CN105223290B - A kind of method and application measuring hemoglobin alpha and beta globin chain ratio - Google Patents
A kind of method and application measuring hemoglobin alpha and beta globin chain ratio Download PDFInfo
- Publication number
- CN105223290B CN105223290B CN201510618319.5A CN201510618319A CN105223290B CN 105223290 B CN105223290 B CN 105223290B CN 201510618319 A CN201510618319 A CN 201510618319A CN 105223290 B CN105223290 B CN 105223290B
- Authority
- CN
- China
- Prior art keywords
- globin chain
- beta
- peptide fragment
- fragment
- chain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to field of biochemistry detection, and in particular to a kind of method measuring hemoglobin alpha and beta globin chain ratio and its application in detecting beta Thalassemia.The method includes the following steps：Hemoglobin samples are taken to be cracked, obtain the crack fragment of the crack fragment and beta globin chain of alpha globin chain, a crack fragment is chosen respectively as mark peptide fragment, using the concentration of the mark peptide fragment of the concentration and beta globin chain of the mark peptide fragment of mass spectroscopy alpha globin chain, the ratio of alpha globin chain and beta globin chain is to be indicated by the concentration ratio of the mark peptide fragment of the mark peptide fragment concentration and beta globin chain of alpha globin chain.The present invention by quantitative analysis α, beta globin chain ratio, can EARLY RECOGNITION different type beta Thalassemia, severe, moderate and mild beta thalassaemia can be identified.
Description
Technical field
The invention belongs to field of biochemistry detection, and in particular to a kind of hemoglobin alpha and beta globin chain ratio of measuring
Method and its application in detecting beta Thalassemia.
Background technology
Hemoglobin (Hemoglobin, Hb) is made of a globin and four ferrohemes (also known as ferroprotoporphyrin),
Each ferroheme forms a ring by four pyroles subunits again, and ring center is a ferrous ion.Each globin then has four
Polypeptide chain composition, four peptide chains of globin are different.Adult can be made of 2 α chains and 2 β chains, claim HbA, fetus
It is made of 2 α chains and 2 γ chains, claims HbF, replaced soon by HbA after birth.Every polypeptide chain of globin and one it is blood red
Element connection, constitutes a monomer of hemoglobin, in other words subunit's (i.e. subunit).
Thalassemia, also known as thalassemia are the common diseases of Chinese children, account for about all anaemias
30-40%, be distributed widely in the multiple areas in the world, it is especially common with provinces and cities such as Chongqing, Sichuan, Guangdong, Guangxi in China,
It is the important social concern highly paid close attention to.This disease is since genetic defect causes hemoglobin alpha, the synthesis of beta globin chain to lack
It loses or insufficient, α, beta globin chain proportional imbalance, the one kind in turn resulting in red blood cell life span shortening or dysfunction is congenital poor
Blood.Thalassemia is broadly divided into alpha Thalassemia and beta Thalassemia two according to the difference of the globin chain of unconventionality expression
Major class.Currently as diagnosis after the onset, hypochromic microcytic anemia is generally found to be by blood routine examination and excludes to lack
When iron anaemia, the patient of doubtful thalassemia is carried out in conjunction with hemoglobin electrophoresis and gene diagnosis technology
It makes a definite diagnosis, test operation is complicated, somewhat expensive, and it is universal to be unfavorable for screening.Currently, still lacking quick and high pass to thalassemia
The detection method of amount.
Invention content
Thalassemia is since genetic defect leads to hemoglobin alpha, beta globin chain synthesis missing or insufficient, α, β pearl egg
White chain proportional imbalance in turn results in a kind of congenital anemia of red blood cell life span shortening or dysfunction.Present inventor
Think, since thalassemia is caused by α, beta globin chain proportional imbalance, whether we can be blood red according to quantitative analysis
α in albumen, beta globin chain ratio realize EARLY RECOGNITION different type beta ThalassemiaHow the blood red egg of quantitative analysis
The ratio of α, beta globin chain in whiteFurther how about realize high-throughput detectionPresent inventor is exactly in solution
It states during problem to realize the present invention.
In view of this, the purpose of the present invention is to provide a kind of alpha globin chains measuring hemoglobin and beta globin chain ratio
The method of rate since the crack fragment using hemoglobin is tested as mark peptide fragment, therefore does not require the structure of hemoglobin
As complete, the hemoglobin concentration of blood preparation that is micro and being preserved through the long period can be measured, and this method is easy to operate, institute
Need reagent cost low.
