CN101344503B - Method for protein example enrichment and combination with capillary electrophoretic analysis - Google Patents

Abstract

The invention discloses a coupling method of protein sample concentration and capillary electrophoretic analysis, which comprises the protein sample concentration, pushing of enriched samples, sample introduction under the wicking action of a separating capillary and the capillary electrophoretic analysis; a coupling system comprises a constant flow pump, a membrane interface, a polyfluortetraethylene pipe, a silicon rubber and the separating capillary; the constant flow pump is used for continuously pushing the samples into the membrane interface in the sample concentration; the samples enter the polyfluortetraethylene pipe after the sample concentration is finished; the separating capillary is inserted into the polyfluortetraethylene pipe, and the samples enter a sample introducing end of the separating capillary under the wicking action; when the sample introduction is finished, two ends of the separating capillary are inserted into a buffering liquid, and voltage is applied so as to implement electrophoresis. The coupling method of protein sample concentration and capillary electrophoretic analysis has the following advantages: compared with completely offline methods, the coupling method has reduced operation steps, lowered sample loss, good concentration effect of the low-concentration protein samples, little dead volume at the membrane interface, reduced sample consumption, simple and convenient method, simple operations and low cost.

Description

Translated fromChinese

一种用于蛋白质样品富集及与毛细管电泳分析联用的方法A method for protein sample enrichment and combined use with capillary electrophoresis analysis

技术领域technical field

本发明涉及低丰度蛋白质样品富集与检测方法，具体地说是一种用于蛋白质样品富集与毛细管电泳分析联用的方法。The invention relates to a low-abundance protein sample enrichment and detection method, in particular to a method for combining protein sample enrichment and capillary electrophoresis analysis.

背景技术Background technique

毛细管电泳现在已经成为一种常规的分离方法，在化学、生物学等领域应用广泛。但是由于毛细管的光程短，所以毛细管电泳的检测灵敏度较低。尽管采用激光诱导荧光检测可以降低毛细管电泳的检测限，但是蛋白质的荧光标记过程繁琐，而且仪器价格昂贵。因此，发展用于蛋白质样品富集与毛细管电泳分析联用的方法，对于提高毛细管电泳的检测灵敏度是非常必要的。Capillary electrophoresis has now become a routine separation method, widely used in chemistry, biology and other fields. However, due to the short optical path of the capillary, the detection sensitivity of capillary electrophoresis is low. Although laser-induced fluorescence detection can reduce the detection limit of capillary electrophoresis, the fluorescent labeling process of proteins is cumbersome and the instrument is expensive. Therefore, it is very necessary to develop a method for the combination of protein sample enrichment and capillary electrophoresis analysis to improve the detection sensitivity of capillary electrophoresis.

目前，已报道了多种毛细管电泳的样品富集方法。其中场放大进样(FASS)[1，2]是基于样品缓冲液和电泳缓冲溶液电导率的差异而产生的样品区带压缩。但是，当样品缓冲液的电导率高于电泳缓冲液时，样品则无法通过场放大进样富集。等速电泳(ITP)是将样品区带夹在前导缓冲液与尾随缓冲液之间的一种样品富集方法[3，4]。但是，ITP不能同时对阳离子与阴离子进行富集与分离。此外，还有等电聚焦[5]、基于半通透性中空纤维膜的电堆积[6]和基于刻蚀毛细管的电堆积[7]等方法。Currently, a variety of capillary electrophoresis sample enrichment methods have been reported. Among them, field amplified sampling (FASS) [1, 2] is based on the sample zone compression caused by the difference in conductivity between the sample buffer and the electrophoresis buffer solution. However, when the conductivity of the sample buffer is higher than that of the electrophoresis buffer, the sample cannot be enriched by field amplification injection. Isotachophoresis (ITP) is a sample enrichment method in which the sample band is sandwiched between a leading buffer and a trailing buffer [3, 4]. However, ITP cannot simultaneously enrich and separate cations and anions. In addition, there are isoelectric focusing [5], electro deposition based on semi-permeable hollow fiber membrane [6] and electro deposition based on etched capillary [7].

[1]Burgi，D.S.，Chien，R.L.Anal.Chem.1992，64，1046-1050.[1] Burgi, D.S., Chien, R.L.Anal.Chem.1992, 64, 1046-1050.

