WO2018090651A1 - 一种离体体液中蛋白质的预处理方法 - Google Patents

一种离体体液中蛋白质的预处理方法 Download PDF

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WO2018090651A1
WO2018090651A1 PCT/CN2017/092553 CN2017092553W WO2018090651A1 WO 2018090651 A1 WO2018090651 A1 WO 2018090651A1 CN 2017092553 W CN2017092553 W CN 2017092553W WO 2018090651 A1 WO2018090651 A1 WO 2018090651A1
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protein
body fluid
surfactant
solid phase
reducing agent
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张丽华
袁辉明
随志刚
杨开广
张玉奎
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中国科学院大连化学物理研究所
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    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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    • G01N1/40—Concentrating samples
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2001/4061—Solvent extraction
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    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
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    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

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  • the invention relates to a rapid pretreatment method for exogenous protein secretion, which is a method for collective liquid separation, protein extraction, purification and enzymatic hydrolysis.
  • Body fluids usually carry and transmit important signaling molecules.
  • proteins in urine are mainly derived from glomeruli, renal tubules, prostate, and bladder cells. Therefore, efficient extraction of ultra-low levels of protein in body fluids and analysis of their proteome can directly reflect human tissue.
  • the existing body fluid protein processing methods usually require cumbersome operations, and multi-step sample transfer is not only time-consuming and laborious, but also has a serious loss, and it is difficult to achieve efficient treatment of low-abundance proteins. These problems severely affect the accuracy, sensitivity, and throughput of humoral proteome analysis.
  • the object of the present invention is to provide a method for extracting, purifying and enzymatically hydrolyzing collective liquid, and the whole process can be completed in the same centrifuge tube, thereby avoiding the loss and pollution caused by the transfer of the sample during the treatment.
  • the method can directly process body fluid proteins in cell culture fluids and body fluids without complicated and cumbersome operations, while maintaining a high degree of continuity and high throughput throughout the process.
  • Adding a polymer solution to extract protein in a body fluid under low temperature conditions wherein the polymer is one or two of polyethylene glycol, polyetherimide and polyvinyl alcohol having a polymerization degree of 500-10000.
  • the mass concentration of the polymer solution is 1%-50%; the volume ratio of the polymer solution to the ex vivo blood and/or urine is 1:1-10; the separation temperature range is -20-10 °C ;
  • the surfactant solution and the protein reducing agent are added to denature the body fluid protein under high temperature conditions; wherein the surfactant type can be an anionic surfactant (specifically, sodium lauryl polyoxyethylene ether sulfate, dodecane Alkylbenzenesulfonic acid, sodium lauryl sulfate, sodium fatty acid isethionate, one or more of ammonium lauryl sulfate, cationic surfactant (specifically octadecyltrimethyl) Ammonium chloride, cetyltrimethylammonium chloride, one or more of distearylhydroxyethylmethylammonium methyl sulfate, zwitterionic surfactant (specifically dodecane a betaine, a cocoyl imidazoline fatty alcohol, a polyoxyethylene ether, one or more of the sulfosuccinate disodium salt) and a nonionic surfactant (specifically coconut fatty acid diethanolamide,
  • the microspheres or the surface are covalently bonded to the iodoacetic acid-N-succinamide ester silicon sphere, and the mass ratio of the protein to the solid phase alkylating agent is 1/1 to 1-5; the polymer microsphere matrix material is a polyacrylate.
  • the protein enzyme used may be one or two or three of trypsin, lysine protease and protease V8; body fluid protein and The enzyme addition mass ratio is 1/1-1/5, the enzymatic hydrolysis temperature is 25-37 ° C, and the enzymatic hydrolysis time is 0.5-4 hours;