To achieve the above object, the technical scheme is that：
A method of the alpha globin chain and beta globin chain ratio measuring hemoglobin, include the steps that carrying out as follows：
(1) it takes hemoglobin samples to be cracked, obtains the crack fragment of globin polypeptide chain；The globin polypeptide chain
Crack fragment includes the crack fragment of the crack fragment and beta globin chain of alpha globin chain；
(2) using any bar crack fragment in alpha globin chain crack fragment as the mark peptide fragment of alpha globin chain, with β pearls
Mark peptide fragment of any bar crack fragment as beta globin chain in protein chain crack fragment, utilizes mass spectroscopy alpha globin chain
Mark peptide fragment concentration and beta globin chain mark peptide fragment concentration, the ratio of alpha globin chain and beta globin chain is by α pearls
The mark peptide fragment concentration of protein chain and the concentration ratio of the mark peptide fragment of beta globin chain indicate；The mark peptide fragment of the alpha globin chain
Concentration indicated by the integrating peak areas of the mark peptide fragment mass spectrograph response intensity of alpha globin chain, the mark of the beta globin chain
The concentration of will peptide fragment is indicated by the integrating peak areas of the mark peptide fragment mass spectrograph response intensity of beta globin chain.
In the step (1), cutting cracking, such as pancreas are carried out to what hemoglobin samples carried out that cracking can be used enzyme selectivity
Protease can cut off the carboxyl side in lysine and arginine residues in polypeptide chain, specific acquisition lysine or arginine
The peptide fragments of ending.Chromatographic separation technology progress, such as liquid chromatogram, thin-layer chromatography can be used in separation to crack fragment,
Or it is detached using electrophoretic techniques.After separation, any bar generated after the cracking of globin polypeptide chain or a plurality of feature are chosen
Property polypeptide fragment can be used as mark peptide fragment.
Further, the method for the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin, the step
Suddenly in (1), hemoglobin samples are preferably cracked using trypsase, and the crack fragment of alpha globin chain has 14, sequence
Row such as SEQ ID NO：Shown in 1-14；The crack fragment of beta globin chain has 15, sequence such as SEQ ID NO：Shown in 15-29.
Further, the method for the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin, the step
Suddenly in (2), using mark of the high crack fragment of response as alpha globin chain when Mass Spectrometer Method in alpha globin chain crack fragment
Peptide fragment, using mark peptide of the high crack fragment of response as beta globin chain when Mass Spectrometer Method in beta globin chain crack fragment
Section.
Further, the method for the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin, alpha globin
The sequence such as SEQ ID NO of the mark peptide fragment of chain：Shown in 1, the sequence such as SEQ ID NO of the mark peptide fragment of beta globin chain：15 institutes
Show.
Further, the method for the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin, the matter
Spectrum is measured and is measured using Liquid Chromatography-Tandem Mass Spectrometry, particular by liquid chromatogram mobile input mode fast sample, then
Quantitative detection is carried out by tandem mass spectrometer, realizes high-throughput detection.
Further, the method for the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin, passes through matter
When spectrometer MRM pattern Quantitative detections, each feature peptide fragment of alpha globin chain and beta globin chain is by its stable isotope
The internal standard of label is corrected and quantifies, and eliminates systematic error and matrix effect.
Further, the method for the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin, is passing through
When mass spectrograph MRM pattern Quantitative detections, the different fragment ions of the mark peptide fragment of alpha globin chain are to combination and β pearl eggs
The different fragment ion of mark peptide fragment of white chain is used equally for detecting to combination (parent ion/daughter ion combines).
Further, since the implementation of the method for the present invention does not depend on the complete conformation of hemoglobin, therefore the sample of acquisition is protected
It deposits of less demanding, directly whole blood sample can be taken to be analyzed, blood can be also made to dry blood spot sample as hemoglobin
The preservation form of sample is convenient for long-term preservation and long distance transportation, and at low cost.
The present invention also provides alpha globin chains and beta globin chain ratio based on a kind of measurement hemoglobin
Application of the method in detecting beta Thalassemia.
To achieve the above object, the technical scheme is that：
Measure method the answering in detecting beta Thalassemia of the alpha globin chain and beta globin chain ratio of hemoglobin
With the application is specially：According to the side of the alpha globin chain and beta globin chain ratio of a kind of measurement hemoglobin
Method measures known normal person, the alpha globin chain of known different type beta Thalassemia person and unknown sample and β pearls respectively
Protein chain ratio α/β reference value, is judged according to α/β reference value.