[2]Burgi，D.S.Anal.Chem.1993，65，3726-3729.[2] Burgi, D. S. Anal. Chem. 1993, 65, 3726-3729.

[3]Krivankova，L.；Gebauer，P.；Bocek，P.J.Chromatogr.A1995，716，35-48.[3] Krivankova, L.; Gebauer, P.; Bocek, P.J. Chromatogr.A1995, 716, 35-48.

[4]Foret，F.；Szoko，E.；Karger，B.L.Electrophoresis，1993，14，417-428.[4] Foret, F.; Szoko, E.; Karger, B.L. Electrophoresis, 1993, 14, 417-428.

[5]Hjerten，S.；Liao，J.L.；Zhang，R.J.Chromatogr.A 1994，676，409-420.[5] Hjerten, S.; Liao, J.L.; Zhang, R.J. Chromatogr.A 1994, 676, 409-420.

[6]Wu，X.Z.；Hosaka，A.；Hobo，T..Anal.Chem.1998，70，2081-2084.[6] Wu, X.Z.; Hosaka, A.; Hobo, T..Anal.Chem.1998, 70, 2081-2084.

[7]Wei W.，Edward，S.Y.，Anal.Chem.2002，74，3899-3905.[7] Wei W., Edward, S.Y., Anal.Chem.2002, 74, 3899-3905.

发明内容Contents of the invention

本发明的目的在于提供一种用于蛋白质样品富集及与毛细管电泳分析联用的方法。利用此方法可以对低浓度蛋白质样品进行富集，并通过毛细作用的进样方法实现样品富集与毛细管电泳分离分析的联用。The purpose of the present invention is to provide a method for protein sample enrichment and combined use with capillary electrophoresis analysis. This method can be used to enrich low-concentration protein samples, and realize the combination of sample enrichment and capillary electrophoresis separation and analysis through capillary action sampling method.

一种用于蛋白质样品富集及与毛细管电泳分析联用的方法，其特征在于：所述的用于蛋白质样品富集及与毛细管电泳分析联用的方法，包括蛋白质样品的富集(图1，A)、富集后样品的推出(图1，B)、分离毛细管在毛细作用下进样(图1，C)和毛细管电泳分析(图1，D)；A method for protein sample enrichment and combined use with capillary electrophoresis analysis, characterized in that: the method for protein sample enrichment and combined use with capillary electrophoresis analysis includes the enrichment of protein samples (Fig. 1 , A), push-out of enriched sample (Fig. 1, B), separation capillary under capillary action (Fig. 1, C) and capillary electrophoresis analysis (Fig. 1, D);

联用系统包括恒流泵(A0)、膜接口(A1)、聚四氟乙烯管(A2)、硅橡胶(A3)和分离毛细管(A4)；用于蛋白质样品富集的膜接口(图1，A1；图2)包括连接毛细管(1)、中空纤维膜(2)、盛装缓冲液的离心管(3)、固定连接毛细管的聚四氟乙烯管(4)以及加入与取出缓冲液的开口(5)；The combined system includes constant flow pump (A0), membrane interface (A1), polytetrafluoroethylene tube (A2), silicone rubber (A3) and separation capillary (A4); membrane interface for protein sample enrichment (Figure 1 , A1; Fig. 2) includes a connecting capillary (1), a hollow fiber membrane (2), a centrifuge tube (3) containing a buffer, a Teflon tube (4) fixedly connecting the capillary, and openings for adding and removing the buffer (5);

富集样品时，先将膜接口(A1)的中空纤维膜与连接毛细管(1)中充满样品，再将膜接口(A1)出口端的连接毛细管(1)用硅橡胶堵死，最后使用恒流泵将样品不断推入到膜接口(A1)中；由于蛋白质大分子无法透过中空纤维膜(2)而达到富集效果；富集结束后，取下硅橡胶(A3)，用恒流泵将富集后的样品从膜接口(A1)中推出，样品进入到聚四氟乙烯管中；将分离毛细管(A4)插入到聚四氟乙烯管(A2)，样品在毛细作用下进入分离毛细管(A4)的进样端；进样结束后，将分离毛细管(A4)两端插入到缓冲液中，施加电压，进行电泳。When enriching the sample, first fill the hollow fiber membrane and the connecting capillary (1) of the membrane interface (A1) with the sample, then block the connecting capillary (1) at the outlet of the membrane interface (A1) with silicone rubber, and finally use a constant flow The pump continuously pushes the sample into the membrane interface (A1); because the protein macromolecules cannot pass through the hollow fiber membrane (2), the enrichment effect is achieved; after the enrichment, remove the silicone rubber (A3) and use a constant flow pump The enriched sample is pushed out from the membrane interface (A1), and the sample enters the polytetrafluoroethylene tube; the separation capillary (A4) is inserted into the polytetrafluoroethylene tube (A2), and the sample enters the separation capillary under capillary action (A4) sampling end; after the sampling is completed, insert the two ends of the separation capillary (A4) into the buffer, apply voltage, and perform electrophoresis.

