CN105223290A - A kind of method of Measuring hemoglobin α and beta globin chain ratio and application - Google Patents
A kind of method of Measuring hemoglobin α and beta globin chain ratio and application Download PDFInfo
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Abstract
The invention belongs to field of biochemistry detection, be specifically related to a kind of method of Measuring hemoglobin α and beta globin chain ratio and detecting the application in beta Thalassemia.Described method comprises the following steps: get hemoglobin samples and carry out cracking, obtain the crack fragment of alpha globin chain and the crack fragment of beta globin chain, choose a crack fragment respectively as mark peptide section, utilize the concentration of the concentration of the mark peptide section of mass spectroscopy alpha globin chain and the mark peptide section of beta globin chain, namely the ratio of alpha globin chain and beta globin chain is represented by the concentration ratio of the mark peptide section concentration of alpha globin chain and the mark peptide section of beta globin chain.The present invention, can the dissimilar beta Thalassemia of EARLY RECOGNITION by quantitative test α, beta globin chain ratio, can identify severe, moderate and mild beta thalassaemia.
Description
Technical field
The invention belongs to field of biochemistry detection, be specifically related to a kind of method of Measuring hemoglobin α and beta globin chain ratio and detecting the application in beta Thalassemia.
Background technology
Haemoglobin (Hemoglobin, Hb) is made up of a globin and four protohemes (also known as ferroprotoporphyrin), and each protoheme forms a ring by four pyroles subunits again, and ring center is a ferrous ion.Each globin is then made up of four polypeptied chains, and four peptide chains of globin are different.Adult can be made up of 2 α chains and 2 β chains, and claim HbA, fetus is made up of 2 α chains and 2 γ chains, claims HbF, is replaced soon after birth by HbA.Every bar polypeptied chain of globin is connected with a protoheme, forms a monomer of haemoglobin, subunit's (i.e. subunit) in other words.
Thalassemia, also known as thalassemia, is the common disease of Chinese children, account for the 30-40% of all anaemias, be distributed widely in multiple area, the world, in China, especially common with provinces and cities such as Chongqing, Sichuan, Guangdong, Guangxi, be the important social concern highly paid close attention to.This disease is because genetic defect causes hemoglobin alpha, beta globin chain to synthesize disappearance or not enough, α, beta globin chain proportional imbalance, and then causes shorten or parafunctional a kind of congenital anemia red blood cell life span.Thalassemia, according to the difference of the globin chain of unconventionality expression, is mainly divided into alpha Thalassemia and the large class of beta Thalassemia two.Current as the diagnosis after morbidity, when being generally found to be hypochromic microcytic anemia by blood routine examination and getting rid of hypoferric anemia, in conjunction with HE and gene diagnosis technology, the patient to doubtful thalassemia makes a definite diagnosis, test operation is complicated, somewhat expensive, and be unfavorable for that examination is popularized.At present, quick and high-throughout detection method is still lacked to thalassemia.
Summary of the invention
Thalassemia is because genetic defect causes hemoglobin alpha, beta globin chain to synthesize disappearance or not enough, α, beta globin chain proportional imbalance, and then causes shorten or parafunctional a kind of congenital anemia red blood cell life span.Present inventor thinks, can we realize the dissimilar beta Thalassemia of EARLY RECOGNITION according to the ratio of α, beta globin chain in quantitative test haemoglobin since thalassemia is caused by α, beta globin chain proportional imbalance? the how ratio of α, beta globin chain in quantitative test haemoglobin? how about realize high flux further to detect? present inventor achieves the present invention in the process solved the problem just.
In view of this, the object of the present invention is to provide a kind of alpha globin chain of Measuring hemoglobin and the method for beta globin chain ratio, test as mark peptide section owing to adopting the crack fragment of haemoglobin, therefore do not require that the conformation of haemoglobin is complete, trace can be measured and the hemoglobin concentration of blood preparation of preserving through the long period, and the method is simple to operate, required reagent cost is low.
