CN111398596A - Complete equipment and method for identifying haptoglobin phenotype - Google Patents
Complete equipment and method for identifying haptoglobin phenotype Download PDFInfo
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Abstract
The invention discloses a complete equipment and a method for identifying haptoglobin phenotype, which firstly discloses the application of an MA L DI-TOF mass spectrometer in identifying haptoglobin phenotype, and further discloses the complete equipment and the method for identifying haptoglobin phenotype.
Description
Technical Field
The invention relates to the field of biotechnology. And more particularly to a kit and method for identifying haptoglobin phenotypes.
Background
Haptoglobin (Hp), commonly known as Haptoglobin, is an acute phase protein that is predominantly distributed in the plasma and other body fluids of mammals. In plasma, haptoglobin can combine with free Hemoglobin (Hb) in plasma to form Hp-Hb (haptoglobin-Hemoglobin) compound, and can be recognized and phagocytized by CD163 receptors in macrophages, so that free Hemoglobin in blood circulation is eliminated, and iron or heme generated by Hemoglobin in a hemolysis process is prevented from causing oxidative damage to an organism.
Haptoglobin is an acidic glycoprotein consisting of 347/406 amino acid residues encoded by two alleles Hp1 and Hp2 at position 16q22 of the autosome, in which the linkage between the light chain and the light chain (chain α) and between the light chain and the heavy chain (chain β) is effected by disulfide bonds, the light chain comprising α1Chain sum α2Two chains consisting of α 1 chain 84 amino acid residues with a molecular weight of about 9kDa and α2The chain consists of 143 amino acid residues, and has a molecular weight of about 16 kDa.the heavy chain, β chain, consists of only one amino acid (about 245 amino acid residues), and has a molecular weight of about 40 kDa.the thiol group at cysteine Cys at position 15 of chain α 1 forms a disulfide bond with the thiol group of cysteine in the other chain α, while the thiol group at cysteine 72 forms a disulfide bond with the thiol group of chain β. α2Both the cysteine thiol groups at positions 15 and 74 of the chain can form disulfide bonds with the cysteine thiol group of the other α chain, while the thiol group of the cysteine at position 131 forms a disulfide bond with the thiol group of the β chain, it is known that α1Only one cysteine thiol group is present in the chain which can be linked to the light chain, and α2Haptoglobin proteins share 3 phenotypes encoded by two alleles, Hp1-1, Hp2-1 and Hp2-2, and therefore the multimeric forms formed by αβ differ among the three haptoglobin phenotypes, Hp1-1, expressing only α 1 light chain and thus Hp only as a dimer (α)1β)2The form of (A) is represented by (B) α in the Hp2-1 phenotype1And α2The light chains were all expressed and thus Hp was expressed (α)1β)2、(α1β)2(α2β)nThe Hp2-2 phenotype expresses only α2Light chain, therefore Hp to (α)2β)nIn the form of a dimer, trimer, tetramer, etc.
Researches find that the three different phenotypes of the haptoglobin have certain correlation with the onset risks of diseases, such as coronary heart disease, essential hypertension, schizophrenia, diabetes and the like. Among the three haptoglobin phenotypes, Hp2-2 has the weakest ability to bind hemoglobin, thus causing the worst ability to clear free hemoglobin in plasma, and when hemolysis occurs, it may cause oxidative damage of iron ions to organs such as heart and kidney. Particularly, in the Hp2-2 population with diabetes, the life span of red blood cells is shortened to promote the increase of free hemoglobin in blood plasma, and the haptoglobin of Hp2-2 phenotype has weaker capability of removing hemoglobin, so that the risk of cardiovascular diseases, nephropathy and other complications of the patients with the diabetes is increased. Moreover, for diabetic patients, haptoglobin of different phenotypes will affect the therapeutic efficacy of the drug against the disease. For this reason, the identification of plasma haptoglobin phenotype will be a target in the prevention and treatment of diseases, and in particular, will serve the establishment of a personalized medical model when a new medical age leading to "precision medicine" comes.