本发明提供的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述恒流泵(A0)的流量为0.5-2.5μL/min。The method for protein sample enrichment combined with capillary electrophoresis analysis provided by the present invention is characterized in that: the flow rate of the constant flow pump (A0) is 0.5-2.5 μL/min.

本发明提供的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的膜接口(A1)的中空纤维膜(2)为醋酸纤维素膜，内径为100～400μm，截留分子量为3000～8000Da，有效长度为2cm-4cm。The method for combining protein sample enrichment and capillary electrophoresis analysis provided by the present invention is characterized in that: the hollow fiber membrane (2) of the membrane interface (A1) is a cellulose acetate membrane with an inner diameter of 100-400 μm, The molecular weight cut-off is 3000~8000Da, and the effective length is 2cm-4cm.

本发明提供的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的膜接口(A1)的连接毛细管(1)内径与外径要与醋酸纤维素膜内径相匹配，以保证连接毛细管(1)可以穿入膜中。The method for the combination of protein sample enrichment and capillary electrophoresis analysis provided by the present invention is characterized in that: the inner diameter and outer diameter of the connecting capillary (1) of the membrane interface (A1) must match the inner diameter of the cellulose acetate membrane , to ensure that the connecting capillary (1) can penetrate into the membrane.

本发明提供的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的聚四氟乙烯管(A2)的内径接近连接毛细管(1)的外径，聚四氟乙烯管(A2)与连接毛细管(1)为密封连接。The method for protein sample enrichment combined with capillary electrophoresis analysis provided by the present invention is characterized in that: the inner diameter of the polytetrafluoroethylene tube (A2) is close to the outer diameter of the connecting capillary (1), and the polytetrafluoroethylene tube (A2) The tube (A2) is tightly connected to the connecting capillary (1).

本发明提供的一种用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述硅橡胶(A3)也能是其他的能够提供较高反压的材料，如毛细管聚合物整体柱，或者毛细管硅胶整体柱，或者毛细管填充柱等等。A method for protein sample enrichment combined with capillary electrophoresis analysis provided by the present invention is characterized in that: the silicone rubber (A3) can also be other materials that can provide higher back pressure, such as capillary polymer Monolithic column, or capillary silica gel monolithic column, or capillary packed column, etc.

本发明提供的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的分离毛细管的内径为50或者75μm。。The method for combining protein sample enrichment and capillary electrophoresis analysis provided by the present invention is characterized in that: the inner diameter of the separation capillary is 50 or 75 μm. .

本发明采用的技术方案为：The technical scheme adopted in the present invention is:

使用具有一定截留分子量的中空纤维膜，利用小分子可以自由通过，而大分子蛋白质被截留的原理来实现蛋白质的富集。在富集时，膜接口的出口端用硅橡胶堵死，并用恒流泵将样品不断推入到膜接口中进行富集。膜接口制作时，使用环氧树脂胶将毛细管与中空纤维膜的连接处密封，可避免在富集过程中样品通过毛细管与中空纤维膜的接口处漏出。富集结束后，取下硅橡胶用恒流泵将富集后的样品从膜接口中推入聚四氟乙烯管中。将分离毛细管插入聚四氟乙烯管中，利用毛细作用将聚四氟乙烯管中的样品转移到分离毛细管的进样端。进样结束后，将分离毛细管两端插入到缓冲液中，施加电压，进行电泳分离。Using a hollow fiber membrane with a certain molecular weight cut-off, the enrichment of proteins is achieved by utilizing the principle that small molecules can pass through freely, while large molecular proteins are blocked. During enrichment, the outlet end of the membrane interface is blocked with silicon rubber, and a constant flow pump is used to continuously push the sample into the membrane interface for enrichment. When making the membrane interface, use epoxy glue to seal the connection between the capillary and the hollow fiber membrane, which can prevent the sample from leaking through the interface between the capillary and the hollow fiber membrane during the enrichment process. After enrichment, remove the silicone rubber and use a constant flow pump to push the enriched sample from the membrane interface into the polytetrafluoroethylene tube. Insert the separation capillary into the polytetrafluoroethylene tube, and use capillary action to transfer the sample in the polytetrafluoroethylene tube to the sampling end of the separation capillary. After the sample injection, insert the two ends of the separation capillary into the buffer, apply voltage, and perform electrophoresis separation.