For achieving the above object, technical scheme of the present invention is:
The alpha globin chain of Measuring hemoglobin and a method for beta globin chain ratio, comprise the step of carrying out as follows:
(1) get hemoglobin samples and carry out cracking, obtain the crack fragment of globin polypeptied chain; The crack fragment of described globin polypeptied chain comprises the crack fragment of alpha globin chain and the crack fragment of beta globin chain;
(2) the mark peptide section using arbitrary crack fragment in alpha globin chain crack fragment as alpha globin chain, mark peptide section using arbitrary crack fragment in beta globin chain crack fragment as beta globin chain, utilize the concentration of the concentration of the mark peptide section of mass spectroscopy alpha globin chain and the mark peptide section of beta globin chain, namely the ratio of alpha globin chain and beta globin chain is represented by the concentration ratio of the mark peptide section concentration of alpha globin chain and the mark peptide section of beta globin chain; The concentration of the mark peptide section of described alpha globin chain is represented by the integrating peak areas of the mark peptide section mass spectrometer response intensity of alpha globin chain, and the concentration of the mark peptide section of described beta globin chain is represented by the integrating peak areas of the mark peptide section mass spectrometer response intensity of beta globin chain.
In described step (1), the carrying out cutting cracking that cracking can adopt enzyme selectivity is carried out to hemoglobin samples, as trypsase can cut off the carboxyl side in lysine in polypeptied chain and arginine residues, the peptide fragments of specific acquisition lysine or arginine ending.To the separation of crack fragment, chromatographic separation technology can be adopted to carry out, as liquid chromatography, thin-layer chromatography etc., or adopt electrophoretic techniques to be separated.After separation, the wantonly one or more characteristic polypeptide fragment produced after choosing the cracking of globin polypeptied chain can be used as mark peptide section.
Further, the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, in described step (1), hemoglobin samples preferably adopts trypsase to carry out cracking, the crack fragment of alpha globin chain has 14, and its sequence is as shown in SEQIDNO:1-14; The crack fragment of beta globin chain has 15, and its sequence is as shown in SEQIDNO:15-29.
Further, the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, in described step (2), the crack fragment that during Mass Spectrometer Method, response is high in alpha globin chain crack fragment is as the mark peptide section of alpha globin chain, and the crack fragment that during Mass Spectrometer Method, response is high in beta globin chain crack fragment is as the mark peptide section of beta globin chain.
Further, the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, the sequence of the mark peptide section of alpha globin chain is as shown in SEQIDNO:1, and the sequence of the mark peptide section of beta globin chain is as shown in SEQIDNO:15.
Further, the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, described mass spectroscopy adopts Liquid Chromatography-Tandem Mass Spectrometry to measure, particular by liquid chromatogram mobile input mode fast sample, carry out Quantitative detection by tandem mass spectrometer again, realize high flux and detect.
Further, the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, during by mass spectrometer MRM pattern Quantitative detection, each feature peptide section of alpha globin chain and beta globin chain carries out correction with quantitative by marking in its cold labeling, eliminates systematic error and matrix effect.
Further, the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, when by mass spectrometer MRM pattern Quantitative detection, the fragmention that the different fragmention of the mark peptide section of the alpha globin chain mark peptide section to combination and beta globin chain is different all can be used for detecting to combination (parent ion/daughter ion combination).
Further, enforcement due to the inventive method does not rely on the complete conformation of haemoglobin, therefore it is less demanding to the Sample preservation gathered, directly can get whole blood sample analysis, also blood can be made the preservation form of dry blood spot sample as haemoglobin sample, be convenient to long-term preservation and long distance transportation, and cost is low.
Present invention also offers and detecting the application in beta Thalassemia based on the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio.
For achieving the above object, technical scheme of the present invention is:
The alpha globin chain of Measuring hemoglobin and the method for beta globin chain ratio are detecting the application in beta Thalassemia, described application is specially: measure known normal person, the alpha globin chain of known dissimilar beta Thalassemia person and unknown sample and beta globin chain ratio α/β reference value respectively according to the alpha globin chain of described a kind of Measuring hemoglobin and the method for beta globin chain ratio, judge according to α/β reference value.
