood quality control, buffer, matrix solution, time-of-flight mass spectrometry based thalassemia detection software, a specific target plate for thalassemia mass spectrometry detection. The kit can be used for providing standard data or curve comparison when a sample to be detected is subjected to mass spectrometry so as to judge whether the sample to be detected is a thalassemia patient or whether a medicine and a treatment method are effective.

In another embodiment, the kit may contain software or a chip of the standard database of the 3 characteristic protein polypeptides, which can be used to provide standard data or curve comparison when performing mass spectrometry on a sample to be tested, so as to determine whether the sample to be tested is a thalassemia patient or whether the drug and the treatment method are effective.

The fourth invention of the invention is to provide the application of the polypeptide marker composition or the mass spectrum model in preparing products for diagnosing thalassemia.

In one embodiment, the product comprises: diagnostic reagent, special target plate for detection, chip, carrier, kit and the like.

The fifth invention aim of the invention is to provide a construction method for preparing the mass spectrum model, which comprises the following steps:

1) collecting whole blood of a plurality of clinically confirmed patients and whole blood of normal contrast personnel as two groups of whole blood samples, and performing low-temperature freezing for later use;

2) pre-mass spectrometric pretreatment of the whole blood protein polypeptide and addition of the standard apophysemoglobin (Apomyoglobin) to the sample;

3) performing mass spectrometry detection reading on the two groups of the pretreated whole blood polypeptides to obtain fingerprint spectrums of the two groups of the whole blood polypeptides;

4) carrying out standardized processing on the fingerprint spectrums of the whole blood polypeptides of all cancer patients and normal persons, and collecting data;

5) the obtained data are subjected to experimental quality control treatment, and the following mass spectrum peaks (m/z) are shared: 5042, 5809, 5933, 6986, 7562, 7667, 7932, 8085, 8475, 8580, 15120, 15329, 15859, 15989

6) Screening 3 polypeptide peaks with the following mass-to-charge ratios, which are characteristic of thalassemia, from step 5): 15120m/z, 15859m/z, 15989m/z, and establishing a mass spectrum model for diagnosing thalassemia according to the protein polypeptide markers and the change of mass-to-charge ratio peaks, wherein the polypeptide sequences of the 3 thalassemia characteristic proteins are normal control proteins, and the sequences are respectively shown in SEQ ID No. 1-3.

In one embodiment, the allowable range of m/z deviation of the three protein peaks is ± 0.15%, and thus, the peaks of the healthy population characteristic protein (SEQ ID NOs: 1-3) have the characteristic mass-to-charge ratios of: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment), characterized mass to charge ratios of the patient characteristic protein (SEQ ID NOS: 1-3) peaks: 15120m/z (. alpha. -globin fragment), 15859m/z (. beta. -globin fragment), 15989m/z (. v. -globin fragment).

In any of the above embodiments, in step 2), during the sample processing, 1 to 3 internal standard protein standards, preferably Apomyoglobin (Apomyoglobin), close to the detection range of the characteristic protein are added in advance for relative quantification, so that hemoglobin can be quantified relatively more accurately, that is, by comparing the peak intensities of the internal standards, the peak absolute intensity of the characteristic protein and the variation value of up-regulation or down-regulation can be directly converted. In a preferred embodiment, wherein the standard is Apomyoglobin (Apomyoglobin), m/z is 169952.

The sixth invention of the present invention is to provide a use of a characteristic protein composition or a mass spectrum model for characterizing thalassemia for screening a drug for thalassemia or evaluating a treatment method for thalassemia.

In one embodiment, the signature protein marker composition consists of 3 signature protein fragments, wherein the sequences of the signature protein fragments are shown in SEQ ID Nos. 1-3, respectively.

In a specific embodiment, the peaks of the healthy population characteristic proteins (SEQ ID NOS: 1-3) are characterized by mass-to-charge ratios of: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment), wherein the deviation tolerance range of m/z related to the invention is +/-0.15%. Wherein the 15120m/z peak is used to determine the intensity of change in up-or down-regulation of 15859m/z or 15989 m/z. See normal human map (figure 1).

In another preferred embodiment, the peaks of the healthy population characteristic proteins (SEQ ID NOS: 1-2) are characterized by mass-to-charge ratios of: 15119m/z (. alpha. -globin fragment), 15867m/z (. beta. -globin fragment).