The present invention by quantitative analysis α, beta globin chain ratio, can EARLY RECOGNITION different type beta Thalassemia, can
Severe, moderate and mild beta thalassaemia are identified.The patient that screening of the present invention goes out, at least can be from following side
Face is benefited：For would develop into the infant of β thalassemia major, prevent the sense that complication and treatment of blood transfusion may be brought early
Dye, can improve the living environment of infant；For the infant of light-duty beta Thalassemia, instruct its avoid for a long time contact low pressure,
The environment of anoxic can avoid the appearance of Anemia；Before pregnant, the detection of antenatal parental generation beta Thalassemia, contribute to genetic counselling
And fertility decision, the birth of β thalassemia major infant is reduced or avoided, achievees the purpose that prenatal and postnatal care.The present invention has
Wide application prospect and huge social benefit.
The present invention also provides a kind of kit of detection beta Thalassemia, which can conveniently realize
Extraction, denaturation and the cracking of hemoglobin are conducive to commercial applications.
To achieve the above object, the technical scheme is that：
A kind of kit of detection beta Thalassemia, the kit includes corpuscular hemoglobin extractant, albumen
Denaturant, protein cleavage agent and crack protein double solvents, the corpuscular hemoglobin extractant are water, the protein denaturant
For the mixed liquor of acetonitrile and formic acid, the protein cleavage agent is the ammonium bicarbonate soln containing protease, and the crack protein is multiple
Solvent is the acetonitrile solution containing formic acid.
Further, a kind of kit of detection beta Thalassemia, the kit further include alpha globin chain and β
The Isotopic Internal Standard object of each feature peptide fragment of globin chain, and normally mark product, beta Thalassemia heterozygote mark product, the Mediterranean β are poor
Blood homozygote.
The method have the benefit that：
(1) method of the alpha globin chain and beta globin chain ratio of measurement hemoglobin of the invention, due to using blood red
The crack fragment of albumen is tested as mark peptide fragment, therefore does not require the conformation of hemoglobin complete, can be measured micro and be passed through
The hemoglobin concentration for the blood preparation that long period preserves, and this method is easy to operate, required reagent cost is low.The present invention's
Method can be used dry blood spot sample and be detected, and facilitates preservation and delivers over long distances.
(2) a kind of alpha globin chain of measurement hemoglobin and the method for beta globin chain ratio of the invention can be applied to examine
Survey beta Thalassemia.Measure known normal person, known different type beta Thalassemia person and not respectively according to this method
The α/β reference value for knowing sample, is judged according to α/β reference value, can EARLY RECOGNITION different type beta Thalassemia, energy
It is enough that severe, moderate and mild beta thalassaemia are identified.
(3) kit of detection beta Thalassemia of the invention, can conveniently realize hemoglobin extraction,
Denaturation and cracking, are conducive to commercial applications, have wide clinical conversion and application prospect.
Description of the drawings
Fig. 1 Liquid Chromatography-Tandem Mass Spectrometries screen the mass spectrogram of hemoglobin alpha globin chain mark peptide fragment；
Fig. 2 Liquid Chromatography-Tandem Mass Spectrometries screen the mass spectrogram of hemoglobin beta globin chain logo peptide fragment；
The integrogram (wherein, (1) of the automatic integrating peak areas quantitative analysis mark peptide fragments of Fig. 3 and its isotope tag object
Indicate that peptide fragment integrogram, (2) are that α T3 indicate peptide fragment internal standard α T3 for α T3ISIntegrogram).
Fig. 4 is the mass spectrum MRM Pattern recognition collection of illustrative plates that alpha globin chain α T1 (729.4m/z) indicate peptide fragment.
Fig. 5 is the mass spectrum MRM Pattern recognition collection of illustrative plates that beta globin chain β T1 (952.3m/z) indicate peptide fragment.
Specific implementation mode
The preferred embodiment of the present invention is described in detail referring to the drawings.Illustrated embodiment is in order to preferably right
Present disclosure illustrates, but is not that present disclosure is only limitted to illustrated embodiment.So being familiar with the skill of this field
Art personnel carry out nonessential modifications and adaptations according to foregoing invention content to embodiment, still fall within the protection model of the present invention
It encloses.
Instrument, reagent and consumptive material needed for following embodiment are：
(1) instrument needed for
Connect triple level four bars mass spectrographs (API3200) (AB companies, the U.S.), Shimadzu liquid chromatograph (SHIMADZU
LC-20AD) (chromatographic column：XSelect CSH130 C183.5 μm), 3.2mm perforating pliers, centrifuge (Gnencompany
1580R), computer, pipettor (EPPENDORF).
(2) reagent needed for
Acetonitrile, formic acid, TPCK-Treated trypsase, ammonium hydrogen carbonate, all liq reagent are that HPLC or mass spectrum are pure
Grade.