The present invention, can the dissimilar beta Thalassemia of EARLY RECOGNITION by quantitative test α, beta globin chain ratio, can identify severe, moderate and mild beta thalassaemia.The patient that examination of the present invention goes out, at least can be benefited from the following aspects: for developing into the infant of β thalassemia major, prevents the infection that complication and treatment of blood transfusion may be brought early, can improve the living environment of infant; For the infant of light-duty beta Thalassemia, instruct it to avoid the environment of Long contact time low pressure, anoxic, the appearance of Anemia can be avoided; Before pregnant, the detecting of antenatal parental generation beta Thalassemia, contribute to genetic counselling and fertility decision-making, reduce or avoid the birth of β thalassemia major infant, reach the object of prenatal and postnatal care.The present invention has broad application prospects and huge social benefit.
Present invention also offers a kind of kit detecting beta Thalassemia, this kit conveniently can realize the extraction of haemoglobin, sex change and cracking, is beneficial to commercial applications.
For achieving the above object, technical scheme of the present invention is:
A kind of kit detecting beta Thalassemia, described kit comprises corpuscular hemoglobin extractant, protein denaturant, protein cleavage agent and crack protein double solvents, described corpuscular hemoglobin extractant is water, described protein denaturant is the mixed liquor of acetonitrile and formic acid, described protein cleavage agent is the ammonium bicarbonate soln containing proteinase, and described crack protein double solvents is the acetonitrile solution containing formic acid.
Further, described a kind of kit detecting beta Thalassemia, described kit also comprises the Isotopic Internal Standard thing of alpha globin chain and beta globin chain each feature peptide section, and normally marks product, beta Thalassemia heterozygote mark product, beta Thalassemia homozygote.
Advantageous Effects of the present invention is:
(1) the alpha globin chain of Measuring hemoglobin of the present invention and the method for beta globin chain ratio, test as mark peptide section owing to adopting the crack fragment of haemoglobin, therefore do not require that the conformation of haemoglobin is complete, trace can be measured and the hemoglobin concentration of blood preparation of preserving through the long period, and the method is simple to operate, required reagent cost is low.Method of the present invention can adopt dry blood spot sample to detect, and convenient preservation and long distance are sent.
(2) the alpha globin chain of a kind of Measuring hemoglobin of the present invention and the method for beta globin chain ratio can be applicable to detect beta Thalassemia.The α/β reference value of known normal person, known dissimilar beta Thalassemia person and unknown sample is measured respectively according to the method, carry out judging according to α/β reference value, can the dissimilar beta Thalassemia of EARLY RECOGNITION, can identify severe, moderate and mild beta thalassaemia.
(3) kit of detection beta Thalassemia of the present invention, conveniently can realize the extraction of haemoglobin, sex change and cracking, be beneficial to commercial applications, has wide clinical conversion and application prospect.
Accompanying drawing explanation
The mass spectrogram of Fig. 1 Liquid Chromatography-Tandem Mass Spectrometry screening hemoglobin alpha globin chain mark peptide section;
The mass spectrogram of Fig. 2 Liquid Chromatography-Tandem Mass Spectrometry screening haemoglobin beta globin chain logo peptide section;
(wherein, (1) is α T3 mark peptide section integrogram to the integrogram of Fig. 3 automatic integrating peak areas quantitative analysis mark peptide section and isotope tag thing thereof, and (2) mark α T3 for α T3 indicates in peptide section
iSintegrogram).
Fig. 4 is the mass spectrum MRM Pattern recognition collection of illustrative plates that alpha globin chain α T1 (729.4m/z) indicates peptide section.
Fig. 5 is the mass spectrum MRM Pattern recognition collection of illustrative plates that beta globin chain β T1 (952.3m/z) indicates peptide section.
Embodiment
Referring to accompanying drawing, the preferred embodiments of the present invention are described in detail.Illustrated embodiment is to be described content of the present invention better, but is not that content of the present invention is only limitted to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.
Instrument, reagent and consumptive material required in following examples are:
(1) required instrument
To connect triple level Four bar mass spectrometer (API3200) (AB company, the U.S.), Shimadzu liquid chromatograph (SHIMADZULC-20AD) (chromatographic column: XSelectCSH130C
183.5 μm), 3.2mm perforating plier, hydro-extractor (Gnencompany1580R), computing machine, pipettor (EPPENDORF).