In other preferred embodiments, the peaks of the thalassemia patient characteristic proteins (SEQ ID NOS: 1-3) are characterized by mass-to-charge ratios of: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment) of the three protein peaks, and m/z deviation allowed within 0.15%. . Wherein, in the detected sample, when the expression of 15859m/z is down-regulated or the expression of 15989m/z is up-regulated in the population over 2 years old, the detection is indicated as a thalassemia patient or a potential patient; after the thalassemia patient is treated, when 15989m/z (gamma globin fragment) is up-regulated relative to the original expression level, the curative effect of the medicine of the detected person is better, and the medicine can be continuously used for treatment. Wherein the 15120m/z peak is used to determine the intensity of change in up-or down-regulation of 15859m/z or 15989 m/z. See also map of thalassemia patients (fig. 2).

In one embodiment, the method for constructing a mass spectrometry model comprises:

2) performing pre-mass spectrometric pretreatment on the whole blood protein polypeptide, and adding a standard product Apomyoglobin (Apomyoglobin) into a sample, wherein the peak value m/z of a protein spectrum of the Apomyoglobin is 169952;

In any of the above embodiments, wherein 1 to 3 internal standard protein standards, preferably Apomyoglobin (Apomyoglobin), are added in advance to perform relative quantification during the sample processing in step 2), which is close to the detection range of the characteristic protein, the hemoglobin can be relatively quantified more accurately. In a preferred embodiment, wherein the standard is Apomyoglobin (Apomyoglobin), m/z is 169952.

In any of the above embodiments, the method for screening a drug comprises determining whether the drug has an effect on the protein of interest by a mass spectrometry model for diagnosing thalassemia, thereby determining whether the drug is a desired drug having activity against thalassemia.

In any of the embodiments above, the method of treatment comprises hematopoietic stem cell transplantation and gene editing. In a preferred embodiment, the hematopoietic stem cell transplantation method is a method of treating thalassemia by transplanting hematopoietic stem cells in children of 2 to 6 years old. In another preferred embodiment, the gene editing method is to perform gene editing on umbilical cord blood of a newborn with thalassemia, perform mass spectrometry detection, and judge through a mass spectrometry model for thalassemia diagnosis, so as to screen whether the editing is effective. In a more preferred embodiment, the composition proved to be therapeutically effective in the therapeutic method may be judged to be a desired drug having activity against thalassemia, which is selected, wherein the composition may be a therapeutic composition introduced into a human body in a gene editing method, or hematopoietic stem cells introduced in a hematopoietic stem cell transplantation method.

The invention screens out corresponding thalassemia markers and establishes a detection model for analysis and detection by combining a bioinformatics method, wherein the bioinformatics method comprises the steps of carrying out standardization processing on a polypeptide fingerprint, carrying out experimental quality control processing on obtained data, screening whole blood characteristic polypeptides of expected thalassemia patients, establishing a mass spectrum model, and optionally establishing and verifying the mass spectrum model by combining a genetic algorithm with a nearest neighbor algorithm and the like. In the present invention, the coefficient of variation is preferably 6%.

Technical effects

The mass spectrum is used for detecting the characteristic polypeptide in the whole blood of the thalassemia patient, can be used for establishing a whole blood characteristic polypeptide mass spectrum model and screening and diagnosing the thalassemia, and can be used for screening and diagnosing the thalassemia.

Compared with other detection methods for thalassemia, the method has the following advantages:

firstly, the invention adopts a plurality of characteristic polypeptide combinations with significant differences between the thalassemia patients and normal people to detect the thalassemia whole blood, and adopts a method combining traditional statistics and modern bioinformatics methods to process data, thereby obtaining polypeptide fingerprint detection models of the thalassemia patients and the healthy people whole blood, and a series of discovered polypeptide quality-to-charge ratio peaks provide basis and resources for searching new more ideal thalassemia markers.

Second, compared with the conventional whole blood detection method, the method has higher sensitivity and specificity, and can be used for screening the medicament for resisting the thalassemia.

Thirdly, the construction method of the model is reasonable and feasible in design, provides a new screening method for providing the clinical cure rate of the thalassemia, and provides a new idea for exploring the mechanism of occurrence and development of the thalassemia.

