CN107045064A - A kind of pre-treating method of polypeptide based on derivatization in plasma sample - Google Patents
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- 238000001212 derivatisation Methods 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 42
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 39
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 32
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 6
- 230000008014 freezing Effects 0.000 claims abstract 2
- 238000007710 freezing Methods 0.000 claims abstract 2
- 238000010200 validation analysis Methods 0.000 claims abstract 2
- 210000002381 plasma Anatomy 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 2
- 238000004451 qualitative analysis Methods 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 claims 1
- 150000001768 cations Chemical class 0.000 claims 1
- 125000005439 maleimidyl group Chemical class C1(C=CC(N1*)=O)=O 0.000 claims 1
- 238000002552 multiple reaction monitoring Methods 0.000 claims 1
- 230000035484 reaction time Effects 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 17
- 238000004458 analytical method Methods 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 7
- 238000004949 mass spectrometry Methods 0.000 abstract description 5
- 238000002203 pretreatment Methods 0.000 abstract description 4
- 150000003923 2,5-pyrrolediones Chemical class 0.000 abstract description 3
- 239000002244 precipitate Substances 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 abstract 2
- OHAOZJBBOUNZKA-UHFFFAOYSA-N 3-[4-(dimethylamino)phenyl]pyrrole-2,5-dione Chemical compound CN(C1=CC=C(C=C1)C1=CC(=O)NC1=O)C OHAOZJBBOUNZKA-UHFFFAOYSA-N 0.000 abstract 1
- IYMZEPRSPLASMS-UHFFFAOYSA-N 3-phenylpyrrole-2,5-dione Chemical class O=C1NC(=O)C(C=2C=CC=CC=2)=C1 IYMZEPRSPLASMS-UHFFFAOYSA-N 0.000 abstract 1
- 102000004506 Blood Proteins Human genes 0.000 abstract 1
- 108010017384 Blood Proteins Proteins 0.000 abstract 1
- 125000001841 imino group Chemical group [H]N=* 0.000 abstract 1
- 238000005457 optimization Methods 0.000 abstract 1
- 229940057874 phenyl trimethicone Drugs 0.000 abstract 1
- -1 phenyl trimethicone ammonium iodides Chemical class 0.000 abstract 1
- 239000013595 supernatant sample Substances 0.000 abstract 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 7
- 125000003396 thiol group Chemical group [H]S* 0.000 description 6
- 230000006920 protein precipitation Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 238000006845 Michael addition reaction Methods 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- BFXYMHXQRLNFAF-UHFFFAOYSA-M 4-(N-Maleimido)phenyltrimethylammonium iodide Chemical compound [I-].C1=CC([N+](C)(C)C)=CC=C1N1C(=O)C=CC1=O BFXYMHXQRLNFAF-UHFFFAOYSA-M 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007810 chemical reaction solvent Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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Abstract
Description
技术领域：Technical field:
本发明涉及血浆样品衍生化技术，具体涉及多肽样品在血浆中衍生化的前处理技术。The invention relates to plasma sample derivatization technology, in particular to the pretreatment technology of polypeptide sample derivatization in plasma.
背景技术：Background technique:
在生物样本分析中，前处理技术是建立可靠，灵敏度高的分析方法的基础。常见的衍生化过程，均需除去生物样本中的蛋白后进行测定，衍生化过程和条件根据不同的反应多有不同。具体过程为，生物样品利用蛋白沉淀法(包括有机溶剂蛋白沉淀法，酸沉淀法，热变性法等)或固相萃取法进行处理后，挥干样品后，用反应溶剂进行复溶，一定条件下进行衍生化反应。这种方法对于蛋白结合率高和吸附性强的样品分析具有一定限制。且对于反应条件苛刻的衍生化，过程复杂，耗时长，成本高，使用溶剂对环境污染大。并且多肽的蛋白结合率高及非特异性吸附比较严重，使得其血浆样品前处理面临更加巨大挑战。In the analysis of biological samples, pretreatment technology is the basis for establishing reliable and sensitive analytical methods. The common derivatization process needs to remove the protein in the biological sample before measurement, and the derivatization process and conditions vary according to different reactions. The specific process is that biological samples are processed by protein precipitation method (including organic solvent protein precipitation method, acid precipitation method, thermal denaturation method, etc.) or solid phase extraction method, and after the sample is evaporated to dryness, it is redissolved with a reaction solvent under certain conditions. Under the derivatization reaction. This method has certain limitations for the analysis of samples with high protein binding rate and strong adsorption. Moreover, for derivatization with harsh reaction conditions, the process is complicated, time-consuming, high in cost, and the use of solvents causes great environmental pollution. Moreover, the high protein binding rate and serious non-specific adsorption of peptides make the pretreatment of plasma samples face even greater challenges.