(2) required reagent
Acetonitrile, formic acid, TPCK-Treated trypsase, ammonium bicarbonate, all liq reagent is HPLC or the pure level of mass spectrum.
Water used is deionized water.
Interior mark: the Isotopic Internal Standard thing of each feature peptide section, normal concentration is known.
Quality-control product: yin and yang attribute tester, and normally mark product, beta Thalassemia heterozygote mark product, beta Thalassemia homozygote.
(3) required consumptive material
Common 96 hole reaction plates, the 96 hole ELISA Plate with 0.22 μm of bore filter function, pipettor gun head, 96 orifice plate aluminum foil special overlay films, 96 orifice plate sealed membranes, identification bar code.
Detect the process of sample: get peripheral blood or venous blood sample one, about 50 μ l, instil in Whatman
filter paper, natural diffuseness also dries, and make dry blood spot sample, sealed membrane is sealed up for safekeeping, 4 DEG C preserve for a long time, to be measured.Or directly get whole blood sample analysis.When doing security protection with zip lock bag, distance express transportation can be grown, solve the inconvenience of directly sending blood preparation.
The experimental technique of unreceipted actual conditions in following preferred embodiment, conveniently condition is carried out.
The foundation of embodiment 1 detection method
(1) preparation of reagent
Corpuscular hemoglobin extractant (reagent A): deionized water; For the extraction of corpuscular hemoglobin.
Protein denaturant (reagent B): acetonitrile: formic acid solution (12g/L) volume ratio is 3:1; For protein denaturation.
Protein cleavage agent (reagent C): TPCK-Treated trypsase freeze-dried powder is dissolved in 1mol/L ammonium bicarbonate soln, makes the lysate that concentration is 5g/L; For the hydrolysis of protein.
Crack protein double solvents (reagent D): acetonitrile solution (acetonitrile and water volume ratio are 1:1), the formic acid wherein also containing 1.2g/L.
(2) extraction of haemoglobin and cracking
1. dry blood spot sample standard hole device is got diameter 3.2mm circular filter paper blood sheet (if directly get whole blood sample analysis, be equivalent to the sample of 3.2 μ l whole bloods) in 96 hole filter plates, add reagent A 200 μ l, room temperature low speed jolting 15 minutes, abundant lysed blood.
2. get hemolysate 100 μ l, be transferred to 96 new orifice plates, add reagent B40 μ l, quick jolting 1 minute, static 5 minutes of room temperature, makes albuminous degeneration.
3. reagent C 10 μ l is added in the mixed liquor 2. obtained in step, low speed jolting mixes, sealed membrane is sealed up for safekeeping, puts in 37 DEG C of incubators and hatches 2 hours, and the crack fragment of haemoglobin globin polypeptied chain comprises the crack fragment of alpha globin chain and the crack fragment of beta globin chain; The crack fragment of alpha globin chain has 14, respectively called after α T1-α T14, and be shown in Table 1, its sequence is as shown in SEQIDNO:1-14; The crack fragment of beta globin chain has 15, respectively called after β T1-β T15, and be shown in Table 2, its sequence is as shown in SEQIDNO:15-29.
4. get 96 hole filters, add successively reagent D 108 μ l and step 3. in hatch after sample liquid 12 μ l, slightly shake redissolution, brief centrifugation, leave and take lower plate sample, to be measured.
The crack fragment of table 1. alpha globin chain
The crack fragment of table 2. beta globin chain
(3) α, beta globin chain logo peptide section mass spectroscopy
1. the activation of Liquid Chromatography-Tandem Mass Spectrometry coupling and establishment
A, liquid phase chromatogram condition: arrange automatic sampler parameter by table 3, arrange sampling time program by table 4.
B, Mass Spectrometry Conditions: by table 5, correlation parameter is set.
The setting of these parameters can make the crack fragment of haemoglobin well be separated, and obtains good response after making to enter mass spectrum, optionally carries out quantitative test to selected mark peptide section.The setting of correlation parameter is not limited to this, according to the different crack fragments obtained, and the difference of the mark peptide section chosen, can the corresponding adjustment carrying out liquid chromatography and mass spectrometer parameters.