具体方法包括：蛋白质样品的富集(图1，A)，富集后样品的推出(图1，B)，分离毛细管插入进样(图1，C)，毛细管电泳分析(图1，D)。此联用系统包括恒流泵(A0)、膜接口(A1)、聚四氟乙烯管(A2)、硅橡胶(A3)和分离毛细管(A4)。用于蛋白质样品富集的膜接口(图1，A1；图2)包括中空纤维膜(2)、连接毛细管(1)、装有缓冲液的离心管(3)以及固定连接毛细管的聚四氟乙烯管(4)。在富集时，先将膜接口的出口端用硅橡胶封住，再用恒流泵将样品不断推入到膜接口中进行富集。在膜接口制作时，使用环氧树脂胶将毛细管与中空纤维膜连接，并在连接处密封，从而避免在富集过程中样品在接口处漏出。富集结束后，取下硅橡胶，用恒流泵将富集后的样品从膜接口中推入到聚四氟乙烯管中。将分离毛细管插入聚四氟乙烯管，富集后的样品在毛细作用下进入分离毛细管的进样端。进样结束后，将分离毛细管两端插入到缓冲液中，施加电压，进行电泳分离。Specific methods include: enrichment of protein samples (Fig. 1, A), push out of enriched samples (Fig. 1, B), separation capillary insertion and injection (Fig. 1, C), capillary electrophoresis analysis (Fig. 1, D) . The combined system includes constant flow pump (A0), membrane interface (A1), polytetrafluoroethylene tube (A2), silicone rubber (A3) and separation capillary (A4). The membrane interface for protein sample enrichment (Fig. 1, A1; Fig. 2) consists of a hollow fiber membrane (2), connecting capillary (1), centrifuge tube (3) filled with buffer, and PTFE that fixes the connecting capillary Vinyl tubing (4). When enriching, first seal the outlet end of the membrane interface with silicone rubber, and then use a constant flow pump to continuously push the sample into the membrane interface for enrichment. When making the membrane interface, use epoxy glue to connect the capillary to the hollow fiber membrane, and seal the connection, so as to avoid the leakage of the sample at the interface during the enrichment process. After enrichment, remove the silicone rubber, and use a constant flow pump to push the enriched sample from the membrane interface into the Teflon tube. The separation capillary is inserted into the polytetrafluoroethylene tube, and the enriched sample enters the sampling end of the separation capillary under capillary action. After the sample injection, insert the two ends of the separation capillary into the buffer, apply voltage, and perform electrophoresis separation.

样品富集时，可以通过改变样品进入膜接口的流速与富集时间来改善样品的富集效果。在毛细管进样时，可以通过调节分离毛细管插入聚四氟乙烯管的深度来控制进入分离毛细管的样品量。When the sample is enriched, the enrichment effect of the sample can be improved by changing the flow rate of the sample entering the membrane interface and the enrichment time. During capillary sampling, the amount of sample entering the separation capillary can be controlled by adjusting the depth at which the separation capillary is inserted into the polytetrafluoroethylene tube.

本发明的优点：Advantages of the present invention:

可实现蛋白质样品富集与毛细管电泳分析的联用，降低毛细管电泳分析的检测限；与完全离线的方法相比，减少了操作步骤，降低了样品的损失。对低浓度蛋白质样品具有很好的富集效果；膜接口处死体积小(1μL)，减少了样品消耗；方法简便、操作简单、成本低廉；具有良好的通用性，实用性强，具有较高的推广价值。The combination of protein sample enrichment and capillary electrophoresis analysis can be realized, and the detection limit of capillary electrophoresis analysis can be reduced; compared with the completely offline method, the operation steps are reduced and the loss of samples is reduced. It has a good enrichment effect on low-concentration protein samples; the membrane interface has a small dead volume (1μL), which reduces sample consumption; the method is simple, easy to operate, and low in cost; it has good versatility, strong practicability, and high Promote value.