Water used is deionized water.
Internal standard：The Isotopic Internal Standard object of each feature peptide fragment, known to normal concentration.
Quality-control product：Yin and yang attribute reference material, and normally mark product, beta Thalassemia heterozygote mark product, beta Thalassemia homozygosis
Son.
(3) consumptive material needed for
Common 96 hole reaction plate, 96 hole elisa Plates with 0.22 μm of bore filter function, pipettor gun head, 96 orifice plates are special
With aluminium foil overlay film, 96 orifice plate sealed membranes, identification bar code.
Detect the processing of sample：Peripheral blood or vein blood specimen one is taken to drip, about 50 μ l instil in WhatmanFilter paper
Piece, natural diffuseness simultaneously dry, and dry blood spot sample is made, and sealed membrane is sealed up for safekeeping, 4 DEG C of long-term preservations, to be measured.Or directly take whole blood
Sample is analyzed.In the case of doing security protection with zip lock bag, can long range express transportation, solve directly delivering blood
The inconvenience of sample.
Test method without specific conditions in preferred embodiment below is carried out according to normal condition.
The foundation of 1 detection method of embodiment
(1) preparation of reagent
Corpuscular hemoglobin extractant (reagent A)：Deionized water；Extraction for 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 powders are dissolved in 1mol/L ammonium bicarbonate solns,
The lysate of a concentration of 5g/L is made；Hydrolysis for protein.
Crack protein double solvents (reagent D)：(acetonitrile is 1 with water volume ratio to acetonitrile solution：1), wherein also containing 1.2g/
The formic acid of L.
(2) extraction and cracking of hemoglobin
1. dry blood spot sample is got diameter 3.2mm circular filter papers Blood piece (if directly taken complete with standard hole device
Blood specimen is analyzed, and the sample of 3.2 μ l whole bloods is equivalent to) in 96 hole filters plates, 200 μ l of reagent A are added, room temperature is low
Speed shaking 15 minutes, abundant lysed blood.
2. taking 100 μ l of hemolysate, 96 new orifice plates are transferred to, 40 μ l of reagent B are added, quickly shaking 1 minute, room temperature is quiet
Only 5 minutes, make albuminous degeneration.
3. 10 μ l of reagent C are added in 2. mixed liquor that step obtains, low speed shakes mixing, and sealed membrane is sealed up for safekeeping, sets 37 DEG C
It is incubated 2 hours in incubator, the crack fragment of hemoglobin globin polypeptide chain includes the crack fragment and β pearl eggs of alpha globin chain
The crack fragment of white chain；The crack fragment of alpha globin chain has 14, is respectively designated as α T1- α T14, is shown in Table 1, sequence
Such as SEQ ID NO：Shown in 1-14；The crack fragment of beta globin chain has 15, is respectively designated as β T1- β T15, is shown in Table 2,
Its sequence such as SEQ ID NO：Shown in 15-29.
4. taking 96 hole filters, the 12 μ l of sample liquid after being incubated in sequentially adding 108 μ l of reagent D and step 3. are slight to shake
Redissolution is swung, brief centrifugation leaves and takes lower plate sample, to be measured.
The crack fragment of 1. alpha globin chain of table
The crack fragment of 2. beta globin chain of table
(3) α, beta globin chain logo peptide fragment mass spectroscopy
1. activation associated with Liquid Chromatography-Tandem Mass Spectrometry and establishment
A, liquid phase chromatogram condition：Autosampler parameter is set by table 3, sampling time program is set by table 4.
B, Mass Spectrometry Conditions：By table 5, relevant parameter is set.
The setting of these parameters can be such that the crack fragment of hemoglobin detaches well, and make to obtain well after entering mass spectrum
Response, quantitative analysis optionally is carried out to selected mark peptide fragment.The setting of relevant parameter is not limited to this, according to
The different crack fragments of acquisition, and the difference of mark peptide fragment chosen, can accordingly carry out liquid chromatogram and mass spectrometer parameters
Adjustment.
3. autosampler parameter of table
4. sampling time of table program (Time Program)
Note:Binary Flow 0.15ml/min；Pressure limits 0-15Mpa.
5. mass spectrometer parameters of table
2. mass spectrograph multiple reaction monitors the optimization of (MRM) mode parameter.