Table 3. automatic sampler parameter
Table 4. sampling time program (TimeProgram)
Note: BinaryFlow0.15ml/min; Pressurelimits0-15Mpa.
Table 5. mass spectrometer parameters
2. the optimization of mass spectrometer multiple reaction monitoring (MRM) mode parameter.
Sample extract is through trypsinization, α, beta globin chain hydrolysate produce the polypeptide fragment of different mass-to-charge ratio, as shown in Table 1 and Table 2, in principle, alpha globin chain crack fragment all can be used as the mark peptide section of alpha globin chain, and beta globin chain crack fragment all can be used as the mark peptide section of beta globin chain.According to selected mark peptide section, set up the parameter of mass spectrometer MRM detecting pattern, comprise DP (removing a bunch voltage), EP (injecting voltage), CE (collision voltage), CEP (collision cell entrance potential), CXP (collision cell exit potential) etc., and revise optimization range, the step-length of parameter as required.Set Q1 parent ion and Q3 daughter ion sweep parameter respectively.Particularly, the MRM sweep parameter of the mark peptide section optimization of part α, beta globin chain is in table 6.
Table 6. part α, beta globin chain logo peptide section MRM sweep parameter
3. the mensuration of the mark peptide section of alpha globin chain and the mark peptide section of beta globin chain and quantitative test
Sample extract is through trypsinization, α, beta globin chain hydrolysate produce the polypeptide fragment of different mass-to-charge ratio, as shown in Table 1 and Table 2, in principle, alpha globin chain crack fragment all can be used as the mark peptide section of alpha globin chain, beta globin chain crack fragment all can be used as the mark peptide section of beta globin chain, and these mark peptides Duan Jun can be used for diagnosis index design.
For alpha globin chain, hydrolysate produces the polypeptide fragment of different mass-to-charge ratio, its mass spectrogram as shown in Figure 1, concrete sequence and mass-to-charge ratio as shown in table 1, totally 14 sequences, are numbered α T1-α T14, these fragments are when the enough sensitivity of mass spectrometer, all can be used as the analysis (numbering α T8 be an amino acid, when remove sample Free Amino Acids interference, also can as indicate peptide section) of mark peptide section for haemoglobin.
For beta globin chain, hydrolysate produces the polypeptide fragment of different mass-to-charge ratio, its mass spectrogram as shown in Figure 2, concrete sequence and mass-to-charge ratio as shown in table 2, totally 15 sequences, are numbered β T1-β T15, these fragments are when the enough sensitivity of mass spectrometer, all can be used as the analysis (numbering β T8 be an amino acid, when remove sample Free Amino Acids interference, also can as indicate peptide section) of mark peptide section for haemoglobin.
Data conversion and quantitative analysis method:
What mass spectrometer raw data showed is the response intensity (Intensity) of instrument to variable concentrations polypeptide fragment, utilizes MultiQuant2.0.2 software, carries out automatic integration analysis, calculate peak area score value with integrating peak areas method to mark peptide section.The ratio of α, beta globin chain, is represented by the ratio of α, beta globin chain logo peptide section, and indicates that the concentration level of peptide section can directly be represented by the integrating peak areas size of mass spectrometer response intensity.Indicate peptide section for α T3, α T3 indicates that the concentration of peptide section carrys out direct representation by the integrating peak areas size of mass spectrometer response intensity, as (1) figure in Fig. 3.
Further, in order to eliminate systematic error and matrix effect, often kind of feature peptide section all carries out correction with quantitative by mark in its cold labeling; Preparation and the term of validity of interior mark liquid are controlled, with error between guaranteeing batch in the scope required.Utilize MultiQuant2.0.2 software, with integrating peak areas method, respectively automatic integration analysis is carried out to mark peptide section and isotope tag thing thereof, calculate peak area score value.Indicate peptide section for α T3, α T3 indicates that the concentration of peptide section need through the isotope tag thing α T3 of its concentration known
iScarry out correction with quantitative, as shown in Figure 3.(1) figure in Fig. 3 is that α T3 indicates peptide section integrating peak areas, and (2) figure in Fig. 3 is the isotope-labeled interior mark α T3 that α T3 indicates peptide section
iSintegrating peak areas.α T3 indicates that the computing formula of peptide section concentration (nmol/ml) is as follows:
α T3 indicates that peptide section concentration=(α T3 indicates peptide section integrating peak areas/interior mark α T3
iSintegrating peak areas) mark α T3 in *
iSconcentration.