Currently, there are two main categories of haptoglobin phenotype identification techniques: DNA-based PCR techniques and protein-based electrophoresis techniques. However, both of the two technologies have the characteristics of complex operation, long time consumption, large sample amount, low flux, high technical requirements on operators and the like, are not favorable for clinical popularization of haptoglobin phenotype identification projects, and fully play the guidance role of haptoglobin phenotypes in the disease diagnosis and treatment process, particularly the reference value in the aspect of diabetes complication prevention and treatment.
Therefore, there is a need to provide a method for identifying haptoglobin phenotypes at high throughput and rapidly, which addresses at least one of the above problems.
Disclosure of Invention
It is a first object of the invention to provide the use of a MA L DI-TOF mass spectrometer for identifying haptoglobin phenotypes.
It is a second object of the present invention to provide a kit for identifying a haptoglobin phenotype.
The third purpose of the invention is to provide a method for identifying haptoglobin phenotype, which has the characteristics of small sample demand, easiness in obtaining, simplicity in pretreatment, high flux and the like.
To achieve the above object, the present invention firstly provides the use of a MA L DI-TOF mass spectrometer for identifying haptoglobin phenotypes.
The haptoglobin phenotype of the invention is Hp1-1 type (only α is expressed)1Light chain), Hp2-2 type (only α is expressed)2Light chain) and Hp2-1 type (simultaneously expressing α)1Light chain sum α2Light chain).
The invention further provides a kit for identifying a haptoglobin phenotype, the kit comprising a MA L DI-TOF mass spectrometer, a reagent for MA L DI-TOF mass spectrometry detection and a readable carrier;
the readable carrier is described as follows:
if the reduced sample to be detected has peaks with mass-to-charge ratios of 9180-9200 and no peaks with mass-to-charge ratios of 15935-15955 in a protein spectrogram obtained by detection of an MA L DI-TOF mass spectrometer, identifying the haptoglobin phenotype of the sample to be detected as Hp1-1 type, if the reduced sample to be detected has peaks with mass-to-charge ratios of 15935-15955 and no peaks with mass-to-charge ratios of 9180-9200 in the protein spectrogram obtained by detection of the MA L DI-TOF mass spectrometer, identifying the haptoglobin phenotype of the sample to be detected as Hp2-2 type, and if the reduced sample to be detected has peaks with mass-to-charge ratios of 9180-9200 and peaks with mass-to-charge ratios of 15935-15955 in the protein spectrogram obtained by detection of the MA L DI-TOF mass spectrometer, identifying the haptoglobin phenotype of the sample to be detected as Hp2-1 type.
Further, the mass-to-charge ratio of 15935 to 15955 is preferably 15947, and the mass-to-charge ratio of 9180 to 9200 is preferably 9196 to 9197.
Further, the MA L DI-TOF mass spectrometry detection reagent comprises sinapic acid matrix.
Further, the reduced sample to be detected is obtained by reducing the sample to be detected by using a dithiothreitol reduction reagent.
Further, reducing the sample to be detected and the dithiothreitol reduction reagent at 95 ℃ for 5min according to the volume ratio of 1: 5-1: 19; preferably, the volume ratio of the sample to be detected to the dithiothreitol reduction reagent is 1: 9.
Further, the content of dithiothreitol in the dithiothreitol reduction reagent is 80-120 mM, and the content of ammonium bicarbonate is 25 mM; preferably, the content of dithiothreitol in the dithiothreitol reduction reagent is 100mM, and the content of ammonium bicarbonate is 25 mM; wherein, the solvent of the dithiothreitol reduction reagent is water.
The invention further provides a method of identifying a haptoglobin phenotype.
The method for identifying the haptoglobin phenotype comprises the following steps of detecting a reduced sample to be detected by using an MA L DI-TOF mass spectrometer to obtain a protein spectrogram of the reduced sample to be detected, identifying the haptoglobin phenotype of the sample to be detected according to mass-to-charge ratios appearing in the protein spectrogram, wherein if a peak of a mass-to-charge ratio of 9180-9200 does not appear in the protein spectrogram, the haptoglobin phenotype of the sample to be detected is identified as an Hp1-1 type, if a peak of a mass-to-charge ratio of 15935-15955 appears in the protein spectrogram, a peak of a mass-to-charge ratio of 9180-9200 does not appear in the protein spectrogram, the haptoglobin phenotype of the sample to be detected is identified as an Hp2-2 type, and if a peak of a mass-to-charge ratio of 9180-9200 and a peak of a mass-to-charge ratio of 15935-15955 appear in the protein spectrogram, the haptoglobin phenotype of the sample to be detected is identified as an Hp 2.