Fourth, 300 whole blood samples were analyzed using the present invention, 200 of which were training groups (100 of thalassemia patients and 100 of healthy population), and 100 of which were testing groups (50 of thalassemia patients and 50 of healthy population). The verification result shows that 49 cases of the thalassemia are judged correctly, and the detection rate reaches 98%. The detection rate of the normal group is 100 percent. Therefore, the invention can diagnose the thalassemia at an early stage, evaluate the curative effect of the treatment method and the medicament and is beneficial to providing excellent medicaments and treatment methods for patients.

Fifthly, the detection range of the proximity characteristic protein can be used, which is beneficial to detecting 1-3 internal standard proteins in MALDI mass spectrum, the internal standard proteins can have different molecular weight ranges, so that the protein samples to be detected with different molecular weights can be corrected by the corresponding internal standard proteins close to the molecular weight ranges, and the peak absolute intensity of the characteristic protein and the change value of the up-regulation or down-regulation of the peak absolute intensity can be directly converted by comparing the peak intensity of the internal standard proteins. Wherein the internal standard protein is preferably apomyoglobin.

Drawings

The following experimental spectra were all MALDI-TOF-MS spectra.

FIG. 1 is a protein polypeptide map of a part of healthy human whole blood, wherein 1-A, 1-B and 1-C are protein polypeptide maps of 3 healthy human whole blood.

FIG. 2 is a polypeptide map of whole blood protein of thalassemia patients.

FIG. 3 is a whole blood protein polypeptide map of a newborn umbilical cord blood sample.

Figure 4 is a comparison of fingerprints for healthy adults and healthy neonates.

FIG. 5 is a comparison of fingerprints of healthy people and patients with thalassemia.

FIG. 6 is a fingerprint of whole blood of 2 thalassemia patients after the addition of an internal standard.

FIG. 7 is a graph of AUC for determining diagnostic sensitivity after the addition of an internal standard.

FIG. 8 is an evaluation fingerprint of the therapeutic effect of the drug administered to the thalassemia patient detected by a fingerprint mass spectrum model.

FIG. 9 is a comparison graph of the differences in the expression levels of beta-globin and gamma-globin detected in blood cells after gene editing in vitro according to the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example I Mass Spectrometry detection of Mediterranean anemia Whole blood Polypeptides and establishment of Mediterranean anemia screening model

1. Sample and instrument:

selected from 300 whole blood samples, 150 of which were from thalassemia patients, another 150 from healthy people, and in addition 10 neonatal cord blood samples. Thalassemia patients were identified by postoperative pathology reports. All whole blood samples were drawn on the early morning fasting (cord blood for newborn) and stored in a-80 ℃ cryo-refrigerator after whole blood isolation.

The matrix-assisted laser desorption time of flight mass spectrum Clin-TOF is developed by Beijing Yixinbo Biotechnology Limited. DP data analysis software developed by companies was used for data processing.

2. The technical route is as follows:

collection of whole blood: blood samples were collected in BD tubes to avoid hemolysis. The tube was slowly shaken up and down five times to mix the clots in the blood. The blood was coagulated at room temperature (25 ℃) for 1 hour and placed vertically. Where blood must be clotted exactly for one hour, otherwise different peptide profiles result due to different clotting times of the sample. SST tubes (vacuum blood collection tubes, BD Co.) were centrifuged at 1.400-2.000g for ten minutes at room temperature using a clinical centrifuge. Whole blood (supernatant) was aspirated into the corresponding labeled tube. The sample was labeled into a clean 0.5ml centrifuge tube, and the same whole blood sample was dispensed into a 50ul tube and multiple tubes. Whole blood samples were immediately cryopreserved at-80 ℃. Due to the fact that polypeptide precipitation is easily caused by repeated freezing and thawing of a whole blood sample, partial polypeptide is lost in a peptide spectrum, and repeated freezing and thawing should be avoided. Frozen whole blood is divided into permanent storage and to be subpackaged. The whole blood can be stored for years at-80 ℃ after being subpackaged.

Treatment of whole blood samples: before performing the polypeptide extraction experiment, 1 tube of each of the dispensed whole blood samples was taken from a low temperature refrigerator and placed on wet ice. Thawing for 60-90 min. And (3) sucking 2 mu L of the whole blood sample, adding the whole blood sample into 1ml of the sample treatment solution, and mixing for 30s by using a 4-position vortex mixer to obtain the whole blood treatment solution.

Sucking 5 μ L of whole blood treatment solution and 10 μ L of SA matrix, and mixing well (15 times per sample by pipette suction); sucking 1 mu L of matrix sample mixed solution to spot a target plate; then carrying out MALDI-TOF-MS detection; and finally, judging the detection result by using thalassemia identification software.