  • the obtained enzymatic hydrolyzed product is directly subjected to liquid chromatography-mass spectrometry;
  • the rapid pretreatment method of body fluid protein developed is used for rapid pretreatment of exogenous protein in clinical diagnosis, protein proteomics research and tumor marker screening.
  • the high molecular weight polymer is used to extract body fluid protein at low temperature, which improves the extraction efficiency of medium and low abundance body fluid protein;
  • the solid phase alkylation reagent is used to covalently bind body fluid protein, which simplifies the steps of purifying body fluid protein, and improves the recovery rate and pretreatment flux of body fluid protein;
  • Protein enzymatic products can be directly analyzed by liquid chromatography-mass spectrometry to provide technical support for high-throughput clinical humoral proteome analysis.
  • Figure 1 Schematic diagram of pretreatment of body fluid protein samples.
  • FIG. 1 Schematic diagram of body fluid protein pretreatment method is shown in Figure 1.
  • a final concentration of 10% (w/v) polyethylene glycol solution was added to 5 mL human urine, and allowed to stand at 0 ° C for 1 hour, centrifuged at 16000 rpm for 30 min to obtain urine.
  • the body fluid was added to a final concentration of 4% (w/v) anionic surfactant SDS and 50 mM tris(2-carboxyethyl)phosphine in a 95 ° C water bath for 30 min, and the surface covalently bonded iodoacetic acid-N-succinamide was added.
  • the ester silicon ball was shaken for 1.5 hours, the supernatant was removed by centrifugation, and the surface of the silicon sphere was washed by adding 50% methanol and 50 mM ammonium hydrogencarbonate solution respectively. Finally, the body fluid protein to enzyme mass ratio was 1:1 and trypsin was incubated at 37 ° C for 30 min. After centrifuging, the supernatant was subjected to liquid chromatography-mass spectrometry, and the results of the analysis are shown in Fig. 2.
  • a 1% human urine was added with a final concentration of 10% (w/v) polyethylene glycol and a final concentration of 20% (w/v) polyetherimide mixed solution, allowed to stand at 4 ° C for 0.5 hours, centrifuged at 16000 rpm for 30 min.
  • the body fluid in the urine was obtained, and a final concentration of 4% (w/v) cationic surfactant CTAB and 50 mM tris(2-carboxyethyl)phosphine in a 95 ° C water bath were added for 30 min, and the surface covalently bonded iodoacetic acid-N was added.
  • the succinamide ester polymer microspheres were shaken for 1.5 hours, the supernatant was removed by centrifugation, and the surface of the silicon sphere was washed by adding 50% methanol and 50 mM ammonium hydrogencarbonate solution respectively, and finally the pancreas was added in a mass ratio of body fluid to enzyme of 1:2.
  • the protease and lysine protease were incubated at 37 ° C for 1 min, and the supernatant was centrifuged for liquid chromatography-mass spectrometry. The results of the analysis are shown in FIG.
  • a final concentration of 50% (w/v) polyethylene glycol and a final concentration of 20% (w/v) polyetherimide mixed solution were added to 1 mL of human blood, and allowed to stand at -20 ° C for 0.5 hour and centrifuge at 16000 rpm for 30 minutes.
  • a 50% (w/v) polyethylene glycol and a final concentration of 20% (w/v) polyetherimide mixed solution were added to 50 mL of human urine, and allowed to stand at 0 ° C for 0.5 hour and centrifuge at 16000 rpm for 30 minutes.
  • a 50% final concentration of 10% (w/v) polyethylene glycol and a final concentration of 10% (w/v) polyvinyl alcohol and 20% polyetherimide mixed solution were added to the urine at -5 °C. After hourly, centrifugation at 16000 rpm for 30 min to obtain body fluid, adding a final concentration of 10% (w/v) SDS and 50 mM dithiothreitol in a water bath at 80 ° C for 30 min, adding a silicon sphere covalently bonded with iodoacetic acid-N-succinamide ester.
  • the reaction was shaken for 1.5 hours, the supernatant was removed by centrifugation, and the surface of the silicon sphere was washed by adding 50% methanol and 0.1% formic acid respectively. Finally, the protease V8 was added at a mass ratio of body fluid to enzyme of 1:5, and incubated at 37 ° C for 30 min, and the supernatant was centrifuged. After that, liquid chromatography-mass spectrometry was carried out, and good analytical results were obtained.