Further, the mass-to-charge ratio of 15935 to 15955 is preferably 15947, and the mass-to-charge ratio of 9180 to 9200 is preferably 9196 to 9197.
Further, the setting parameters of the MA L DI-TOF Mass spectrometer are that the laser frequency is 1000Hz, the laser energy is 5.2 muJ, the detector voltage is-0.75 kV, the Focus Mass is 10000Da, and the collection Mass-to-charge ratio range is 5000Da-20,000 Da.
Further, the reduced sample to be detected is obtained by reducing the sample to be detected by using a dithiothreitol reduction reagent.
Further, the volume ratio of the sample to be detected to the dithiothreitol reduction reagent is 1: 5-1: 19, and the sample is reduced for 5min at 95 ℃. Preferably, the volume ratio of the sample to be detected to the dithiothreitol reduction reagent is 1: 9.
Further, the content of dithiothreitol in the dithiothreitol reduction reagent is 80-120 mM, and the content of ammonium bicarbonate is 25 mM; preferably, the content of dithiothreitol in the dithiothreitol reduction reagent is 100mM, and the content of ammonium bicarbonate is 25 mM; wherein, the solvent of the dithiothreitol reduction reagent is water.
Further, the method for detecting the reduced sample to be detected by using the MA L DI-TOF mass spectrometer comprises the following steps:
mixing the reduced sample to be detected with the erucic acid matrix to obtain a mixed solution;
placing the erucic acid matrix points in each hole of a target plate of an MA L DI-TOF mass spectrometer, and drying and crystallizing the erucic acid matrix to obtain a prefabricated target plate;
the mixture was spotted onto each well of the pre-fabricated target plate, dried and then placed on a MA L DI-TOF mass spectrometer for analysis.
Further, the volume ratio of the reduced sample to be detected to the sinapic acid substrate is 1: 5-1: 19, the sinapic acid content in the sinapic acid substrate is 10-20mg/m L, the acetonitrile content is 30-40% by volume, the ethanol content is 10-20% by volume, preferably, the volume ratio of the reduced sample to the sinapic acid substrate is 1:9, the sinapic acid content in the sinapic acid substrate is 10mg/m L, the acetonitrile content is 40% by volume, and the ethanol content is 10% by volume, wherein the solvent of the sinapic acid substrate is water.
The sample to be detected is plasma, and in a specific embodiment of the invention, the sample to be detected is fingertip plasma.
The invention has the following beneficial effects:
the method for identifying the haptoglobin phenotype in the plasma only needs 2 microliters of terminal fingertip plasma, can be used for spotting and carrying out MA L DI-TOF mass spectrometry detection after short-time high-temperature reduction (about 5min), can quickly identify three phenotypes of the haptoglobin in the human plasma, and is expected to provide reference for diseases related to the haptoglobin phenotype in clinic, particularly diabetic complications.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a typical protein profile of human plasma Hp1-1 haptoglobin.
FIG. 2 is a typical protein profile of human plasma Hp2-1 haptoglobin.
FIG. 3 is a typical protein profile of human plasma Hp2-2 haptoglobin.
FIG. 4 is a typical protein profile of a haptoglobin standard.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
EXAMPLE 1 identification of haptoglobin phenotype in human plasma
Firstly, reagent preparation:
① preparation of DTT reducing reagent (DTT reducing reagent), accurately weighing 0.154g DTT and 0.098g ammonium bicarbonate (chromatogram pure) in a 50m L volumetric flask, adding a proper amount of deionized water to fully dissolve solid powder (ultrasonic if necessary), and fixing the volume to scale with deionized water to obtain the DTT reducing reagent with DTT concentration of 100mM and ammonium bicarbonate concentration of 25 mM.