In addition, an appropriate amount (e.g., 5, 10 or 20nmol) of Apomyoglobin (Apomyoglobin) is selected and completely dissolved in an appropriate amount (e.g., 600, 800 or 1000. mu.l) of the diluent to obtain a standard for protein internal standard. Then MALDI-TOF-MS detection was performed in parallel as described above: that is, 5 μ L of whole blood (thalassemia patients) treatment solution, 5 μ L of protein internal standard and 10 μ LSA matrix are sucked and mixed uniformly (15 times per sample by pipette suction); sucking 1 mu L of matrix sample mixed solution to spot a target plate; then carrying out MALDI-TOF-MS detection; and finally, judging the detection result by using thalassemia identification software.

3. Bioinformatics method

(I) Mass Spectrometry data acquisition

A Clin-TOF mass spectrometer was used. And (3) removing impurities from 10shot at 60% of laser energy, collecting a certain point of a sample crystallization point from 50shot at 36%, and collecting 400shot at each sample crystallization point for 8 times on average. Laser frequency: 30 Hz. Data collection range: 4-18 KDa. External standard calibration with standards was performed before data was collected at every 8 sample crystallization points with average molecular weight deviation less than 200 ppm.

Experiment quality control: (1) for each collected original atlas, the quantity of peaks with S/N > being 5 is set as a standard for judging the quality of the atlas; (2) for the whole experimental operation, the peak list is shown in table 1, the intra-group variation coefficient of Sigma whole blood is adopted to ensure the experimental consistency, the variation coefficient of the method of the example is 6%, the allowable range of consistency is met, and the experimental consistency is good.

TABLE 1 thalassemia mass spectrum peak List

(II) preprocessing of raw data

The original data is processed by data DP analysis software of Beijing Yixinbo Biotechnology GmbH, and then normalized.

(III) selection of Mediterranean anemia-characteristic Polypeptides

The relative importance of each polypeptide peak to the distinction of various samples is different, and the method of T test P value and peak frequency is comprehensively applied to evaluate the relative importance of each polypeptide peak.

(IV) logistic regression algorithm

Regression analysis is a statistical analysis method for determining the quantitative relationship of interdependence between two or more variables. The application is very wide, and regression analysis is divided into unitary regression analysis and multiple regression analysis according to the number of related variables; according to the number of independent variables, simple regression analysis and multiple regression analysis can be divided; according to the type of relationship between independent variables and dependent variables, linear regression analysis and nonlinear regression analysis can be classified. If a regression analysis includes only one independent variable and one dependent variable and the relationship between the independent variable and the dependent variable can be approximated by a straight line, the regression analysis is called a univariate linear regression analysis. If two or more independent variables are included in the regression analysis and there is a linear correlation between the independent variables, it is referred to as a multiple linear regression analysis. In big data analysis, regression analysis is a predictive modeling technique that studies the relationship between dependent variables (targets) and independent variables (predictors). This technique is commonly used for predictive analysis, time series modeling, and discovering causal relationships between variables. The method adopts a logistic regression method, and the classification function adopts a linear classification algorithm.

A cross validation process is introduced in the classification process of the logistic regression algorithm set linear classification algorithm, half of patients and normal samples are randomly selected to establish a model in all samples, and the rest samples are used as validations. The method can supervise the training process, and avoid the phenomenon of 'over-learning' that the established model has good performance on a modeling sample and has poor performance on a prediction sample.

After a mass spectrum data classification model is established for a training sample by using a logistic regression algorithm and a linear classification algorithm, the classification capability of the established model is checked by using a verification sample.

Finally, fig. 1-6 are obtained, wherein:

FIGS. 1-A, 1-B and 1-C are polypeptide maps of 3 healthy human whole blood proteins. Display of protein polypeptide peaks in all modeled samples, arrows pointing to characteristic polypeptide peaks of thalassemia with charge-to-mass ratios 15120m/z, 15859 for the modeled samples; wherein the measured α globin peaks m/z are 15119m/z, 15122m/z, 15119m/z, respectively, and the β globin peaks m/z are 15867m/z, 15869m/z, 15865m/z, respectively, all falling within a range of ± 0.15% of the allowed deviation;