Sample extract liquor is digested through pancreatin, and α, beta globin chain hydrolysate generate the polypeptide fragment of different mass-to-charge ratioes, such as table
1 and table 2 shown in, in principle, alpha globin chain crack fragment can be used as the mark peptide fragment of alpha globin chain, the cracking of beta globin chain
Segment can be used as the mark peptide fragment of beta globin chain.According to selected mark peptide fragment, the ginseng of mass spectrograph MRM detection patterns is set up
Number, including DP (removing cluster voltage), EP (injecting voltage), CE (collision voltage), CEP (collision cell entrance potential), CXP (collision cells
Exit potential) etc., and optimization range, the step-length of parameter are changed as needed.Q1 parent ions and the scanning of Q3 daughter ions is set separately
Parameter.Specifically, the MRM sweep parameters of the mark peptide fragment optimization of part α, beta globin chain are shown in Table 6.
6. part α of table, beta globin chain logo peptide fragment MRM sweep parameters
3. measurement and quantitative analysis of the mark peptide fragment of alpha globin chain with the mark peptide fragment of beta globin chain
Sample extract liquor is digested through pancreatin, and α, beta globin chain hydrolysate generate the polypeptide fragment of different mass-to-charge ratioes, such as table
1 and table 2 shown in, in principle, alpha globin chain crack fragment can be used as the mark peptide fragment of alpha globin chain, the cracking of beta globin chain
Segment can be used as the mark peptide fragment of beta globin chain, these mark peptide fragments are used equally for diagnosis index to design.
By taking alpha globin chain as an example, hydrolysate generates the polypeptide fragment of different mass-to-charge ratioes, and mass spectrogram is as shown in Figure 1, tool
Body sequence and mass-to-charge ratio are as shown in table 1, totally 14 sequences, and number is α T1- α T14, these segments are in the enough sensitivity of mass spectrograph
In the case of, can be used as mark peptide fragment for hemoglobin analysis (number α T8 be an amino acid, in removing sample
In the case that free amino acid interferes, mark peptide fragment can also be used as).
By taking beta globin chain as an example, hydrolysate generates the polypeptide fragment of different mass-to-charge ratioes, and mass spectrogram is as shown in Fig. 2, tool
Body sequence and mass-to-charge ratio are as shown in table 2, totally 15 sequences, and number is β T1- β T15, these segments are in the enough sensitivity of mass spectrograph
In the case of, can be used as mark peptide fragment for hemoglobin analysis (number β T8 be an amino acid, in removing sample
In the case that free amino acid interferes, mark peptide fragment can also be used as).
Data conversion and quantitative analysis method：
Response intensity (Intensity) of the instrument to various concentration polypeptide fragment is shown in mass spectrograph initial data, profit
With MultiQuant2.0.2 softwares, automatic integration analysis is carried out to mark peptide fragment with integrating peak areas method, calculates peak area point
Value.The ratio of α, beta globin chain are represented by the ratio of α, beta globin chain logo peptide fragment, and indicate that the concentration level of peptide fragment can be straight
The integrating peak areas size by mass spectrograph response intensity is connect to indicate.By taking α T3 mark peptide fragments as an example, the concentration of α T3 mark peptide fragments
It is directly indicated by the integrating peak areas size of mass spectrograph response intensity, such as (1) figure in Fig. 3.
Further, in order to eliminate systematic error and matrix effect, each feature peptide fragment is all by its stable isotope labeling
Internal standard is corrected and quantifies；The preparation of internal standard solution and the term of validity are controlled, to ensure that error is in the range of requiring between criticizing.It utilizes
MultiQuant2.0.2 softwares carry out automatic integration respectively with integrating peak areas method to mark peptide fragment and its isotope tag object
Analysis calculates peak area score value.By taking α T3 mark peptide fragments as an example, the concentration of α T3 mark peptide fragments need to be through the isotope of its known concentration
Marker α T3ISIt is corrected and quantifies, as shown in Figure 3.(1) figure in Fig. 3 is α T3 mark peptide fragment integrating peak areas, in Fig. 3
(2) figure be α T3 mark peptide fragment isotope labelling internal standard α T3ISIntegrating peak areas.α T3 mark peptide fragment concentration (nmol/
Ml calculation formula) is as follows：
α T3 mark peptide fragments concentration=(α T3 mark peptide fragments integrating peak areas/internal standard α T3ISIntegrating peak areas) * internal standards α
T3ISConcentration.
Therefore, mark peptide fragment concentration computation formula is：
4. the ratio calculation of α, beta globin chain
The ratio of α, beta globin chain are represented by α, beta globin chain logo peptide fragment concentration ratio, and globin chain mark peptide fragment is dense
The calculating of degree step 3. in introduced.Below with response high cracking piece when Mass Spectrometer Method in alpha globin chain crack fragment
Mark peptide fragments of the section α T1 as alpha globin chain, with response high cracking piece when Mass Spectrometer Method in beta globin chain crack fragment
Mark peptide fragments of the section β T1 as beta globin chain, is introduced.