Therefore, indicate that peptide section concentration computation formula is:
4. the ratio of α, beta globin chain calculates
The ratio of α, beta globin chain, is represented by α, beta globin chain logo peptide section concentration ratio, the calculating of globin chain mark peptide section concentration step 3. in introduce.Below using the crack fragment α T1 that in alpha globin chain crack fragment, during Mass Spectrometer Method, response is high as the mark peptide section of alpha globin chain, the crack fragment β T1 that during Mass Spectrometer Method, response is high in beta globin chain crack fragment, as the mark peptide section of beta globin chain, is introduced.
As shown in Figure 1, the α T1 fragment response of alpha globin chain is higher, and namely Mass Spectrometer Method is comparatively sensitive.For α chain α T1 (729.4m/z), under tandem mass spectrum MRM detecting pattern, by its its fragmention of Isotopic Internal Standard quantitative test to composition, the content situation of indirect analysis alpha globin chain, the content of alpha globin chain is indicated the concentration approximate representation of peptide section by it.α T1 (729.4m/z) indicates that the right composition of peptide section fragmention is as shown in table 7, when analysis mark peptide section fragmention is to composition, it is arbitrary to the combination of parent ion/daughter ion that each mark peptide section can be chosen wherein, as α chain α T1 (729.4/430.2), and do internal control with Isotopic Internal Standard.α T1 (729.4m/z) indicates that Fig. 4 is shown in by the mass spectrum MRM Pattern recognition collection of illustrative plates of peptide section.
As shown in Figure 2, the β T1 fragment response of beta globin chain is higher, and namely Mass Spectrometer Method is comparatively sensitive.For the β T1 (952.3m/z) of β chain, under tandem mass spectrum MRM detecting pattern, by its its fragmention of Isotopic Internal Standard quantitative test composition, the content situation of indirect analysis beta globin chain, the content of beta globin chain is indicated the concentration approximate representation of peptide section by it.β T1 (952.3m/z) indicates that the right composition of peptide section fragmention is as shown in table 8, when analysis mark peptide section fragmention is to composition, each mark peptide section can choose that it is arbitrary to the combination of parent ion/daughter ion, as the β T1 (952.3/502.3) of β chain, and do internal control with Isotopic Internal Standard.β T1 (952.3m/z) indicates that Fig. 5 is shown in by the mass spectrum MRM Pattern recognition collection of illustrative plates of peptide section.
Table 7. hemoglobin alpha globin chain T1 (729.4m/z) indicates that peptide section fragmention forms
Table 8. haemoglobin beta globin chain T1 (952.3m/z) indicates that peptide section fragmention forms
The ratio of α, beta globin chain, is represented by the ratio of α, beta globin chain feature peptide section; And the concentration level of feature peptide section can be represented by the integrating peak areas size of mass spectrometer response intensity; In order to eliminate systematic error and matrix effect, often kind of feature peptide section all carries out correction with quantitative by mark in its cold labeling; Preparation and the term of validity of interior mark liquid are controlled, with error between guaranteeing batch in the scope required.
In principle, all feature peptide Duan Jun can be used for the calculating of α, beta globin chain ratio, and be the design that mass spectrometer response is high, the sensitiveer mark peptide section of reaction representatively carries out technical scheme selected in scheme, part is as shown in table 9; Each mark peptide section chooses a pair parent ion/daughter ion combination, as α T1 (729.4/430.2) and β T1 (952.3/502.3), and does internal control with Isotopic Internal Standard; The equation of calculating α, beta globin chain ratio is as follows:
Note: for α T1 (729.4/430.2) and β T1 (952.3/502.3), in formula, α T is the integrating peak areas of α T1 (729.4/430.2); α T
iSfor the corresponding integrating peak areas of the Isotopic Internal Standard of mark peptide section α T1; β T is the integrating peak areas of mark peptide section β T1 (952.3/502.3); β T
iSfor the corresponding integrating peak areas of the Isotopic Internal Standard of mark peptide section β T1; R is constant, for marking α T in stable isotope
iSwith β T
iSthe ratio of concentration.