FIG. 2 is a whole blood protein polypeptide profile of thalassemia patients, wherein 2-A, 2-B, 2-C are whole blood protein polypeptide profiles of 3 thalassemia patients, as shown in the figure, m/z of the measured alpha globin peak and beta globin peak are within ± 0.15% of the allowable deviation;

FIG. 3 is a whole blood protein polypeptide map of a newborn umbilical cord blood sample, wherein 3-A, 3-B and 3-C are the whole blood protein polypeptide maps of 3 newborn umbilical cord blood samples. In fetal and neonatal populations, alpha and gamma proteins play a major physiological role, with very little beta protein content; wherein the measured α globin peaks have m/z 15120m/z 15122m/z 15119m/z, respectively, the β globin peaks have m/z 15859m/z 15869m/z 15858m/z, respectively, and the γ globin peaks have m/z 15989m/z 15988m/z 15987m/z, respectively, all falling within a tolerance of ± 0.15%;

figure 4 is a comparison of fingerprints for healthy adults and healthy neonates. The physiological role of gamma protein after fetal birth is gradually replaced by beta protein. The gamma protein peak intensity is therefore very low in healthy adults. But gamma compensation peaks appear in partially beta-poor patients. Therefore, abnormal upregulation of the gamma peak can be one of the indicators of thalassemia;

FIG. 5 is a fingerprint comparison of healthy population and thalassemia patients, wherein the beta globin expression intensity of the healthy population and the thalassemia patients is obviously different.

Based on the MALDI mass spectrum platform, the ratio of beta peak to alpha peak of the thalassemia patient group and the normal group is obviously different, and the mean value of the ratio of beta peak to alpha peak of 100 normal persons is 0.88. However, the serious α -thalassemia patients are misjudged as normal persons and need to be further judged. Thus, the addition of an internal standard to the test sample can play a critical role. For example, FIG. 6 is a fingerprint of whole blood from 2 thalassemia patients with an internal standard. Wherein the m/z stability of the internal standard protein standard is 169952 m/z, and the internal standard protein standard is close to the detection range of the characteristic protein and has obvious distinction from three characteristic proteins. The peak absolute intensity of the characteristic protein and the change value of the up-regulation or the down-regulation of the peak absolute intensity of the characteristic protein can be directly converted by comparing the peak intensity of the internal standard protein, which shows that the internal standard protein can verify the experimental error of a system, and can accurately quantify the peak absolute intensity of the characteristic protein and the change value of the up-regulation or the down-regulation of the peak absolute intensity of the characteristic protein, thereby completing the diagnosis of whether the thalassemia or the curative effect is generated after treatment.

EXAMPLE II identification of characteristic proteins

Magnetic bead enrichment method

1. And after the peak to be identified is determined by analysis, back-checking the sample with the highest intensity of the peak to be identified in the pre-processed sample.

2. The magnetic beads used in the previous experiments were ascertained. This sample was processed in 20 portions in parallel. The enrichment is a tube.

3. Centrifuge 1300rmp for 5 min. And taking the supernatant from the magnetic frame. Avoid leaving the magnetic bead to influence later stage experiment.

4. Spin dry the liquid and mark.

A Nano liquid UPLC liquid phase system manufactured by Waters company is adopted: the parameters were set as follows, trapping the column:

C18,5μm,180μm×20mm,nanoAcquity^TMcolumn; and (3) analyzing the column:

C₁₈,1.7μm,75μm×150mm,nanoAcquity^TMcolumn; mobile phase A: 5% acetonitrile, 0.1% formic acid in water; mobile phase B: 95% acetonitrile, 0.1% formic acid in water, all solutions being HPLC grade. The trapping flow rate is 15 mul/min, the trapping time is 3min, and the analysis flow rate is 300 ml/min; the analysis time is 60min, and the temperature of a chromatographic column is 35 ℃; the Partial Loop mode was injected in a volume of 18. mu.l.

Gradient elution program settings:

the method adopts a ThermoFisher LTQ Obitrap XL (Thermo) mass spectrum system, a Nano electrospray ion source (Michrom), and the spraying voltage is 1.4 kV; scanning time of the mass spectrum is 60 min; the experimental mode is Data dependence (Data dependence) and Dynamic Exclusion (Dynamic Exclusion), and each parent ion is excluded for 60 seconds after 2 MS/MS times; the scanning range is 400-2000 m/z; one-stage scanning (MS) uses Obitrap, resolution settingDefined as 1000009 (at m/z 400); CID and secondary scanning use LTQ; selecting single isotope of 10 ions with the strongest intensity as parent ion to carry out MS/MS (single charge exclusion without being used as parent ion) in MS spectrogram. Sequest was performed using the data analysis software Bioworks browser 3.3.1 SP1^TMThe search was performed by searching the database to IPI Human (version 3.45, entry 71983) and appending the inverse to the end of the database to reduce false positives. The parent ion error was set to 50ppm, the fragment ion error was set to 1Da, and the enzyme digestion was performed in the non-digestion mode. Search result parameter is set to deltacn>0.10, two charges Xcorr2.0, three charges Xcorr 2.5, three or more charges Xcorr3.0, peptide adaptability<1 e-003. The peptide fragment and polypeptide result displayed under the parameter condition has high accuracy and is set according to the literature and international polypeptide group regulations.

Data analysis was performed on samples of healthy persons and thalassemia patients in example 1, and 3 thalassemia-characteristic polypeptide peaks having the following mass-to-charge ratios were selected: 15120m/z, 15859m/z, 15989m/z, see Table 2.

Table 2 comparison of three polypeptide peaks for modeling compared to healthy human and thalassemia patients

And (3) performing polypeptide identification on all the polypeptides which show differences between the normal group and the thalassemia group by using a magnetic bead enrichment method. The results are shown in Table 3.

TABLE 3 characterization of the polypeptide sequences

Mass to charge ratio (m/z)	Polypeptide sequence
		15120	SEQ ID NO:1
15859	SEQ ID NO:2
		15989	SEQ ID NO:3

Wherein, SEQ ID No. 1:

SEQ ID No.2：

SEQ ID No.3：

MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDAIKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH

EXAMPLE III Blind selection test of Mediterranean anemia screening model

200 of 300 whole blood samples (150 from thalassemia patients and 150 from healthy people) were selected as training samples for modeling (100 thalassemia patients and 100 healthy people). The thalassemia patients are determined by gene detection. All whole blood samples were drawn on an empty stomach in the early morning and stored in a-80 ℃ cryostat after whole blood isolation.

The remaining 100 samples were alternatively used as test samples for blind selection tests, of which 50 were known from thalassemia patients and 50 were from healthy populations. The processing method is the same as above.

And (3) establishing a mass spectrum model of the thalassemia polypeptide by using the characteristic polypeptide peak of the thalassemia screened in the example 1-2. The model is defined to use 3 input variables, which are: 15120m/z, 15859m/z, 169952 m/z.

The recognition rate of model training is 100%. And cross validation is carried out by adopting a random selection method, and the validation result is 99%. The model has good prediction capability.

TABLE 4 model training results

Sample(s)	Number of examples	Prediction of thalassemia group	Predicting normal group	Prediction rate%
					Group of poverty and poverty	100	100	0	100
Normal group	100	0	100	100
					Total of	200	100	100

From table 4, it can be seen that the results for the training samples are: 100 of the 100 normal groups are judged correctly, and the specificity is 100%; 100 of the 100 thalassemias were judged correct with 100% sensitivity.

After the model training is completed, the model is used to blindly select and predict 100 verification samples and determine the sample types, the method is as described in example 1, and an internal standard protein sample is added to carry out detection together.

The results showed that 49 of 50 thalassemia patients were accurately predicted and 50 of 50 normal groups were accurately predicted, as detailed in table 5.

Table 5 validation of sample prediction results

Sample(s)	Number of examples	Prediction of thalassemia group	Predicting normal group	Prediction rate%
					Group of poverty and poverty	50	49	1	98
Normal group	50	0	50	100

From table 5, it can be seen that the results of validating the samples are: 50 of the 50 normal groups are judged correctly, and the specificity is 100%; 49 of the 50 thalassemia groups were judged correct with a sensitivity of 98%.

As can be seen from table 5: the blind selection detection accuracy of the invention for the thalassemia group is basically the same as that of model training, but the prediction result of the normal group reaches 100%, which indicates that in the result after model training, the experimenter can fully and effectively eliminate false positive result through fine optimization, which indicates that the diagnosis result of the positive result is true and credible, and the missed diagnosis is avoided to the maximum extent, thus having positive significance.

As a further proof, the discrimination of the positive and negative measurement results of the present invention was evaluated simultaneously, and the ROC graph was drawn by connecting the intersections of the sensitivity and the false negative rate calculated by different threshold values to each other in a graph with the false positive probability as the horizontal axis and the true positive (i.e., sensitivity) as the vertical axis.