As shown in Figure 1, the α T1 segment responses of alpha globin chain are higher, i.e., Mass Spectrometer Method is more sensitive.With α chain α T1
For (729.4m/z), under tandem mass spectrum MRM detection patterns, by its Isotopic Internal Standard quantitative analysis its fragment ion to group
At the content situation of indirect analysis alpha globin chain, the content of alpha globin chain is indicated the concentration approximate representation of peptide fragment by it.αT1
(729.4m/z) indicates that the composition of peptide fragment fragment ion pair is as shown in table 7, when analysis mark peptide fragment fragment ion is to composition, often
A mark peptide fragment can choose it is therein it is any parent ion/daughter ion is combined, such as α chain α T1 (729.4/430.2), same position is used in combination
Internal control is denoted as in element.The mass spectrum MRM Pattern recognition collection of illustrative plates of α T1 (729.4m/z) mark peptide fragments is shown in Fig. 4.
As shown in Figure 2, the β T1 segment responses of beta globin chain are higher, i.e., Mass Spectrometer Method is more sensitive.With the β T1 of β chains
For (952.3m/z), under tandem mass spectrum MRM detection patterns, pass through its fragment ion group of its Isotopic Internal Standard quantitative analysis
At the content situation of indirect analysis beta globin chain, the content of beta globin chain is indicated the concentration approximate representation of peptide fragment by it.βT1
(952.3m/z) indicates that the composition of peptide fragment fragment ion pair is as shown in table 8, when analysis mark peptide fragment fragment ion is to composition, often
A mark peptide fragment can choose its it is any parent ion/daughter ion is combined, such as the β T1 (952.3/502.3) of β chains, isotope is used in combination
Inside it is denoted as internal control.The mass spectrum MRM Pattern recognition collection of illustrative plates of β T1 (952.3m/z) mark peptide fragments is shown in Fig. 5.
Table 7. hemoglobin alpha globin chain T1 (729.4m/z) mark peptide fragment fragment ion compositions
Table 8. hemoglobin beta globin chain T1 (952.3m/z) mark peptide fragment fragment ion compositions
The ratio of α, beta globin chain are represented by the ratio of α, beta globin chain feature peptide fragment；And the concentration of feature peptide fragment is high
It is low to be indicated by the integrating peak areas size of mass spectrograph response intensity；In order to eliminate systematic error and matrix effect, each is special
Sign peptide fragment is all corrected and is quantified by the internal standard of its stable isotope labeling；The preparation of internal standard solution and the term of validity are controlled, with true
Error is in the range of requiring between guarantor crowd.
All feature peptide fragments are used equally for the calculating of α, beta globin chain ratio in principle, are mass spectrograph selected in scheme
Response is high, relatively sensitively mark peptide fragment is as the design for carrying out technical solution is represented for reaction, and part is as shown in table 9；Each mark
Will peptide fragment chooses a pair of of parent ion/daughter ion combination, and such as α T1 (729.4/430.2) and β T1 (952.3/502.3), same position is used in combination
Internal control is denoted as in element；It is as follows to calculate α, the equation of beta globin chain ratio：
Note：By taking α T1 (729.4/430.2) and β T1 (952.3/502.3) as an example, α T are α T1 (729.4/430.2) in formula
Integrating peak areas；αTISFor the correspondence integrating peak areas of the Isotopic Internal Standard of mark peptide fragment α T1；β T are mark peptide fragment β T1
The integrating peak areas of (952.3/502.3)；βTISFor the correspondence integrating peak areas of the Isotopic Internal Standard of mark peptide fragment β T1；R is normal
Number is stable isotope internal standard α TISWith β TISThe ratio between concentration.
The calculating of 9. α of table, beta globin chain ratio
Note formula in table：α T represent the integrating peak areas of alpha globin chain logo peptide fragment α T；
αTISRepresent the correspondence integrating peak areas of the Isotopic Internal Standard of alpha globin chain logo peptide fragment α T；
β T represent the integrating peak areas of beta globin chain logo peptide fragment β T；
βTISRepresent the correspondence integrating peak areas of the Isotopic Internal Standard of beta globin chain logo peptide fragment β T；
R is constant, is stable isotope internal standard α TISWith β TISThe ratio between concentration.
Embodiment 2
Using above-mentioned established detection scheme, by liquid phase systems Flow Sampling mode fast sample, then pass through mass spectrum
Instrument is used for quickly detecting, and realizes that the high-throughput sample introduction of detection sample, each sample sample detection time are less than 1 minute, primary real
It tests, it can be with batch detection at least 192 samples.