附图说明Description of drawings

图1为本发明的操作流程图；Fig. 1 is the operation flowchart of the present invention;

图2为膜接口(图1，A1)的放大图；Figure 2 is an enlarged view of the membrane interface (Figure 1, A1);

图3为三种蛋白质的混合液(Cytochrome C，0.0333mg/mL；lysozyme，0.0333mg/mL；trypsin inhibitor，0.167mg/mL)经过膜接口富集、进样和电泳分离所得的谱图；Figure 3 is the chromatogram of the mixture of three proteins (Cytochrome C, 0.0333mg/mL; lysozyme, 0.0333mg/mL; trypsin inhibitor, 0.167mg/mL) after membrane interface enrichment, sample injection and electrophoresis separation;

考察了在相同富集时间(20min)、不同富集流速(0.5μL/min，1μL/min，2μL/min)下蛋白质的富集分离效果；样品溶解在10mM磷酸盐缓冲液(pH3.0)中，缓冲液池中装满10mM磷酸盐缓冲液(pH3.0)，电泳缓冲液为20mM磷酸盐(pH3.0)；每个峰对应的蛋白质为：1，Cytochrome C；2，lysozyme；3，trypsin inhibitorThe effect of protein enrichment and separation under the same enrichment time (20min) and different enrichment flow rates (0.5μL/min, 1μL/min, 2μL/min) was investigated; the sample was dissolved in 10mM phosphate buffer (pH3.0) In, the buffer pool is filled with 10mM phosphate buffer (pH3.0), and the electrophoresis buffer is 20mM phosphate (pH3.0); the protein corresponding to each peak is: 1, Cytochrome C; 2, lysozyme; 3 , trypsin inhibitor

a：富集前样品；b：0.5μL/min流速下富集所得样品；c：1μL/min流速下富集所得样品；d：2μL/min流速下富集所得样品；a: Sample before enrichment; b: Sample enriched at a flow rate of 0.5 μL/min; c: Sample enriched at a flow rate of 1 μL/min; d: Sample enriched at a flow rate of 2 μL/min;

图4为胰岛素溶液(0.027mg/mL)经过膜接口富集前的谱图；Fig. 4 is the spectrogram before the enrichment of insulin solution (0.027mg/mL) through membrane interface;

图5为胰岛素溶液(0.027mg/mL)经过膜接口富集后的谱图。Fig. 5 is the spectrogram of the insulin solution (0.027 mg/mL) enriched through the membrane interface.

具体实施方式Detailed ways

实施例1：Example 1:

请参阅图1与图2，富集样品时，先将膜接口(A1)的中空纤维膜(2)与连接毛细管(1)中充满样品，再将膜接口出口端的连接毛细管用硅橡胶(A3)堵死，最后使用恒流泵(A0)将样品不断推入到膜接口中进行蛋白质的富集。富集结束后，取下硅橡胶，用恒流泵将富集后的样品从膜接口中推入到聚四氟乙烯管(A2)中。分离毛细管(A4)插入聚四氟乙烯管中，利用毛细作用样品转移到分离毛细管(A4)的进样端。进样结束后，将分离毛细管两端插入到缓冲液中，施加电压，进行电泳分离。Please refer to Figure 1 and Figure 2. When enriching the sample, first fill the hollow fiber membrane (2) and the connecting capillary (1) of the membrane interface (A1) with the sample, and then cover the connecting capillary at the outlet of the membrane interface with silicone rubber (A3 ) to block, and finally use the constant flow pump (A0) to continuously push the sample into the membrane interface for protein enrichment. After enrichment, remove the silicone rubber, and use a constant flow pump to push the enriched sample from the membrane interface into the Teflon tube (A2). The separation capillary (A4) is inserted into the polytetrafluoroethylene tube, and the sample is transferred to the injection end of the separation capillary (A4) by capillary action. After the sample injection, insert the two ends of the separation capillary into the buffer, apply voltage, and perform electrophoresis separation.