The calculating of table 9. α, beta globin chain ratio
Formula in note table: α T represents the integrating peak areas of alpha globin chain logo peptide section α T;
α T
iSrepresent the corresponding integrating peak areas of the Isotopic Internal Standard of alpha globin chain logo peptide section α T;
β T represents the integrating peak areas of beta globin chain logo peptide section β T;
β T
iSrepresent the corresponding integrating peak areas of the Isotopic Internal Standard of beta globin chain logo peptide section β T;
R is constant, for marking α T in stable isotope
iSwith β T
iSthe ratio of concentration.
Embodiment 2
Utilize the above-mentioned detection scheme established, by liquid phase systems Flow Sampling mode fast sample, then detected fast by mass spectrometer, realize the high flux sample introduction detecting sample, each sample sample detection time is less than 1 minute, once tests, can with batch detection at least 192 samples.
According to technique scheme of the present invention, analyze 200 routine normal controls (June to 40 at age year old) and 358 routine beta Thalassemia patients and 16 routine HbE type Anemic patients, set up reference value interval (1st-99th) as shown in table 10.Reference interval may because of different experiments room, different experiments personnel, different reagent lot and slightly difference, advises that oneself reference value is set up in each laboratory interval.
Table 10. normal person, beta Thalassemia people carrier, patient α: β ratio reference interval
Method evaluation:
According to the distribution of above-mentioned reference interval and sample, method described in the invention is to the average recall rate about 77% of above-mentioned 9 kinds of beta Thalassemia heterozygotes; 95% is greater than to the recall rate of above-mentioned 13 kinds of beta Thalassemia homozygotes and β sudden change double heterozygote; To 15 kinds of α, β compound poor recall rate be greater than 95%; 95% is greater than to 2 kinds of compound thalassemic recall rates of HbE, β; Be less than 15% to the recall rate of HbE sickle-cell anaemia, namely the method for the invention is not suitable for the detection of sickle-cell anaemia.
Choose a routine normal specimen, a routine β respectively
+ground poor (CD17), a routine β
0ground poor (CD27/28 & IVS-II-654), in doing batch and batch between revision test, computation of mean values and standard deviation, precision and the repeatability of display screening method are higher, and result is as shown in table 11.
In technical scheme described in table 11. batch, batch between repeatability and Precision Analyze
The present invention, can the dissimilar beta Thalassemia of EARLY RECOGNITION by quantitative test α, beta globin chain ratio, can identify severe, moderate and mild beta thalassaemia.Detection method of the present invention is greater than 95% to the suddenly change recall rate of double heterozygote of 13 kinds of beta Thalassemia homozygotes and β, to 15 kinds of α, β compound poor recall rate be greater than 95%, and method has higher precision and repeatability.The patient that examination of the present invention goes out, at least can be benefited from the following aspects: for developing into the infant of β thalassemia major, prevents the infection that complication and treatment of blood transfusion may be brought early, can improve the living environment of infant; For the infant of light-duty beta Thalassemia, instruct it to avoid the environment of Long contact time low pressure, anoxic, the appearance of Anemia can be avoided; Before pregnant, the detecting of antenatal parental generation beta Thalassemia, contribute to genetic counselling and fertility decision-making, reduce or avoid the birth of β thalassemia major infant, reach the object of prenatal and postnatal care.The present invention has broad application prospects and huge social benefit.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (10)
1. the alpha globin chain of Measuring hemoglobin and a method for beta globin chain ratio, is characterized in that, comprise the step of carrying out as follows:
(1) get hemoglobin samples and carry out cracking, obtain the crack fragment of globin polypeptied chain; The crack fragment of described globin polypeptied chain comprises the crack fragment of alpha globin chain and the crack fragment of beta globin chain;
(2) the mark peptide section using arbitrary crack fragment in alpha globin chain crack fragment as alpha globin chain, mark peptide section using arbitrary crack fragment in beta globin chain crack fragment as beta globin chain, utilize tandem mass spectrometer to measure the concentration of mark peptide section of alpha globin chain and the concentration of the mark peptide section of beta globin chain, namely the ratio of alpha globin chain and beta globin chain is represented by the concentration ratio of the mark peptide section concentration of alpha globin chain and the mark peptide section of beta globin chain; The concentration of the mark peptide section of described alpha globin chain is represented by the integrating peak areas of the mark peptide section mass spectrometer response intensity of alpha globin chain, and the concentration of the mark peptide section of described beta globin chain is represented by the integrating peak areas of the mark peptide section mass spectrometer response intensity of beta globin chain.