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Abstract

一种离体体液中蛋白质的预处理方法，通过一定比例的高分子聚合物溶液在低温条件下从离体体液(血液和尿液等)中提取出蛋白质，加入一定浓度的表面活性剂和蛋白质还原剂在高温条件下使蛋白质快速变性，加入可与蛋白质巯基快速反应的固相烷基化试剂，该试剂可选择性与蛋白质上的巯基反应，离心后，反复清洗固相颗粒表面去除其它干扰小分子(如糖、盐、表面活性剂、脂类等)，获得高纯度的蛋白质，最后加入蛋白酶将体液蛋白质酶解成肽段后，离心后获得的产物可直接采用液相色谱-质谱系统进行分析。与传统的体液蛋白质预处理方法相比，该方法具有操作简单、重复性好、预处理通量高等优点。

Description

一种离体体液中蛋白质的预处理方法 技术领域
本发明涉及一种外体泌蛋白质快速预处理方法，是一种集体液分离，蛋白质提取，纯化及酶解的方法。
背景技术
体液通常携带和传递着重要的信号分子。例如，研究显示，尿液中的蛋白质主要来源于肾小球、肾小管、前列腺以及膀胱细胞，因此，对体液中超低含量的蛋白质进行高效提取，并对其蛋白质组进行分析可以直接反映人体组织、器官的生理和病理状态。现有的体液蛋白质处理方法通常需要繁琐的操作，多步的样品转移，不仅费时费力，而且损失严重，难以实现低丰度蛋白质的高效处理。这些问题严重影响了体液蛋白质组分析的准确度、灵敏度和通量。
发明内容
为了解决上述问题，本发明的目的在于提供一种集体液蛋白质提取，纯化及酶解的方法，整个过程可在同一个离心管中完成，避免了样品在处理过程中转移造成的丢失和污染。该方法可以直接处理细胞培养液和体液中的体液蛋白质，不需要复杂和繁琐的操作，同时整个处理过程保持高度的连续性和高通量性。
为了实现该目的，本发明的技术方案是：
1、加入高分子聚合物溶液在低温条件下提取体液中的蛋白质，其中高分子聚合物为聚合度500-10000的聚乙二醇、聚醚酰亚胺和聚乙烯醇中的一种或两种以上，高分子聚合物溶液的质量浓度为1％-50％；高分子聚合物溶液与离体血液和/或尿液的体积比1：1-10；分离温度范围为-20-10℃；
2、加入表面活性剂溶液和蛋白质还原剂在高温条件下使体液蛋白质变性；其中表面活性剂的种类可为阴离子表面活性剂(具体为十二烷基醇聚氧乙烯醚硫酸钠,十二烷基苯磺酸,十二烷基硫酸钠,脂肪醇羟乙基磺酸钠,十二烷基硫酸铵中的一种或两种以上)、阳离子表面活性剂(具体为十八烷基三甲基氯化铵,十六烷基三甲基氯化铵,二硬脂基羟乙基甲基硫酸甲脂铵中的一种或两种以上)、两性离子表面活性剂(具体为十二烷基甜菜碱,椰油基咪唑啉脂肪醇,聚氧乙烯醚,磺基琥珀酸二钠盐中的一种或两种以上)和非离子表面活性剂(具体为椰油脂肪酸二乙醇酰胺,壬基酚聚氧乙烯醚羟基合成醇,聚氧乙烯醚,C12-14烷基糖苷中的一种或两种以上)中的一种或两种以上，浓度为4％-10％(质量g体积ml比)；表面活性剂溶液与步骤1)获得溶液的体积比1：1-10；蛋白质还原剂为二硫苏糖醇或三(2-羧乙基)膦中一种或两种，还原剂终浓度为10mM-100mM；蛋白质提取温度范围为80-95℃，蛋白质还原剂为二硫苏糖醇或三(2-羧乙基)膦中一种或两种，还原剂终浓度为10mM-100mM；蛋白质提取温度范围为80-95℃；
3、加入可与蛋白质巯基快速反应的固相烷基化试剂使体液蛋白质得到分离，获得体液蛋白质，其中固相烷基化试剂为表面共价键合碘乙酸-N-琥珀酰胺酯的聚合物微球或表面共价键合碘乙酸-N-琥珀酰胺酯硅球，蛋白质与固相烷基化试剂的质量比为1/1-1/5；聚合物微球基质材料为聚丙烯酸酯,聚苯乙烯中的一种或两种以上；
4、加入蛋白酶，并与固载体液蛋白质的微球在一定温度下孵育，采用的蛋白质酶可为胰蛋白酶、赖氨酸蛋白酶和蛋白酶V8中的一种或两种或三种；体液蛋白质与酶加入质量比为1/1-1/5，酶解温度为25-37℃，酶解时间为0.5-4小时；
5、得到的酶解产物直接进行液相色谱-质谱分析；