The evaluation method of the judgment sensitivity of the ROC curve diagram comprises the following steps: the area under the ROC curve is between 1.0 and 0.5. When AUC >0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC has lower accuracy when being 0.5-0.7, AUC has certain accuracy when being 0.7-0.9, and AUC has higher accuracy when being more than 0.9. When AUC is 0.5, the diagnostic method is completely ineffective and is not valuable.

The result is shown in fig. 7, the point at the uppermost left corner of the AOC graph of the present invention is the optimal diagnosis threshold point, the abscissa thereof is less than 0.2, and the AUC >0.9 is calculated, which indicates that the present invention has very high sensitivity and specificity, i.e. it indicates that the false positive result can be effectively excluded by refinement and optimization.

Example four drug or treatment methods for screening thalassemia Using Mass Spectrometry model

10 adult patients with thalassemia were recruited as volunteers and were dosed with dabigatran α (Darbepoetin, DAR)4.0ug/kg by weight 2 times a week for 8 weeks.

Separately, the whole blood samples before and after treatment were drawn on an empty stomach in the early morning, and the whole blood was separated and stored in a low-temperature refrigerator at-80 ℃.

The results of mass spectrometry for detecting characteristic proteins of thalassemia before and after treatment, according to the method of example 1 and the mass spectrometry model of example 2, were shown in fig. 8, for blood samples of the above two patients.

FIGS. 8-1-A and 8-1-B are a comparison of the characteristic protein profiles of patient 1 before and after treatment. Wherein 8-A shows that the expression level of beta-globin is reduced, but an obvious gamma-globin expression compensation peak appears, which indicates that the patient is a more serious thalassemia patient. 1-B shows that the expression level of β -globin is continuously down-regulated, but the relative expression up-regulation of γ -globin compared to the initial γ -globin expression compensation peak is already over 0.1 (15120 m/z peak compared to standard intensity), indicating that administration of dapoxetine α to patient 1 has effectively alleviated the symptoms of anemia.

FIGS. 8-2-A and 8-2-B are graphs comparing the characteristic protein profiles of patient 2 after treatment with those before treatment. Wherein 2-B shows that the expression level of beta-globin is reduced, but a certain gamma-globin expression compensation peak appears, which indicates that the patient is a less severe thalassemia patient. 2-A shows that the expression level of β -globin is continuously down-regulated, but the relative expression up-regulation of γ -globin compared to the initial γ -globin expression compensation peak is already over 0.1 (compared to the 15120m/z peak at standard intensity), and the compensated peak is already far more than the β -globin down-regulated, indicating that administration of bepotin α to patient 2 is already more effective for anemia symptoms than administration to patient 1.

As a control test, the 10 blood samples were analyzed and tested by blood cell analysis, and as a result, it was found that:

the Hb increase of the patient 1 is improved by 2.8g/dl, the Hb increase of the patient 2 is improved by 3.1g/dl, the Hb increase of the other 6 patients also reaches 2.0-2.5g/dl, the curative effect is related to the Hb and the basic EPO level of the patient before treatment, and the increase amount (>2.0-2.5g/dl) of the hemoglobin exceeds the judgment standard that the clinical medicine has the curative effect on the thalassemia. From this, it was determined that dabbepotein α is an effective drug for treating thalassemia.

As a further demonstration, 5 cases of blood cells of standard thalassemia were selected and cultured, and then corrected by CRISPR-Cas9 system editing, and then the difference in expression amount before and after gene editing of β -globin and γ -globin was detected by transient mass spectrometry. The results are shown in FIG. 9.

Wherein the content of the first and second substances,

FIG. 9-1 shows that after in vitro cell gene editing, the expression level of beta-globin in the cell is obviously increased and the expression level of gamma-globin is reduced compared with that before editing, which indicates that the function of beta-globin is recovered;

FIG. 9-2 shows that after in vitro cells are edited by genes, the expression level of beta-globin in the cells is obviously increased relative to that before editing, the expression level of gamma-globin is not changed greatly, and the recovery level of beta-globin is better than that in FIG. 9-1;

FIG. 9-3 shows that after in vitro cells are edited by genes, the expression levels of beta-globin and gamma-globin in the cells are obviously increased relative to those before editing, which indicates that the function recovery function of the hemoglobin is better than that of FIG. 9-2;

the results of fig. 9-4 are similar to fig. 9-1, and the results of fig. 9-5 are similar to fig. 9-3.