② preparation of matrix of sinapic acid (SA matrix) solution comprises accurately weighing 0.250g solid powder of sinapic acid in 25m L volumetric flask, adding 10m L acetonitrile (chromatogram pure) and 2.5m L anhydrous ethanol, diluting with deionized water to desired volume, and performing ultrasonic treatment for 5min to dissolve sinapic acid until no visible solid powder exists in the volumetric flask (ultrasonic treatment time can be prolonged if necessary) to obtain an aqueous solution containing 40% acetonitrile and 10% ethanol and having sinapic acid concentration of 10mg/m L.
Second, plasma sample pretreatment
Collecting about 50 μ L blood from fingertips in a 0.6m L centrifuge tube, centrifuging at 3000rpm/min for 10min to collect the upper plasma sample.
1. Direct dilution
And (3) putting 2.0 mu L of the upper layer plasma sample into a 0.6m L centrifuge tube, adding a 25mM ammonium bicarbonate aqueous solution of 18 mu L, uniformly mixing by vortex, diluting the plasma sample by 10 times, putting 2.0 mu L of the plasma 10-time diluent into a new 0.6m L centrifuge tube, adding an SA matrix solution of 18 mu L, uniformly mixing by vortex, and obtaining a mixed solution 1 (namely the sample before reduction) to be spotted.
2. Reduction treatment
And (3) putting the 2.0 mu L upper layer plasma sample into a 0.6m L centrifuge tube, adding 18 mu L DTT reducing reagent, performing vortex mixing, reducing in a 95 ℃ metal bath or boiling water bath for 5min, taking out, cooling to room temperature to obtain a reduced plasma solution, putting the 2.0 mu L reduced plasma solution into a new 0.6m L centrifuge tube, adding 18 mu L SA matrix solution, performing vortex mixing to obtain a mixed solution 2 (namely a reduced sample), and performing spotting.
Preparation of prefabricated target plate
Placing MA L DI target plate on 42 deg.C heater, uniformly spotting SA matrix solution of 2.5 μ L on each well (diameter is 3mm) of MA L DI target plate, drying and crystallizing matrix to obtain prefabricated target plate, and waiting for use, wherein each well on the target plate has diameter of 3mm, and if the hole diameter of target plate is changed, the matrix is required to be re-spotted
And (3) respectively dropping the mixed solution 1 and the mixed solution 2 obtained in the second step of 2.5 mu L into each hole of the prefabricated target plate obtained in the third step, heating and drying on a heater at 42 ℃, and then waiting for analysis.
1.MA L DI-TOF Mass Spectrometry analysis
The MA L DI-TOF mass spectrometer used by the invention is a Quan-TOF I type MA L DI-TOF mass spectrometer produced by Qingdao Huazhi Biotechnology Limited.
The instrument setup parameters were as follows:
laser frequency: 1000 Hz;
laser energy: 5.2 muJ;
detector voltage: -0.75 kV;
Focus Mass:10000Da;
collection quality range: 5000Da to 20,000 Da.
2. Protein mass spectrogram processing
And (3) performing 100% proportional baseline correction on the spectrogram by adopting a self-contained Default baseline correction mode of the instrument. And (4) selecting a Moving-Average mode to smooth the mass spectrogram after the baseline correction (setting 9 windows). Peak detection parameters: MiniSR: 1; % Height to use: 1. three peak values of m/z28078, m/z14039 and m/z9422 are selected to calibrate peaks on a mass spectrogram in a way of TimeQuadracicOfSqrMass.