According to above-mentioned technical proposal of the present invention, analyze in 200 normal controls (June at age was to 40 years old) and 358 ground β
It is as shown in table 10 to establish reference value section (1st-99th) by extra large Anemic patients and 16 HbE type Anemic patients.Reference interval may
Slightly difference due to different experiments room, different experiments personnel, different reagent lot, it is proposed that the reference value of oneself is established in each laboratory
Section.
10. normal person of table, beta Thalassemia people carrier, patient α:β ratio reference intervals
Method is evaluated：
According to the distribution of above-mentioned reference interval and sample it is found that method described in the invention is poor to above-mentioned 9 kinds of Mediterranean β
The average recall rate about 77% of blood heterozygote；The detection of double heterozygote is mutated to above-mentioned 13 kinds of beta Thalassemia homozygotes and β
Rate is more than 95%；Poor recall rate is more than 95% compoundly to 15 kinds of α, β；Inspection to 2 kinds of compound thalassemias of HbE, β
Extracting rate is more than 95%；15% is less than to the recall rate of HbE sickle-cell anaemias, i.e. the method for the invention is not suitable for reaping hook
The detection of shape cell anemia.
An example normal specimen, an example β are chosen respectively+Ground poor (CD17), an example β0Ground is poor (CD27/28＆IVS-II-654),
It does in batch and repeats to test between batch, calculate mean value and standard deviation, show that the precision of screening method and repeatability are higher, knot
Fruit is as shown in table 11.
In technical solution batch described in table 11., repeatability and Precision Analyze between batch
The present invention by quantitative analysis α, beta globin chain ratio, can EARLY RECOGNITION different type beta Thalassemia, can
Severe, moderate and mild beta thalassaemia are identified.13 kinds of beta Thalassemia homozygotes of detection method pair of the present invention
And the recall rate of β mutation double heterozygotes is more than 95%, poor recall rate is more than 95%, and method compoundly to 15 kinds of α, β
With higher precision and repeatability.The patient that screening of the present invention goes out can at least be benefited from the following aspects：For inciting somebody to action
The infant of β thalassemia major is developed into, prevents the infection that complication and treatment of blood transfusion may be brought early, trouble can be improved
The living environment of youngster；For the infant of light-duty beta Thalassemia, the environment for instructing it to avoid contacting low pressure, anoxic for a long time can
Avoid the appearance of Anemia；Before pregnant, the detection of antenatal parental generation beta Thalassemia, contribute to genetic counselling and fertility decision, subtract
Less or the birth of β thalassemia major infant is avoided, achievees the purpose that prenatal and postnatal care.The present invention has broad application prospects
With huge social benefit.
Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (1)
1. a kind of kit of detection beta Thalassemia, which is characterized in that the kit includes corpuscular hemoglobin extraction
Agent, protein denaturant, protein cleavage agent and crack protein double solvents are taken, the corpuscular hemoglobin extractant is water, described
Protein denaturant is the mixed liquor of acetonitrile and formic acid, and the protein cleavage agent is the ammonium bicarbonate soln containing protease, described
Crack protein double solvents is the acetonitrile solution containing formic acid；The kit further includes alpha globin chain and each spy of beta globin chain
The Isotopic Internal Standard object and normal person for levying peptide fragment mark product, beta Thalassemia heterozygote mark product, beta Thalassemia homozygote.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510618319.5A CN105223290B (en) | 2015-09-24 | 2015-09-24 | A kind of method and application measuring hemoglobin alpha and beta globin chain ratio |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510618319.5A CN105223290B (en) | 2015-09-24 | 2015-09-24 | A kind of method and application measuring hemoglobin alpha and beta globin chain ratio |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105223290A CN105223290A (en) | 2016-01-06 |
| CN105223290B true CN105223290B (en) | 2018-09-11 |
Family
ID=54992361
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510618319.5A Active CN105223290B (en) | 2015-09-24 | 2015-09-24 | A kind of method and application measuring hemoglobin alpha and beta globin chain ratio |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105223290B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110658251A (en) * | 2019-10-01 | 2020-01-07 | 长沙湘华质谱医学科技有限公司 | Use of a characteristic protein composition or mass spectral model for characterizing thalassemia |