6、所发展的体液蛋白质快速预处理方法用于临床诊断、蛋白质质组学研究和肿瘤标志物筛选中的外体泌蛋白质快速预处理方法。
本发明具有如下优点：
1、采用高分子聚合物在低温下提取体液蛋白质，提高了中低丰度体液蛋白质的提取效率；
2、采用固相烷基化试剂共价结合体液蛋白，简化了体液蛋白质纯化的步骤，提高体液蛋白质的回收率和预处理通量；
3、蛋白质酶解产物可直接进行液相色谱-质谱分析，为实现高通量的临床体液蛋白质组分析提供技术支撑。
附图说明
图1、体液蛋白质样品预处理示意图。
图2、尿液蛋白质样品预处理的色谱-质谱分析图。
图3、血液蛋白质样品预处理的色谱-质谱分析图。
具体实施方式
实施例1
体液蛋白质预处理方法示意图如图1所示，在5mL人尿液中加入终浓度10％(w/v)的聚乙二醇溶液,在0℃下静置1小时，16000rpm离心30min获得尿液中的体液，加入终浓度4％(w/v)阴离子表面活性剂SDS和50mM三(2-羧乙基)膦95℃水浴锅反应30min,加入表面共价键合碘乙酸-N-琥珀酰胺酯的硅球振荡反应1.5小时，离心弃上清，分别加入50％甲醇和50mM碳酸氢铵溶液清洗硅球表面，最后按体液蛋白质与酶的质量比为1:1加入胰蛋白酶37℃孵育30min，离心取上清后，进行液相色谱-质谱分析，分析结果如图2所示。
实施例2
在1mL人尿液中加入终浓度10％(w/v)的聚乙二醇和终浓度20％(w/v)聚醚酰亚胺混合溶液,在4℃下静置0.5小时，16000rpm离心30min获得尿液中的体液，加入终浓度4％(w/v)阳离子表面活性剂CTAB和50mM三(2-羧乙基)膦95℃水浴锅反应30min,加入表面共价键合碘乙酸-N-琥珀酰胺酯的聚合物微球振荡反应1.5小时，离心弃上清，分别加入50％甲醇和50mM碳酸氢铵溶液清洗硅球表面，最后按体液蛋白质与酶的质量比为1:2加入胰蛋白酶和赖氨酸蛋白酶37℃孵育1min，离心取上清后，进行液相色谱-质谱分析，分析结果如图3所示。
实施例3
在1mL人血液中加入终浓度50％(w/v)的聚乙二醇和终浓度20％(w/v)聚醚酰亚胺混合溶液,在-20℃下静置0.5小时，16000rpm离心30min获得尿液中的体液，加入终浓度10％(w/v)非离子表面活性剂脂肪醇聚氧乙烯醚和100mM三(2-羧乙基)膦95℃水浴锅反应30min,加入表面共价键合碘乙酸-N-琥珀酰胺酯的聚合物微球振荡反应1.5小时，离心弃上清，分别加入50％甲醇和50mM碳酸氢铵溶液清洗硅球表面，最后按体液蛋白质与酶的质量比为1:3加入胰蛋白酶和赖氨酸蛋白酶37℃孵育1min，离心取上清后，进行液相色谱-质谱分析，获得较好分析结果。
实施例4
在50mL人尿液中加入终浓度50％(w/v)的聚乙二醇和终浓度20％(w/v)聚醚酰亚胺混合溶液,在0℃下静置0.5小时，16000rpm离心30min获得尿液中的体液，加入终浓度10％(w/v)非离子表面活性剂脂肪醇聚氧乙烯醚和100mM三(2-羧乙基)膦95℃水浴锅反应30min,加入表面共价键合碘乙酸-N-琥珀酰胺酯的聚合物微球振荡反应1.5小时，离心弃上清，分别加入50％甲醇和50mM碳酸氢铵溶液清洗硅球表面，最后按体液蛋白质与酶的质量比为1:4加入胰蛋白酶和赖氨酸蛋白酶37℃孵育1min，离心取上清后，进行液相色谱-质谱分析，获得较好分析结果。
实施例5
在50mL尿液中加入终浓度10％(w/v)的聚乙二醇和终浓度10％(w/v)聚乙烯醇和20％聚醚酰亚胺混合溶液,在-5℃下静置1小时，16000rpm离心30min获得体液，加入终浓度10％(w/v)SDS和50mM二硫苏糖醇80℃水浴锅反应30min,加入表面共价键合碘乙酸-N-琥珀酰胺酯的硅球振荡反应1.5小时，离心弃上清，分别加入50％甲醇和0.1％甲酸清洗硅球表面，最后按体液蛋白质与酶的质量比为1:5加入蛋白酶V8，37℃孵育30min，离心取上清后，进行液相色谱-质谱分析，获得了较好分析结果。

Claims (5)

  1. 一种离体体液中蛋白质的预处理方法，其特征在于：
    1)在离体血液和/或尿液中加入高分子聚合物溶液在低温条件下提取体液蛋白质；
    2)加入表面活性剂和蛋白质还原剂在高温条件下使蛋白质快速变性；
    3)再加入可与蛋白质巯基反应的固相烷基化试剂使体液蛋白质得到分离，获得体液蛋白质，最后加入蛋白酶将体液蛋白质酶解成肽段。
  2. 按照权利要求1所述的预处理方法，其特征在于：加入高分子聚合物溶液在低温条件下提取体液中的蛋白质，其中高分子聚合物为聚合度500-10000的聚乙二醇、聚醚酰亚胺和聚乙烯醇中的一种或两种以上，高分子聚合物溶液的质量浓度为1％-50％；高分子聚合物溶液与离体血液和/或尿液的体积比1：1-10；分离温度范围为-20-10℃。
  3. 按照权利要求1所述的预处理方法，其特征在于：加入表面活性剂溶液和蛋白质还原剂在高温条件下使体液蛋白质变性；其中表面活性剂的种类可为阴离子表面活性剂(具体为十二烷基醇聚氧乙烯醚硫酸钠,十二烷基苯磺酸,十二烷基硫酸钠,脂肪醇羟乙基磺酸钠,十二烷基硫酸铵中的一种或两种以上)、阳离子表面活性剂(具体为十八烷基三甲基氯化铵,十六烷基三甲基氯化铵,CTAB，二硬脂基羟乙基甲基硫酸甲脂铵中的一种或两种以上)、两性离子表面活性剂(具体为十二烷基甜菜碱,椰油基咪唑啉脂肪醇,聚氧乙烯醚,磺基琥珀酸二钠盐中的一种或两种以上)和非离子表面活性剂(具体为椰油脂肪酸二乙醇酰胺,壬基酚聚氧乙烯醚羟基合成醇,聚氧乙烯醚,C12-14烷基糖苷中的一种或两种以上)中的一种或两种以上，浓度为4％-10％(质量g体积ml比)；表面活性剂溶液与步骤1)获得溶液的体积比1：1-10；蛋白质还原剂为二硫苏糖醇或三(2-羧乙基)膦中一种或两种，还原剂终浓度为10mM-100mM；蛋白质提取温度范围为80-95℃。
  4. 按照权利要求1所述的预处理方法，其特征在于：加入可与蛋白质巯基快速反应的固相烷基化试剂使体液蛋白质得到分离，获得体液蛋白质，其中固相烷基化试剂为表面共价键合碘乙酸-N-琥珀酰胺酯的聚合物微球或表面共价键合碘乙酸-N-琥珀酰胺酯硅球，蛋白质与固相烷基化试剂的质量比为1/1-1/5；聚合物微球基质材料为聚丙烯酸酯,聚苯乙烯中的一种或两种以上。
  5. 按照权利要求1所述的预处理方法，其特征在于：所采用的蛋白质酶可为胰蛋白酶、赖氨酸蛋白酶和蛋白酶V8中的一种或两种或三种；体液蛋白质与酶加入质量比为1/1-1/5，酶解温度为25-37℃，酶解时间为0.5-4小时。
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