质谱由于其适用范围广，专属性高，灵敏度好等优点，广泛应用于多肽定量分析。但是在分析过程中对于一些离子化效率低的样品，分析方法的灵敏度会受到很大影响。因此需要对样品进行衍生化，提高离子化效率，从而提高多肽测定的灵敏度。Due to its wide application range, high specificity, and good sensitivity, mass spectrometry is widely used in the quantitative analysis of peptides. However, for some samples with low ionization efficiency during the analysis, the sensitivity of the analytical method will be greatly affected. Therefore, it is necessary to derivatize the sample to improve the ionization efficiency, thereby improving the sensitivity of peptide determination.
本发明利用马来酰亚胺衍生物作为衍生化试剂，利用巯基和衍生化试剂的迈克尔加成反应的原理进行衍生化。由于迈克尔加成反应是十分容易进行的反应，对反应条件并无特殊要求。因此建立了一个简单快捷的一步法前处理技术，蛋白沉淀及衍生化一步进行，不仅提高了多肽的质谱响应，建立了灵敏度更高的质谱方法同时提高了多肽在血浆中的提取回收率。The present invention uses maleimide derivatives as derivatization reagents, and utilizes the principle of Michael addition reaction between sulfhydryl and derivatization reagents for derivatization. Since the Michael addition reaction is a very easy reaction, there is no special requirement for the reaction conditions. Therefore, a simple and fast one-step pretreatment technology was established, and protein precipitation and derivatization were carried out in one step, which not only improved the mass spectrometry response of the peptide, established a mass spectrometry method with higher sensitivity, but also improved the extraction recovery rate of the peptide in plasma.
发明内容：Invention content:
本发明提供了一种测定血浆中含巯基多肽样品的衍生化前处理方法。有效的提高了样品测定的灵敏度及回收率。The invention provides a derivatization pretreatment method for measuring thiol-containing polypeptide samples in blood plasma. Effectively improve the sensitivity and recovery rate of sample determination.
一种基于衍生化的多肽在血浆样品中的前处理方法是通过以下方案进行实施的，流程见图1：A pretreatment method based on derivatized polypeptides in plasma samples is implemented through the following scheme, and the flow chart is shown in Figure 1:
称取一定量的衍生化试剂，将其直接溶于乙腈中，配成1.5mg/mL的衍生化试剂。取空白小鼠血浆50μL，加入一定量的含有巯基的待测多肽样品，涡旋混合。加入100μL的衍生化试剂，在40℃条件下涡旋震荡40min。由于乙腈能够对大分子量的蛋白质进行沉淀变性，同时对上清中的多肽样品进行衍生反应。反应结束后对血浆样品进行离心，利用低温冷冻离心机4℃，12000rmp，离心5min.取上清，利用优化好的液质方法对样品进行定性定量的分析。Weigh a certain amount of derivatization reagent, and directly dissolve it in acetonitrile to prepare a 1.5 mg/mL derivatization reagent. Take 50 μL of blank mouse plasma, add a certain amount of polypeptide sample containing thiol to be tested, and vortex to mix. Add 100 μL of derivatization reagent, and vortex shake at 40°C for 40 min. Since acetonitrile can precipitate and denature proteins with large molecular weight, it can also derivatize the peptide samples in the supernatant. After the reaction, the plasma sample was centrifuged and centrifuged at 4°C and 12000rmp for 5min in a low-temperature refrigerated centrifuge. The supernatant was taken, and the qualitative and quantitative analysis of the sample was carried out using the optimized liquid mass method.
本发明有益效果如下：The beneficial effects of the present invention are as follows:
1.本发明中，操作步骤简单，将衍生化反应和蛋白沉淀法同时进行，避免了传统衍生化法和前处理中操作过程繁琐等问题。1. In the present invention, the operation steps are simple, and the derivatization reaction and the protein precipitation method are carried out at the same time, which avoids the problems such as the complicated operation process in the traditional derivatization method and pretreatment.
2.本发明中，反应简单易行，条件温和。和常见衍生化方法相比，条件更加绿色环保，成本低。2. In the present invention, the reaction is simple and easy, and the conditions are mild. Compared with common derivatization methods, the conditions are greener and the cost is lower.
3.多肽衍生化试剂多用于蛋白质组学的研究，能增加多肽丰度提高检测的多肽数量。本发明将衍生化试剂用于多肽体内分析的定量研究。3. Peptide derivatization reagents are mostly used in proteomics research, which can increase the abundance of peptides and increase the number of detected peptides. In the present invention, the derivatization reagent is used in the quantitative research of polypeptide analysis in vivo.
4.本发明中，将季铵基团引入到多肽中有效提高多肽离子化效率，提高样品测定的灵敏度。4. In the present invention, the quaternary ammonium group is introduced into the polypeptide to effectively improve the ionization efficiency of the polypeptide and improve the sensitivity of sample determination.
