Characteristic protein marker composition for mass spectrometric diagnosis of thalassemia and diagnostic product thereof
阅读说明：本技术 用于质谱诊断地中海贫血症的特征蛋白标记组合物及其诊断产品 (Characteristic protein marker composition for mass spectrometric diagnosis of thalassemia and diagnostic product thereof ) 是由 何昆 马庆伟 常亮 向华 梁丽蕴 吕倩 牛燕燕 于 2019-10-01 设计创作，主要内容包括：本发明提供了一种检测地中海贫血症的特征蛋白片段组合物以及评价地中海贫血药物疗效或治疗方法效果评价的质谱模型。其中所述特征蛋白片段组合物的序列分别如SEQ ID No.1-3所示。本发明的特征蛋白片段组合物或质谱模型,可用于地中海贫血症的诊断和筛查以及地中海贫血症的治疗方法和药物疗效评价,方法简单易于操作,准确性高,为地中海贫血症的诊断和筛查、治疗方法、药物疗效评价提供了新的方法和思路。(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.)
1. A characteristic protein marker composition for diagnosing thalassemia, which consists of 3 characteristic protein fragments, wherein the sequences of the characteristic protein fragments are respectively shown as SEQ ID No.1-3,
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%.
2. The composition of claim 1, 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.
3. The composition of claim 2, wherein the peaks of healthy population characteristic proteins (SEQ ID NOS: 1-2) are characterized by mass-to-charge ratios of: 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 (alpha globin fragment) or 15112m/z (alpha globin fragment), 15859m/z (beta globin fragment) or 15853m/z (beta globin fragment).
4. The composition of claim 3 or 4, wherein a downregulation of 15859m/z characterizing the peak of β globin fragment or an upregulation of 15989m/z characterizing the peak of ν globin fragment in a population over 2 years of age is indicative of the patient being tested for thalassemia or a potential patient; after the thalassemia patient is treated, when 15989m/z representing the nuncglobin fragment peak is up-regulated relative to the original expression, the curative effect of the medicine of the detected person is indicated to be good, and the medicine or the method can be continuously used for treatment.
5. The composition of claim 5, wherein the cutoff value for peak intensity at 15859m/z is set at 0.5 with peak intensity at 15120m/z as standard intensity 1; 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.
6. The composition of claims 1 to 5, wherein the signature protein marker composition comprises, in addition to said 3 signature protein fragments, 1 to 3 internal standard protein standards for relative quantification and close to the molecular weight range of the protein to be tested, preferably Apomyoglobin (Apomyoglobin), with a mass spectrum peak m/z of 169952.
7. A diagnostic product for diagnosing thalassemia prepared by the composition of claims 1-6.
8. The diagnostic product of claim 7, wherein said diagnostic product is selected from the group consisting of a whole blood control, a buffer, a matrix solution, time-of-flight mass spectrometry based thalassemia detection software, and a specific target plate for thalassemia mass spectrometry detection, and said kit can be used to provide a standard data or curve comparison when performing mass spectrometry on a sample to be tested to determine whether the sample to be tested is a thalassemia patient or whether a drug and a treatment are effective.
9. The diagnostic product of claim 8, wherein the kit further comprises an internal standard protein Apomyoglobin (Apomyoglobin) for relative quantification, the internal standard protein being close to the characteristic protein detection range, and the absolute peak intensity of the characteristic protein and its up-or down-regulated variation being directly convertible by comparing the peak intensities of the internal standard proteins, wherein the protein profile peak m/z is 169952.
10. Use of a composition according to claims 1-6 for the preparation of a product for the diagnosis of thalassemia, wherein the product comprises: diagnostic reagent, special target plate for detection, chip, carrier, kit and the like.
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
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 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 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.
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.
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.
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 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)
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 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 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.
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 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 one embodiment, the method for constructing a mass spectrometry model comprises:
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) 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;
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)
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 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 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.
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.
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 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 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.
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 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.