In addition, in all the results of fig. 9, the relative proportion of the β peak and the γ peak is significantly different, which further illustrates that the detection method of the combined internal standard protein of the present invention has high sensitivity and specificity, and can effectively eliminate false positive results through refinement and optimization.

In conclusion, the mass spectrum model of the invention can screen the medicine or the treatment method which has curative effect on the thalassemia.

Although the present invention has not been carried out for a diagnostic test of gene editing therapy in human body, the criteria for judging the therapeutic effect of the present invention are similarly to the examination of the increased amount of hemoglobin expression, regardless of the type of hematopoietic stem cell transplantation or drug administration or the treatment of thalassemia by gene editing. Therefore, based on the same principle, the marker or mass spectrum model can also be used for evaluating the treatment method for thalassemia, wherein the treatment method comprises the hematopoietic stem cell transplantation and the gene editing method.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the technical principle of the present invention, and these modifications and improvements should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. Use of a characteristic protein composition or mass spectrometry model characterizing thalassemia for screening a medicament for thalassemia or evaluating a treatment method for thalassemia.

2. The use according to claim 1, said signature protein marker composition consisting of 3 signature protein fragments, wherein the sequences of said signature protein fragments are shown in SEQ ID No.1-3, respectively, and the characterized mass to charge ratios of the three signature protein (SEQ ID NO:1-3) peaks are: 15120m/z (alpha globin fragment), 15859m/z (beta globin fragment), 15989m/z (v globin fragment) of the three protein peaks, and m/z deviation allowed within 0.15%.

3. The use of claim 1, wherein the mass spectrometric model comprises signature protein marker compositions having the sequences shown in SEQ ID nos. 1-3, respectively, wherein the mass spectrometric model is prepared by a method comprising:

2) performing pretreatment before mass spectrum on the whole blood protein polypeptide;

4. The use according to claim 3, wherein 1-3 internal standard protein standards, preferably Apomyoglobin (Apomyoglobin) with a peak m/z of 169952 in the protein spectrum, are added in advance during the sample treatment in step 2) to the range close to the molecular weight of the protein to be detected for relative quantification, and hemoglobin can be quantified relatively more accurately.

5. The use of claim 3 or 4, wherein the characteristic mass to charge ratios of the healthy population characteristic protein (SEQ ID NO:1-2) peaks are: 15120m/z (alpha globin fragment) or 15119m/z (alpha globin fragment), 15859m/z (beta globin fragment) or 15867m/z (beta globin fragment), and/or the characteristic mass to charge ratios of the peaks for the thalassemia patient characteristic protein (SEQ ID NO:1-2) are: 15120m/z (α globin fragment) or 15112m/z (α globin fragment), 15859m/z (β globin fragment) or 15853m/z (β globin fragment), and the 15120m/z peak is used to determine the intensity of change in up or down regulation of 15859m/z or 15989 m/z.

6. The use of claims 1-5, wherein, when 15859m/z, characteristic of the beta globin fragment peak, down-regulates expression or characterizes ν globin in a population over 2 years of age, with 15120m/z peak intensity as standard intensity 1, the cutoff value for the 15859m/z peak intensity is set to 0.5; when the 15859m/z expression is lower than 0.5, the thalassemia patient or the potential patient is detected; in the course of tracking therapeutic effect, when 15989m/z is up-regulated to over 0.1 relative to the original expression level, it indicates that the tested patient is the thalassemia patient or potential patient with the currently adopted therapeutic method or medicine with better therapeutic effect.

7. The use of claims 1 to 6, wherein the method of screening a drug comprises judging whether the drug has an effect on the protein of interest by a mass spectrometry model for diagnosing thalassemia, thereby judging whether it is a desired drug having activity against thalassemia.

8. The use of claims 1-6, wherein the method of treatment comprises hematopoietic stem cell transplantation, comprising the step of transplanting hematopoietic stem cells in children of 2-6 years of age to treat thalassemia.

9. The use of claims 1-6, wherein the treatment comprises gene editing, comprising gene editing of umbilical cord blood of a newborn with thalassemia, mass spectrometric detection, and screening for the effectiveness of the editing as determined by a mass spectrometric model of thalassemia diagnosis.

10. The use of claims 8-9, wherein the composition demonstrating therapeutic efficacy in the treatment method is judged to be among the desired drugs having activity against thalassemia.