5.本发明中，多肽样品在衍生化后对样品的回收率有显著提高。多肽引入衍生化基团后，对原有多肽结构发生部分改变能够有效降低蛋白结合率。5. In the present invention, the recovery rate of the polypeptide sample is significantly improved after derivatization. After the peptide is introduced with a derivatization group, partial changes to the original peptide structure can effectively reduce the protein binding rate.
6.本发明中的方法能够对含有巯基的多肽样品进行测定，具有应用到含有巯基的多肽药物的体内分析的潜能。6. The method of the present invention can measure polypeptide samples containing sulfhydryl groups, and has the potential to be applied to the in vivo analysis of polypeptide drugs containing sulfhydryl groups.
7.本发明中，利用迈克尔加成的原理将马来酰亚胺衍生物和含有巯基的多肽进行反应。该反应原理适用于其他的衍生化试剂，本方法可以根据实际样本进行衍生化反应。7. In the present invention, the principle of Michael addition is used to react the maleimide derivative and the polypeptide containing thiol. This reaction principle is applicable to other derivatization reagents, and this method can be used for derivatization reactions based on actual samples.
附图说明：Description of drawings:
图1为样品前处理过程示意图Figure 1 is a schematic diagram of the sample pretreatment process
图2为HBB与衍生化产物(MPTA-HBB)在相同浓度下的质谱响应图。Figure 2 is the mass spectrum response diagram of HBB and derivatized product (MPTA-HBB) at the same concentration.
具体实施方式：detailed description:
为了更好的理解本发明，下面通过实施例对本发明进一步说明，实施例只用于解释本发明，不会对本发明定构成任何的限定。In order to better understand the present invention, the present invention will be further described through the following examples, which are only used to explain the present invention and will not limit the present invention.
实施例1：利用4-(N-马来酰亚胺基)苯基三甲基碘化铵对多肽HBB小鼠血浆样品进行衍生化的方法建立。Example 1: Establishment of a method for derivatizing polypeptide HBB mouse plasma samples using 4-(N-maleimido)phenyltrimethylammonium iodide.
(1)HBB为血红蛋白β链中的一部分，为15个氨基酸。对其体内药代动进行研究。(1) HBB is a part of hemoglobin β chain, which is 15 amino acids. Study on its pharmacokinetics in vivo.
(2)利用本发明中的衍生化方法，建立测定方法并进行方法学验证。具体过程为：配制1.5mg/mL的衍生化试剂。配制系列浓度的HBB样品，浓度分别为1，3，10，20，50，100和120ng/mL的模拟血浆样品进行分析，选择内标的浓度为50ng/mL。按照上述过程，将衍生化试剂两倍体积加入小鼠血浆中，40℃，涡旋反应40min，利用低温冷冻离心机对样品进行离心，条件为12000rpm，5min。建立测定方法。(2) Utilize the derivatization method in the present invention to establish a measurement method and carry out methodological verification. The specific process is: prepare 1.5mg/mL derivatization reagent. Prepare a series of HBB samples with concentrations of 1, 3, 10, 20, 50, 100 and 120ng/mL simulated plasma samples for analysis, and select the concentration of internal standard as 50ng/mL. According to the above process, add twice the volume of the derivatization reagent to mouse plasma, vortex at 40°C for 40 minutes, and centrifuge the sample in a low-temperature refrigerated centrifuge at 12,000 rpm for 5 minutes. Create a measurement method.
(3)该分析方法与常规蛋白沉淀法比较回收率和响应均提高了很多。其中4-(N-马来酰亚胺基)苯基三甲基碘化铵的测定结果是最好的，HBB的回收率从原来的30％提高到70％左右。同时响应提高了174倍，见图2。显著改善了HBB的质谱响应及回收率。(3) Compared with the conventional protein precipitation method, the recovery rate and response of this analysis method are much improved. Among them, the determination result of 4-(N-maleimido)phenyltrimethylammonium iodide is the best, and the recovery rate of HBB is increased from the original 30% to about 70%. At the same time, the response has been improved by 174 times, as shown in Figure 2. Significantly improved the mass spectrometry response and recovery of HBB.
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| CN115201385A (en) * | 2022-06-22 | 2022-10-18 | 河北医科大学 | A kind of derivatization reagent for electrospray mass spectrometry detection which makes amino small molecule carry two charges and its preparation method and application |
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| CN115201385A (en) * | 2022-06-22 | 2022-10-18 | 河北医科大学 | A kind of derivatization reagent for electrospray mass spectrometry detection which makes amino small molecule carry two charges and its preparation method and application |
| CN115201385B (en) * | 2022-06-22 | 2024-01-09 | 河北医科大学 | Derivatization reagent for electrospray mass spectrometry detection for enabling amino small molecules to carry two charges, and preparation method and application thereof |
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