2. the alpha globin chain of a kind of Measuring hemoglobin according to claim 1 and the method for beta globin chain ratio, it is characterized in that, in described step (1), hemoglobin samples adopts trypsase to carry out cracking, the crack fragment of alpha globin chain has 14, and its sequence is as shown in SEQIDNO:1-14; The crack fragment of beta globin chain has 15, and its sequence is as shown in SEQIDNO:15-29.
3. the alpha globin chain of a kind of Measuring hemoglobin according to claim 1 and the method for beta globin chain ratio, it is characterized in that, in described step (2), the crack fragment that during Mass Spectrometer Method, response is high in alpha globin chain crack fragment is as the mark peptide section of alpha globin chain, and the crack fragment that during Mass Spectrometer Method, response is high in beta globin chain crack fragment is as the mark peptide section of beta globin chain.
4. the alpha globin chain of a kind of Measuring hemoglobin according to claim 3 and the method for beta globin chain ratio, it is characterized in that, the sequence of the mark peptide section of alpha globin chain is as shown in SEQIDNO:1, and the sequence of the mark peptide section of beta globin chain is as shown in SEQIDNO:15.
5. the alpha globin chain of a kind of Measuring hemoglobin according to claim 1 and the method for beta globin chain ratio, it is characterized in that, described mass spectroscopy adopts Liquid Chromatography-Tandem Mass Spectrometry to measure, particular by liquid chromatogram mobile input mode fast sample, carry out Quantitative detection by tandem mass spectrometer again, realize high flux and detect.
6. the alpha globin chain of a kind of Measuring hemoglobin according to claim 5 and the method for beta globin chain ratio, it is characterized in that, during by tandem mass spectrometer MRM pattern Quantitative detection, each feature peptide section of alpha globin chain and beta globin chain carries out correction with quantitative by marking in its cold labeling, eliminates systematic error and matrix effect.
7. the alpha globin chain of a kind of Measuring hemoglobin according to claim 6 and the method for beta globin chain ratio, it is characterized in that, when by mass spectrometer MRM pattern Quantitative detection, the fragmention that the different fragmention of the mark peptide section of the alpha globin chain mark peptide section to combination and beta globin chain is different all can be used for detecting to combination.
8. the alpha globin chain of a kind of Measuring hemoglobin described in any one of claim 1-7 and the method for beta globin chain ratio are detecting the application in beta Thalassemia, it is characterized in that, described application is specially: measure known normal person, the alpha globin chain of known dissimilar beta Thalassemia person and unknown sample and beta globin chain ratio α/β reference value respectively according to the alpha globin chain of a kind of Measuring hemoglobin described in any one of claim 1-7 and the method for beta globin chain ratio, judge according to α/β reference value.
9. one kind is detected the kit of beta Thalassemia, it is characterized in that, described kit comprises corpuscular hemoglobin extractant, protein denaturant, protein cleavage agent and crack protein double solvents, described corpuscular hemoglobin extractant is water, described protein denaturant is the mixed liquor of acetonitrile and formic acid, described protein cleavage agent is the ammonium bicarbonate soln containing proteinase, and described crack protein double solvents is the acetonitrile solution containing formic acid.
10. a kind of kit detecting beta Thalassemia according to claim 9, it is characterized in that, described kit also comprises the Isotopic Internal Standard thing of alpha globin chain and beta globin chain each feature peptide section, and normally marks product, beta Thalassemia heterozygote mark product, beta Thalassemia homozygote.
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