Fifth, haptoglobin phenotypic identification
The haptoglobin phenotype identification is carried out according to a QuandTOF mass spectrogram, as can be seen from figures 1-3, after DTT reduction, if only a peak (specifically 9196.1m/z) with a mass-to-charge ratio of 9180-9200 m/z appears in the mass spectrogram under the assistance of SA matrix, and no peak with a mass-to-charge ratio of 15935-15955 m/z appears in the plasma sample, corresponding to α in haptoglobin1The chain can identify the haptoglobin phenotype of the plasma sample as Hp1-1 type (as shown in figure 1), if the mass spectrogram only has a peak (specifically 15947m/z) with a mass-to-charge ratio of 15935-15955 m/z and no peak with a mass-to-charge ratio of 9180-9200 m/z, the phenotype corresponds to α in the haptoglobin2The chain can identify the phenotype of the haptoglobin of the plasma sample as Hp2-2 type (as shown in figure 3), and if a peak with a mass-to-charge ratio of 9180-9200 m/z (specifically 9196.6m/z, the error of 0.5m/z in figure 1 belongs to a normal error range) and a peak with a mass-to-charge ratio of 15935-15955 m/z (specifically 15947m/z) appear simultaneously in a mass spectrogram, the phenotype of the haptoglobin of the plasma sample corresponds to α in the haptoglobin1Chain sum α2Chains, from which the haptoglobin phenotype in the plasma sample was identified as Hp2-1 (FIG. 2).
Example 2 haptoglobin standards confirm the accuracy of identification of haptoglobin phenotype
First, reagent preparation
The same as in example 1.
Second, pretreatment
The haptoglobin standard substance (purchased from Sigma, H3536-1MG) is derived from mixed human plasma, the purity is more than or equal to 95 percent (SDS-PAGE), the standard substance is a mixture of different haptoglobin serums, and the haptoglobin standard substance comprises three phenotypes of Hp1-1, Hp2-2 and Hp2-1 haptoglobin, 100 mu L deionized water is used for fully dissolving the haptoglobin standard substance freeze-dried powder to prepare 10MG/m L haptoglobin standard substance storage solution, the haptoglobin standard substance storage solution is placed in a freezing storage at the temperature of 20 ℃ below zero after subpackaging, 2 mu L haptoglobin standard substance storage solution is taken out to be placed in a 0.6m L centrifuge tube, 8 mu L deionized water is added, the haptoglobin standard substance solution is obtained after vortex mixing, and pretreatment is respectively carried out according to the:
1. direct dilution
And (3) putting the haptoglobin standard solution with the particle size of 2.0 mu L into a 0.6m L centrifuge tube, adding 25mM ammonium bicarbonate aqueous solution with the particle size of 18 mu L, uniformly mixing in a vortex mode, putting 2.0 mu L of 10-time dilution of the haptoglobin standard solution into a new 0.6m L centrifuge tube, adding SA matrix solution with the particle size of 18 mu L, uniformly mixing in a vortex mode, obtaining mixed solution 1, and carrying out sample application.
2. Reduction treatment
2.0 mu L haptoglobin standard solution is placed in a 0.6m L centrifuge tube, 18 mu L of DTT reducing reagent is added into the centrifuge tube to be uniformly mixed in a vortex mode, the centrifuge tube is placed in a 95 ℃ metal bath or boiling water bath to be reduced for 5min and then taken out, the centrifuge tube is cooled to room temperature, reduced haptoglobin standard solution is obtained, 2.0 mu L of reduced standard solution is placed in a new 0.6m L centrifuge tube, 18 mu L of SA matrix solution is added into the centrifuge tube to be uniformly mixed in a vortex mode, mixed solution 2 is obtained, and sample application is carried out.
Preparation of prefabricated target plate
The same as in example 1.
Tetra, MA L DI-TOF detection
The same as in example 1.
Fifthly, confirmation of light chain in haptoglobin standard product
The standard substance is a mixture of different haptoglobin serums, wherein the haptoglobin serums comprise three phenotypes of Hp1-1, Hp2-2 and Hp 2-1. by comparing mass spectrograms before and after reduction (as shown in figure 4), a peak of 80000-150000 m/z before reduction is a different polymer form formed by αβ in different haptoglobin types, two peaks of 9196m/z and 15947m/z appear after reduction, and the two peaks correspond to two light chains forming the haptoglobin, namely α1And α2。
The feasibility of the method of the invention for identifying haptoglobin phenotypes was further verified by the above-described assay.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (10)
- Use of a MA L DI-TOF mass spectrometer for identifying a haptoglobin phenotype.