样品为三种蛋白质的混合液(Cytochrome C，0.0333mg/mL；lysozyme，0.0333mg/mL；trypsin inhibitor，0.167mg/mL)。样品溶解在10mM磷酸盐缓冲液(pH3.0)中，膜接口中装满10mM磷酸盐缓冲液(pH3.0)，电泳缓冲液为20mM磷酸盐(pH3.0)。膜接口的两端连接毛细管内径均为100μm，外径均为375μm，长度均为10cm。分离毛细管内径为50μm，外径375μm，总长为34cm，有效长度为24cm。毛细管电泳施加电压为-16kv。The sample is a mixture of three proteins (Cytochrome C, 0.0333mg/mL; lysozyme, 0.0333mg/mL; trypsin inhibitor, 0.167mg/mL). The sample was dissolved in 10mM phosphate buffer (pH3.0), the membrane interface was filled with 10mM phosphate buffer (pH3.0), and the electrophoresis buffer was 20mM phosphate (pH3.0). Both ends of the membrane interface are connected capillaries with an inner diameter of 100 μm, an outer diameter of 375 μm, and a length of 10 cm. The inner diameter of the separation capillary is 50 μm, the outer diameter is 375 μm, the total length is 34 cm, and the effective length is 24 cm. The applied voltage of capillary electrophoresis is -16kv.

每次样品富集结束时，关闭恒流泵，稳定1min后取下硅橡胶。用0.5μL/min的流速将富集在膜接口处的样品推出进入到聚四氟乙烯管中。将分离毛细管的进样端插入到聚四氟乙烯管中进行进样，插入深度为4mm。进样结束后，进行电泳分析。考察了富集时间(20min)相同，不同富集流速(0.5μL/min，1μL/min，2μL/min)下的富集效果，如图3所示。At the end of each sample enrichment, the constant flow pump was turned off, and the silicone rubber was removed after 1 min of stabilization. Push the sample enriched at the interface of the membrane into the Teflon tube at a flow rate of 0.5 μL/min. Insert the sampling end of the separation capillary into the polytetrafluoroethylene tube for sampling, the insertion depth is 4mm. After the sample injection, electrophoresis analysis was performed. The enrichment effect under the same enrichment time (20 min) and different enrichment flow rates (0.5 μL/min, 1 μL/min, 2 μL/min) was investigated, as shown in FIG. 3 .

由图3可以看出，在三种富集流速下，富集20min后蛋白质样品均得到了很好的富集。随着富集流速的增大(0.5μL/min-2μL/min)，富集效果也有所提高。富集流速2μL/min，富集时间20min时，富集倍数可以达到21倍。It can be seen from Figure 3 that under the three enrichment flow rates, the protein samples were well enriched after 20 minutes of enrichment. As the enrichment flow rate increases (0.5μL/min-2μL/min), the enrichment effect also increases. When the enrichment flow rate is 2 μL/min and the enrichment time is 20 minutes, the enrichment factor can reach 21 times.

实施例2：Example 2:

样品为胰岛素溶液(0.027mg/mL)。样品溶解在10mM磷酸盐缓冲液(pH3.0)中，膜接口中装满10mM磷酸盐缓冲液(pH3.0)，电泳缓冲液为20mM磷酸盐(pH3.0)。膜接口的两端连接毛细管内径均为100μm，外径均为375μm，长度均为10cm。分离毛细管内径为50μm，外径375μm，总长为34cm，有效长度为24cm。毛细管电泳施加电压为-16kv。The sample was insulin solution (0.027 mg/mL). The sample was dissolved in 10mM phosphate buffer (pH3.0), the membrane interface was filled with 10mM phosphate buffer (pH3.0), and the electrophoresis buffer was 20mM phosphate (pH3.0). Both ends of the membrane interface are connected capillaries with an inner diameter of 100 μm, an outer diameter of 375 μm, and a length of 10 cm. The inner diameter of the separation capillary is 50 μm, the outer diameter is 375 μm, the total length is 34 cm, and the effective length is 24 cm. The applied voltage of capillary electrophoresis is -16kv.