| CN110658252A (en) * | 2019-10-01 | 2020-01-07 | 长沙湘华质谱医学科技有限公司 | Characteristic protein spectrum model for mass spectrum diagnosis of thalassemia and application thereof |
| CN111638261B (en) * | 2020-04-17 | 2023-04-07 | 融智生物科技（青岛）有限公司 | Computing equipment, storage medium and thalassemia screening device and system |
| CN111948404B (en) * | 2020-08-03 | 2023-04-07 | 融智生物科技（青岛）有限公司 | Characteristic protein marker composition for screening thalassemia, mass spectrum model and application of characteristic protein marker composition |
| CN114509526A (en) * | 2021-12-31 | 2022-05-17 | 福建医科大学孟超肝胆医院(福州市传染病医院) | LC-MS/MS-based pure solvent type whole blood immunosuppressant drug concentration joint inspection kit and detection method |
| CN116203144B (en) * | 2022-05-11 | 2023-10-27 | 重庆医科大学附属儿童医院 | Method for determining chain ratio of various globin in hemoglobin and application thereof |
| CN117825542A (en) * | 2022-05-11 | 2024-04-05 | 重庆医科大学附属儿童医院 | Marker composition for hemoglobinopathy, screening reagent and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120889C (en) * | 2000-07-24 | 2003-09-10 | 浙江江南生物科技有限公司 | DNA chip for diagnosing hereditary anaemia related gene mutation |
| CN1570150A (en) * | 2004-05-12 | 2005-01-26 | 刘敬忠 | Alpha-mediterranean anemia gene diagnosis kit and uses |
| KR100773878B1 (en) * | 2007-01-25 | 2007-11-07 | 한국과학기술연구원 | Method for analysis of homocysteine in plasma using gas chromatography-mass spectrometry |
| CN103954717A (en) * | 2014-04-28 | 2014-07-30 | 重庆医科大学附属儿童医院 | Method for testing hemoglobin concentration by utilizing liquid chromatogram tandem mass spectrum |
-
2015
- 2015-09-24 CN CN201510618319.5A patent/CN105223290B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105223290A (en) | 2016-01-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105223290B (en) | A kind of method and application measuring hemoglobin alpha and beta globin chain ratio | |
| CN105842375B (en) | The polypeptide of donkey derived component in one group of discriminating donkey-hide gelatin and its product | |
| CN108088934A (en) | Method and kit a kind of while that detect more kinds of related substanceses of CAH | |
| CN107209189B (en) | Quick screening and evaluation by glycosylated hemoglobin mass spectrum to diabetes and prediabetes | |
| CN109280077A (en) | A kind of identification donkey-hide gelatin and the polypeptide containing pigskin derived component in donkey-hide gelatin product | |
| CN111948404B (en) | Characteristic protein marker composition for screening thalassemia, mass spectrum model and application of characteristic protein marker composition | |
| CN113156018B (en) | Method for establishing liver and gall disease diagnosis model and diagnosis system | |
| CN109187840A (en) | The high flux examination analysis method of organic pollutant in a kind of blood | |
| CN114113381B (en) | Syngnathus schutz characteristic polypeptide, application thereof and method for identifying comfortable Syngnathus schutz | |
| JP4317083B2 (en) | Mass spectrometry method and mass spectrometry system | |
| Thevis et al. | New drugs and methods of doping and manipulation | |
| CN105467022A (en) | Analysis method for neuron injury related differentially expressed protein | |
| CN106908532B (en) | The method for measuring aquatic products Malachite Green, quinolones, sulfa drugs | |
| CN103954717A (en) | Method for testing hemoglobin concentration by utilizing liquid chromatogram tandem mass spectrum | |
| CN102565251A (en) | Method for detecting contents of acylcarnitines in serum or blood scrip | |
| CN109709233A (en) | A method of a variety of mushroom toxin in detection blood, urine | |
| CN102481319A (en) | Apolipoprotein ciii in pre- and type 2 diabetes | |
| CN110045053A (en) | A kind of QuEChERS pre-treating method of the analysis of the amphetamines suitable for blood | |
| CN110133280A (en) | A kind of measuring method of the glycation ratio of hemoglobin of β chain variation | |
| CN111638261B (en) | Computing equipment, storage medium and thalassemia screening device and system | |
| CN109358131B (en) | The rapid analysis method of monoclonal antibody N sugar sugar-type | |
| CN103344710B (en) | Use liquid chromatography-tandem mass spectrometry for criminal investigation object detects the method for diphenoxylate in biological sample | |
| CN112326851A (en) | Mass spectrum quantitative analysis method based on isotope or secondary mass spectrum ion isotope abundance distribution thereof | |
| CN115248277B (en) | Cynanchum paniculatum characteristic polypeptide, application thereof and method for identifying Cynanchum paniculatum | |
| CN109293741A (en) | A kind of identification donkey-hide gelatin and the polypeptide containing horse skin derived component in donkey-hide gelatin product |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |