WO2018090652A1 - 一种高通量体液蛋白质样品预处理装置及其应用 - Google Patents

一种高通量体液蛋白质样品预处理装置及其应用 Download PDF

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WO2018090652A1
WO2018090652A1 PCT/CN2017/092561 CN2017092561W WO2018090652A1 WO 2018090652 A1 WO2018090652 A1 WO 2018090652A1 CN 2017092561 W CN2017092561 W CN 2017092561W WO 2018090652 A1 WO2018090652 A1 WO 2018090652A1
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protein
denaturation
body fluid
hollow fiber
abundance
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French (fr)
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张丽华
袁辉明
戴忠鹏
杨开广
张玉奎
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/02—Hollow fibre modules
    • B01D63/04—Hollow fibre modules comprising multiple hollow fibre assemblies
    • B01D63/046—Hollow fibre modules comprising multiple hollow fibre assemblies in separate housings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins

Definitions

  • the invention relates to a high-flux body fluid protein sample pretreatment device, which is a sample pretreatment system for high-abundance protein removal of collective liquid, on-line denaturation and reduction, desalting and protein enzymatic hydrolysis of medium and low abundance proteins.
  • proteins in body fluids can provide a large amount of information closely related to physiology and pathology
  • humoral proteomics research has become a disease pathogenesis, early diagnosis, classification and individualized treatment. An important means.
  • body fluid samples available in the clinic for basic research are very limited.
  • the treatment of body fluid proteome samples usually adopts an off-line multi-step method to achieve protein denaturation, reduction, alkylation, enzymatic hydrolysis and desalting. Not only is it time-consuming and laborious, but it also causes loss and contamination of protein samples, which in turn affects the accuracy, sensitivity and throughput of quantitative analysis. Therefore, there is an urgent need to develop a highly efficient new method for pretreatment of body fluid proteome samples.
  • sample pretreatment system for high-abundance protein removal of collective liquid, on-line denaturation, reduction, desalting and on-line enzymatic digestion of medium and low abundance proteins.
  • the system can achieve high-throughput, low-loss pretreatment of low-abundance protein samples in body fluids, and has a good application prospect in proteomics research.
  • an object of the present invention is to provide a sample pretreatment system that integrates protein denaturation, reduction, desalting, and in-line enzymatic hydrolysis.
  • the system handles body fluid proteins directly, without the need for complex and cumbersome manual handling, while maintaining a high degree of continuity and high throughput throughout the process.
  • Two or more hollow fiber membranes are used as a transport carrier for low-abundance proteins in body fluids, and are fixed in denaturation and reduction chambers, and high-density denaturing agents and reducing agents are transported to denaturation by liquid chromatography pump or peristaltic pump.
  • the type of the denaturant may be guanidine hydrochloride or urea
  • the concentration is 4-8M
  • the type of the protein reducing agent may be Dithiothreitol, thiol, tris(2-carboxyethyl)phosphine, concentration 5-100 mM, flow rate range of liquid chromatography pump or peristaltic pump 0.1mL/min-5mL/min, heating temperature of temperature control device Range 60-95 ° C;
  • Two or more hollow fiber membranes are used as low-abundance protein transport carriers in body fluids, and fixed in a buffer replacement chamber, and a low-concentration weak alkaline buffer solution is delivered to the buffer through a liquid chromatography pump or a peristaltic pump.
  • the cavity replaces the protein solvent with the exchange liquid to achieve the purpose of protein desalination.
  • the low concentration alkaline buffer solution may be ammonium hydrogencarbonate or ammonium acetate solution, the concentration range is 10-100 mM, and the pH range is 7.5-8.5.
  • the flow rate of the liquid chromatography pump or peristaltic pump is 0.1mL/min-5mL/min;
  • the matrix material of the enzyme reactor is a silica gel particle material having a particle diameter of 10-30 ⁇ m; the protease is immobilized on the surface of the material by covalent bonding, the enzyme is trypsin, and the concentration of the enzyme solution is in the range of 10-50 mg/mL. ;
  • the cluster type high temperature denaturation and reduction device, the solvent displacer and the enzyme reactor are sequentially connected in series to form a bundled protein sample pretreatment device, specifically for the material outflow end of the hollow fiber membrane on the high temperature denaturation and reduction device and the solvent displacer.
  • the inflow end of the hollow fiber membrane is in communication; the material outflow end of the hollow fiber membrane on the solvent displacer is connected to the material inlet of the enzyme reactor;
  • the inlet end of the body fluid high-abundance protein removal antibody column is connected to the liquid chromatography system, and the medium-low abundance protein eluted from the antibody column directly enters the bundled protein sample pretreatment device, and the product is obtained from the enzyme reactor.
  • the material outlet flows out, and the medium-low abundance protein is rapidly cut into polypeptides through the enzyme reactor to achieve medium-low abundance eggs. Rapid conversion of white matter to polypeptide.
  • Polypeptides produced by medium-low abundance protein digestion can be directly detected by mass spectrometry or analyzed by LC/MS system.
  • the system is integrated and the degree of automation is high.
  • FIG. 1 Device diagram of body fluid high-throughput protein sample pretreatment system, including body fluid high-abundance protein removal system (A) and bundled protein sample pretreatment device (B); (1): liquid chromatography pump; (2 ): ten-way valve; (3) high-abundance protein removal antibody column; (4) clustered hollow fiber membrane; (5) denaturation and reduction reaction chamber; (6) temperature control device; (7) denaturing agent and reduction Agent inlet; (8): denaturant and reducing agent outlet; (9) displacement chamber; (10) weakly alkaline buffer solution inlet; (11) weakly alkaline buffer solution outlet; (12): enzyme reactor.
  • A body fluid high-abundance protein removal system
  • B bundled protein sample pretreatment device
  • Sample pretreatment device and LC/MS system for analysis of human plasma low abundance protein a: antibody column for separation of human serum high and low abundance protein chromatogram; b: sample pretreatment device, enzymatic hydrolysate Liquid chromatography-mass spectrometry.
  • FIG. 1 Sample pretreatment device and LC/MS analysis of human urine low abundance protein liquid chromatography-mass spectrometry.
  • the protein sample pretreatment device was used to examine the performance of the bundled protein sample pretreatment device (part B in Figure 1) using transferrin as a sample.
  • 50 hollow fiber membranes were used as transport carriers for low-abundance proteins in body fluids, and fixed in denaturation and reduction chambers to prepare high-temperature denaturing and reducing reactors; 50 hollow fiber membranes were used as low-abundance protein transport carriers in body fluids. And fixed in a buffer replacement cavity to prepare a solvent displacer; the enzyme reactor is prepared by using silica gel particles with a particle diameter of 20 ⁇ m as a matrix material, and the cluster type high temperature denaturation and reduction device, the solvent displacer and the enzyme reactor are sequentially connected in series to form a cluster type. Protein sample pretreatment device.
  • the high-abundance protein removal antibody column was integrated with the cluster protein sample pretreatment system to construct a high-throughput body fluid protein sample pretreatment system (as shown in Figure 1). Show), human plasma as a sample, analysis of human plasma low abundance protein components.
  • the operation steps are as follows: the human plasma sample first removes the high abundance protein through the antibody column (as shown in Figure 3a), and the collected medium and low abundance protein components are introduced into the device (the operation process is the same as in Example 1), and the enzymatic hydrolysis is produced.
  • the polypeptide was captured by a C18 precolumn and then subjected to liquid chromatography as shown in Figure 3b.
  • 100 hollow fiber membranes are used as transport carriers for low-abundance proteins in body fluids, and fixed in denaturation and reduction chambers to prepare high-temperature denaturing and reducing reactors; 50 hollow fiber membranes are used as low-abundance protein transport carriers in body fluids.
  • the solvent displacer is prepared by fixing the buffer displacement chamber;
  • the enzyme reactor is prepared by using the silica gel particles with a particle size of 30 ⁇ m as a matrix material, and the cluster type high temperature denaturation and the reducer, the solvent displacer and the enzyme reactor are connected in series to form a bundle type. Protein sample pretreatment device.
  • a high-throughput protein-liquid sample pretreatment system was constructed by integrating a high-abundance protein removal antibody column with a bundled protein sample pretreatment system.
  • Human urine was used as a sample to analyze low-abundance protein components in human urine.
  • the operation steps are as follows: the human urine sample first removes the high abundance protein through the antibody column, and the collected medium and low abundance protein components are introduced into the device, and the medium and low abundance protein is pushed into the high temperature denaturation and reducer at 50 ⁇ L/min.
  • the temperature is 75 ° C
  • the denaturation and reducing agent is the final concentration of 8 M urea and the final concentration of 50 mM thiol
  • the denatured protein sample is passed to a solvent displacer
  • the exchange liquid is a final concentration of 80 mM ammonium hydrogencarbonate (pH 7.5) (operation process)
  • the polypeptide produced by enzymatic hydrolysis was collected by a C18 precolumn and then subjected to liquid chromatography analysis, as shown in FIG.
  • hollow fiber membranes were used as transport carriers for low-abundance proteins in body fluids, and fixed in denaturation and reduction chambers to prepare high-temperature denaturing and reducing reactors; 100 hollow fiber membranes were used as low-abundance protein transport carriers in body fluids. And fixed in a buffer replacement cavity to prepare a solvent displacer; the enzyme reactor is prepared by using silica gel particles with a particle diameter of 20 ⁇ m as a matrix material, and the cluster type high temperature denaturation and reduction device, the solvent displacer and the enzyme reactor are sequentially connected in series to form a cluster type. Protein sample pretreatment device.
  • a high-throughput protein-liquid sample pretreatment system was constructed by integrating a high-abundance protein removal antibody column with a bundled protein sample pretreatment system.
  • Human serum was used as a sample to analyze low-abundance protein components in human urine. The operation steps are as follows: the human serum sample first removes the high abundance protein through the antibody column, and the collected medium and low abundance protein components are introduced into the device, and the medium and low abundance protein is pushed into the high temperature denaturation and reducer at 100 ⁇ L/min.
  • the temperature was 85 ° C, the denaturing and reducing agent was a final concentration of 4 M urea and a final concentration of 100 mM tris(2-carboxyethyl) phosphine; the denatured protein sample was passed to a solvent displacer with a final concentration of 80 mM ammonium bicarbonate ( pH 8.5), other procedures were the same as in Example 1.
  • the polypeptide produced by enzymatic hydrolysis was captured by a C18 precolumn and then subjected to liquid chromatography analysis, as shown in FIG.

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Abstract

一种高通量体液蛋白质样品预处理装置，是一种集体液(血浆，血清，尿液)高丰度蛋白质去除，中低丰度蛋白质在线变性和还原、除盐以及在线酶解的系统，包括：体液高丰度蛋白质去除抗体柱（3），集束式高温变性和还原器，集束式溶剂置换器以及固定化酶反应器（12）。体液蛋白质样品首先通过抗体柱（3）去除高丰度蛋白质，然后洗脱下来的中低丰度蛋白质再通过集束式高温变性和还原器实现在线、快速变性和还原，通过集束式溶剂置换器将变性剂和还原剂去除，最后通过固定化酶反应器（12）在线酶解，酶解产生的肽段可以通过质谱进行检测。这种高通量体液蛋白质样品预处理装置将体液蛋白质的复杂样品预处理过程进行了集成化。

Description

一种高通量体液蛋白质样品预处理装置及其应用 技术领域
本涉及一种高通量体液蛋白质样品预处理装置，是一种集体液高丰度蛋白质去除，中低丰度蛋白质在线变性和还原、除盐以及蛋白质酶解的样品预处理系统。
背景技术
由于体液(血浆、血清以及尿液等)中的蛋白质可以提供大量与生理、病理密切相关的信息，因此体液蛋白质组研究已成为揭示疾病的发病机制、实现早期诊断、分型及个体化治疗等的重要手段。
由于体液中蛋白质的来源广泛(各种细胞、组织和器官)、种类和数量繁多(数万种以上)、丰度动态范围宽(超过10个数量级)，因此，首要解决的问题就是减少体液中高丰度蛋白质对低丰度蛋白质检测的干扰。
此外，临床提供的可开展基础研究的体液样品量非常有限。目前体液蛋白质组样品的处理通常采用离线多步的方式，实现蛋白质的变性、还原、烷基化、酶解和除盐。不仅费时费力，而且会引起蛋白质样品的损失和污染，进而影响定量分析的准确度、灵敏度和分析通量。因此亟需发展高效的体液蛋白质组样品预处理新方法。
针对传统样品预处理方法存在的问题，我们发展了一种集体液高丰度蛋白质去除，中低丰度蛋白在线变性、还原、除盐以及在线酶解的样品预处理系统。该系统可以实现高通量、低损失的体液中低丰度蛋白质样品预处理，在蛋白质组学研究中具有很好的应用前景。
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蛋白质变性、还原、除盐以及在线酶解于一体的样品预处理系统。该系统可以直接处理体液蛋白质，不需要复杂和繁琐的手工操作，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、采用2根以上中空纤维膜作为体液中低丰度蛋白质的传输载体，并固定于变性和还原反应腔体中，通过液相色谱泵或蠕动泵将高浓度变性剂和还原剂输送至变性和还原反应腔体，并与蛋白质充分混合，在温控系统作用下加热并完成变性还原反应，其中变性剂的种类可以为盐酸胍或尿素，浓度为4-8M，蛋白质还原剂的种类可以为二硫苏糖醇、硫醇、三(2-羧乙基)膦，浓度为5-100mM，液相色谱泵或蠕动泵的流量范围0.1mL/min-5mL/min，温控装置的加热温度范围60-95℃；
2、采用2根以上中空纤维膜作为体液中低丰度蛋白质传输载体，并固定于缓冲液置换腔体中，通过液相色谱泵或蠕动泵将低浓度弱碱性缓冲溶液输送至缓冲液置换腔体，使蛋白质溶剂与交换液发生置换，从而达到蛋白质除盐的目的，其中低浓度碱性缓冲溶液可以为碳酸氢铵或醋酸铵溶液，浓度范围为10-100mM,pH范围为7.5-8.5，液相色谱泵或蠕动泵的流量范围0.1mL/min-5mL/min；
3、酶反应器的基质材料为10-30μm粒径的硅胶颗粒材料；蛋白酶通过共价键合附的方式固定在材料表面，所述酶为胰蛋白酶，酶液浓度范围为10-50mg/mL；
4、将集束式高温变性和还原器，溶剂置换器和酶反应器依次串联构成集束式蛋白质样品预处理装置，具体为高温变性和还原器上的中空纤维膜的物料流出端与溶剂置换器上的中空纤维膜的物料流入端相连通；溶剂置换器上的中空纤维膜的物料流出端与酶反应器的物料入口相连；
5、将体液高丰度蛋白质去除抗体柱的入口端与液相色谱系统相连，从抗体柱上洗脱下来的中低丰度蛋白质直接进入集束式蛋白质样品预处理装置，产物由酶反应器的物料出口流出，中低丰度蛋白质通过酶反应器被迅速切割成多肽，实现中低丰度蛋 白质到多肽的快速转化。
6、中低丰度蛋白质酶解产生的多肽可以通过质谱直接检测或通过液质联用系统进行分析。
本发明具有如下优点：
1、体液蛋白质样品手工操作步骤减少，从而样品损失、污染的可能性也将降低，分析通量也大大提高。
2、系统集成化、自动化程度高。
3、可以与分离鉴定技术在线联用，为实现高通量的体液蛋白质分析提供技术支撑。
附图说明
图1、体液高通量蛋白质样品预处理系统装置图，包括体液高丰度蛋白质去除系统(A)和集束式蛋白质样品预处理装置(B)；其中(1)：液相色谱泵；(2)：十通阀；(3)高丰度蛋白质去除抗体柱；(4)集束式中空纤维膜；(5)变性与还原反应腔体；(6)温控装置；(7)变性剂与还原剂入口；(8)：变性剂与还原剂出口；(9)置换腔体；(10)弱碱性缓冲溶液入口；(11)弱碱性缓冲溶液出口；(12)：酶反应器。
图2、样品预处理装置处理转铁蛋白的质谱图。
图3、样品预处理装置与液质联用系统分析人血浆低丰度蛋白质，a:抗体柱分离人血清高、低丰度蛋白质的色谱图；b:经样品预处理装置后，酶解产物的液相色谱-质谱图。
图4、样品预处理装置与液质联用系统分析人尿液低丰度蛋白质液相色谱-质谱分析图。
图5、样品预处理装置与液质联用系统分析人血清低丰度蛋白质液相色谱-质谱分析图。
具体实施方式
实施例1
采用5根中空纤维膜作为体液中低丰度蛋白质的传输载体，并固定于变性和还原反应腔体中制备高温变性还原反应器；采用5根中空纤维膜作为体液中低丰度蛋白质传输载体，并固定于缓冲液置换腔体，制备溶剂置换器；采用10μm粒径的硅胶颗粒为基质材料制备酶反应器，将集束式高温变性和还原器，溶剂置换器和酶反应器依次串联构成集束式蛋白质样品预处理装置，以转铁蛋白为样品考察集束式蛋白质样品预处理装置(如图1中B部分)的性能。将100μg(0.5mg/mL)转铁蛋白以150μL/min将转铁蛋白推入高温变性和还原器中，其中温度为90℃，变性和还原剂为终浓度6M的盐酸胍和终浓度50mM的二硫苏糖醇；变性后的蛋白质样品通入溶剂置换器，交换液为终浓度50mM碳酸氢铵(pH 8.0),在强制对流作用力下，高浓度的变性剂和还原剂得以去除；最后蛋白质样品通过硅胶基质固定酶反应器(2.0mm i.d×50mm)，在室温下实现在线酶解，酶解产生的肽段通过纳升级液相色谱-质谱进行检测(如图2所示)，从图中可以看出转铁蛋白完全转化成肽段，序列覆盖率为76％。
实施例2
采用50根中空纤维膜作为体液中低丰度蛋白质的传输载体，并固定于变性和还原反应腔体中制备高温变性还原反应器；采用50根中空纤维膜作为体液中低丰度蛋白质传输载体，并固定于缓冲液置换腔体，制备溶剂置换器；采用20μm粒径的硅胶颗粒为基质材料制备酶反应器，将集束式高温变性和还原器，溶剂置换器和酶反应器依次串联构成集束式蛋白质样品预处理装置。将高丰度蛋白质去除抗体柱与集束式蛋白质样品预处理系统集成，构建了高通量体液蛋白质样品预处理系统(如图1所 示)，以人血浆为样品，分析人血浆低丰度蛋白质组分。操作步骤如下：人血浆样品首先通过抗体柱去除高丰度蛋白质(如图3a所示)，收集的中低丰度蛋白质组分通入该装置(操作过程同实施例1)，酶解产生的多肽通过C18预柱捕集，然后进行液质分析，如图3b所示。
实施例3
采用100根中空纤维膜作为体液中低丰度蛋白质的传输载体，并固定于变性和还原反应腔体中制备高温变性还原反应器；采用50根中空纤维膜作为体液中低丰度蛋白质传输载体，并固定于缓冲液置换腔体，制备溶剂置换器；采用30μm粒径的硅胶颗粒为基质材料制备酶反应器，将集束式高温变性和还原器，溶剂置换器和酶反应器依次串联构成集束式蛋白质样品预处理装置。将高丰度蛋白质去除抗体柱与集束式蛋白质样品预处理系统集成，构建了高通量体液蛋白质样品预处理系统。以人尿液为样品，分析人尿液低丰度蛋白质组分。操作步骤如下：人尿液样品首先通过抗体柱去除高丰度蛋白质，收集的中低丰度蛋白质组分通入该装置，以50μL/min将中低丰度蛋白推入高温变性和还原器中，其中温度为75℃，变性和还原剂为终浓度8M尿素和终浓度50mM硫醇；变性后的蛋白质样品通入溶剂置换器，交换液为终浓度80mM碳酸氢铵(pH 7.5)(操作过程同实施例1)，酶解产生的多肽通过C18预柱捕集，然后进行液质分析，如图4所示。
实施例4
采用80根中空纤维膜作为体液中低丰度蛋白质的传输载体，并固定于变性和还原反应腔体中制备高温变性还原反应器；采用100根中空纤维膜作为体液中低丰度蛋白质传输载体，并固定于缓冲液置换腔体，制备溶剂置换器；采用20μm粒径的硅胶颗粒为基质材料制备酶反应器，将集束式高温变性和还原器，溶剂置换器和酶反应器依次串联构成集束式蛋白质样品预处理装置。将高丰度蛋白质去除抗体柱与集束式蛋白质样品预处理系统集成，构建了高通量体液蛋白质样品预处理系统。以人血清为样品，分析人尿液低丰度蛋白质组分。操作步骤如下：人血清样品首先通过抗体柱去除高丰度蛋白质，收集的中低丰度蛋白质组分通入该装置，以100μL/min将中低丰度蛋白推入高温变性和还原器中，其中温度为85℃，变性和还原剂为终浓度4M尿素和终浓度100mM三(2-羧乙基)膦；变性后的蛋白质样品通入溶剂置换器，交换液为终浓度80mM碳酸氢铵(pH 8.5)，其他操作过程同实施例1，酶解产生的多肽通过C18预柱捕集，然后进行液质分析，如图5所示。

Claims (5)

  1. 一种高通量体液蛋白质样品预处理装置，包括：依次串连体液高丰度蛋白质去除系统(A)和集束式蛋白质样品预处理装置(B)；
    其特征在于：
    集束式蛋白质样品预处理装置(B)包括依次串连的集束式高温变性和还原器、集束式溶剂置换器和酶反应器；
    所述集束式高温变性和还原器包括2根以上管状中空纤维膜和变性和还原反应腔体；将2根以上根中空纤维膜集束在聚四氟管中，聚四氟管二端用环氧胶固定于变性和还原反应腔体内，以集束式中空纤维膜为蛋白质传输载体，集束式中空纤维膜两端穿过变性和还原反应腔体侧壁面伸出腔体外，并于变性和还原反应腔体上设有物料进口和出口；变性和还原反应腔体外壁面上设有电加热膜；
    所述集束式溶剂置换器包括2根以上管状中空纤维膜和缓冲液置换腔体；将2根以上根中空纤维膜集束在聚四氟管中，聚四氟管二端用环氧胶固定于变性和还原反应腔体内，以集束式中空纤维膜为蛋白质传输载体，中空纤维膜固定于缓冲液置换腔体中，集束式中空纤维膜两端穿过缓冲液置换腔体侧壁面伸出腔体外，并于缓冲液置换腔体上设有物料进口和出口；
    集束式高温变性和还原器中的集束式中空纤维膜入口端相连；
    体液高丰度蛋白质去除系统(A)包括色谱柱，色谱柱一端经液相色谱泵与体液蛋白质样品储罐相连，色谱柱另一端与集束式高温变性和还原器中的集束式中空纤维膜入口端相连；
    所述高通量体液蛋白质样品预处理装置是将体液高丰度蛋白质去除色谱柱的入口端与体液蛋白质样品储罐相连，从色谱柱上洗脱下来的中低丰度蛋白质直接进入集束式高温变性和还原器，高温变性和还原器上的中空纤维膜的物料流出端与溶剂置换器上的中空纤维膜的物料流入端相连通；溶剂置换器上的中空纤维膜的物料流出端与酶反应器的物料入口相连；产物由酶反应器的物料出口流出，中低丰度蛋白质通过酶反应器被迅速切割成多肽，实现中低丰度蛋白质到多肽的快速转化；
    集束式高温变性和还原器中的物料进口与变性还原反应液相连、出口为废液出口；
    缓冲液置换腔体上设有物料进口与溶剂置换液相连、出口为废液出口。
  2. 按照权利要求1所述的预处理装置，其特征在于：
    所述集束式高温变性和还原器采用2-100根中空纤维膜作为体液中低丰度蛋白质的传输载体，并固定于变性和还原反应腔体中，通过液相色谱泵或蠕动泵将高浓度变性剂和还原剂输送至变性和还原反应腔体，并与蛋白质充分混合，在温控系统作用下加热并完成变性还原反应；
    所述集束式溶剂置换器采用2-100根中空纤维膜作为体液中低丰度蛋白质传输载体，并固定于缓冲液置换腔体中，通过液相色谱泵或蠕动泵将低浓度弱碱性缓冲溶液输送至缓冲液置换腔体，使蛋白质溶剂与交换液发生置换，从而达到蛋白质除盐的目的；
    酶反应器中装填的基质材料为10-30μm粒径的硅胶颗粒材料；蛋白酶通过共价键合附的方式固定在材料表面，所述酶为胰蛋白酶，酶液浓度范围为10-50mg/mL；
    将集束式高温变性和还原器，溶剂置换器和酶反应器依次串联构成集束式蛋白质样品预处理装置。
  3. 按照权利要求1所述的预处理装置，其特征在于：
    液相色谱泵或蠕动泵的流量范围0.1mL/min-5mL/min，温控装置的加热温度范围 60-95℃；
    变性剂的种类可以为盐酸胍或尿素，浓度为4-8M，蛋白质还原剂的种类可以为二硫苏糖醇、硫醇、三(2-羧乙基)膦，浓度为5-100mM；
    低浓度碱性缓冲溶液可以为碳酸氢铵或醋酸铵溶液，浓度范围为10-100mM,pH范围为7.5-8.5。
  4. 按照权利要求1所述的预处理装置，其特征在于：
    所述的体液高丰度蛋白质去除抗体柱的适用的洗脱流量范围为50-1000μL/min,体液蛋白质处理量为0.3-1mg，体液包括血浆、血清或尿液。
  5. 一种权利要求1-4任一所述的高通量体液蛋白质样品预处理装置可用于临床诊断和疾病蛋白质质组学研究中的体液蛋白质快速预处理过程。
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