- 2. A kit for identifying a haptoglobin phenotype, the kit comprising a MA L DI-TOF mass spectrometer, a reagent for MA L DI-TOF mass spectrometry detection, and a readable carrier;the readable carrier is described as follows:if the reduced sample to be detected has peaks of mass-to-charge ratios of 9180-9200 and no peaks of mass-to-charge ratios of 15935-15955 in a protein spectrogram obtained by detection of an MA L DI-TOF mass spectrometer, identifying the haptoglobin phenotype of the sample to be detected as Hp1-1 type, if the reduced sample to be detected has peaks of mass-to-charge ratios of 15935-15955 and no peaks of mass-to-charge ratios of 9180-9200 in the protein spectrogram obtained by detection of the MA L DI-TOF mass spectrometer, identifying the haptoglobin phenotype of the sample to be detected as Hp2-2 type, and if the reduced sample to be detected has peaks of mass-to-charge ratios of 9180-9200 and peaks of mass-to-charge ratios of 15935-15955 in the protein spectrogram obtained by detection of the MA L DI-TOF mass spectrometer, identifying the haptoglobin phenotype of the sample to be detected as Hp2-1 type.
- 3. The kit according to claim 2, wherein the MA L DI-TOF reagent for mass spectrometric detection comprises sinapic acid matrix.
- 4. The plant according to claim 2 or 3, characterized in that: the reduced sample to be detected is obtained by reducing the sample to be detected by using a dithiothreitol reduction reagent.
- 5. The method for identifying the haptoglobin phenotype is characterized by comprising the following steps of detecting a reduced sample to be detected by using an MA L DI-TOF mass spectrometer to obtain a protein spectrogram of the reduced sample to be detected, and identifying the haptoglobin phenotype according to mass-to-charge ratios appearing in the protein spectrogram, wherein if a peak with a mass-to-charge ratio of 9180-9200 does not appear in the protein spectrogram, the haptoglobin phenotype of the sample to be detected is identified as an Hp1-1 type, if a peak with a mass-to-charge ratio of 15935-15955 does not appear in the protein spectrogram, a peak with a mass-to-charge ratio of 9180-9100 does not appear in the protein spectrogram, the haptoglobin phenotype of the sample to be detected is identified as an Hp2-1 type, and if a peak with a mass-to-charge ratio of 9180-9200 and a peak with a mass-to-charge ratio of 15935-15955 simultaneously appear in the protein spectrogram, the haptoglobin phenotype of the sample to be detected is identified as the Hp2-1 type.
- 6. The method according to claim 5, wherein the MA L DI-TOF Mass spectrometer is set according to the following parameters, that is, the laser frequency is 1000Hz, the laser energy is 5.2 muJ, the detector voltage is-0.75 kV, the Focus Mass is 10000Da, and the collection Mass-to-charge ratio range is 5000Da-20,000 Da.
- 7. The method according to claim 5 or 6, characterized in that: the reduced sample to be detected is obtained by reducing the sample to be detected by using a dithiothreitol reduction reagent; preferably, the reduction is at 95 ℃ for 5 min.
- 8. The method of claim 7, wherein: the volume ratio of the sample to be detected to the dithiothreitol reduction reagent is 1: 5-1: 19, the content of dithiothreitol in the dithiothreitol reduction reagent is 80-120 mM, and the content of ammonium bicarbonate is 25 mM.
- 9. The method according to any one of claims 5 to 8, wherein the method for detecting the reduced sample to be tested with a MA L DI-TOF mass spectrometer comprises the steps of:mixing the reduced sample to be detected with the erucic acid matrix to obtain a mixed solution;placing the erucic acid matrix points in each hole of a target plate of an MA L DI-TOF mass spectrometer, and drying and crystallizing the erucic acid matrix to obtain a prefabricated target plate;the mixture was spotted onto each well of the pre-fabricated target plate, dried and then placed on a MA L DI-TOF mass spectrometer for analysis.
- 10. The method according to claim 9, wherein the volume ratio of the reduced sample to be tested to sinapic acid matrix is 1: 5-1: 19, the sinapic acid content in the sinapic acid matrix is 10-20mg/m L, the acetonitrile content is 30-40% by volume, and the ethanol content is 10-20% by volume.
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