每次样品富集结束时，关闭恒流泵，稳定1min后取下硅橡胶。用0.5μL/min的流速将富集在膜接口处的样品推出进入到聚四氟乙烯管中。将分离毛细管的进样端插入到聚四氟乙烯管中进行进样，插入深度为4mm。进样结束后，进行电泳分析。考察了富集时间20min，富集流速1μL/min下的富集效果，如图4、图5所示。At the end of each sample enrichment, the constant flow pump was turned off, and the silicone rubber was removed after 1 min of stabilization. Push the sample enriched at the interface of the membrane into the Teflon tube at a flow rate of 0.5 μL/min. Insert the sampling end of the separation capillary into the polytetrafluoroethylene tube for sampling, the insertion depth is 4mm. After sample injection, electrophoresis analysis was performed. The enrichment effect under the enrichment time of 20 min and the enrichment flow rate of 1 μL/min was investigated, as shown in Fig. 4 and Fig. 5 .

Claims (9)

Translated fromChinese

1.一种用于蛋白质样品富集及与毛细管电泳分析联用的方法，其特征在于：所述的用于蛋白质样品富集及与毛细管电泳分析联用的方法，包括蛋白质样品的富集、富集后样品的推出、分离毛细管在毛细作用下进样和毛细管电泳分析；联用系统包括恒流泵(A0)、膜接口(A1)、聚四氟乙烯管(A2)、硅橡胶(A3)和分离毛细管(A4)；用于蛋白质样品富集的膜接口(A1)包括连接毛细管(1)、中空纤维膜(2)、盛装缓冲液的离心管(3)、固定连接毛细管的聚四氟乙烯管(4)以及加入与取出缓冲液的开口(5)；1. A method for protein sample enrichment and capillary electrophoresis analysis, characterized in that: the method for protein sample enrichment and capillary electrophoresis analysis includes protein sample enrichment, Pushing out of the enriched sample, capillary injection of the separation capillary and capillary electrophoresis analysis; the combined system includes a constant flow pump (A0), a membrane interface (A1), a polytetrafluoroethylene tube (A2), a silicone rubber (A3 ) and separation capillary (A4); the membrane interface (A1) used for protein sample enrichment includes connecting capillary (1), hollow fiber membrane (2), centrifuge tube (3) containing buffer solution, polyfour Vinyl fluoride tube (4) and the opening (5) for adding and removing the buffer solution;富集样品时，先将膜接口(A1)的中空纤维膜与连接毛细管(1)中充满样品，再将膜接口(A1)出口端的连接毛细管(1)用硅橡胶堵死，最后使用恒流泵将样品不断推入到膜接口(A1)中；由于蛋白质大分子无法透过中空纤维膜(2)而达到富集效果；富集结束后，取下硅橡胶(A3)，用恒流泵将富集后的样品从膜接口(A1)中推出，样品进入到联用系统中的聚四氟乙烯管中；将分离毛细管(A4)插入到联用系统中的聚四氟乙烯管(A2)，样品在毛细作用下进入分离毛细管(A4)的进样端；进样结束后，将分离毛细管(A4)两端插入到缓冲液中，施加电压，进行电泳。When enriching the sample, first fill the hollow fiber membrane and the connecting capillary (1) of the membrane interface (A1) with the sample, then block the connecting capillary (1) at the outlet of the membrane interface (A1) with silicone rubber, and finally use a constant flow The pump continuously pushes the sample into the membrane interface (A1); because the protein macromolecules cannot pass through the hollow fiber membrane (2), the enrichment effect is achieved; after the enrichment, remove the silicone rubber (A3) and use a constant flow pump Push the enriched sample out from the membrane interface (A1), and the sample enters the polytetrafluoroethylene tube in the combined system; insert the separation capillary (A4) into the polytetrafluoroethylene tube (A2) in the combined system ), the sample enters the injection end of the separation capillary (A4) under capillary action; after the injection is completed, insert both ends of the separation capillary (A4) into the buffer, apply a voltage, and perform electrophoresis.2.按照权利要求1所述的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述恒流泵(A0)的流量为0.5-2.5μL/min。2. The method for protein sample enrichment combined with capillary electrophoresis analysis according to claim 1, characterized in that: the flow rate of the constant flow pump (A0) is 0.5-2.5 μL/min.3.按照权利要求1所述的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的膜接口(A1)的中空纤维膜(2)为醋酸纤维素膜，内径为100～400μm，截留分子量为3000～8000Da，有效长度为2cm-4cm。3. The method for protein sample enrichment combined with capillary electrophoresis analysis according to claim 1, characterized in that: the hollow fiber membrane (2) of the membrane interface (A1) is a cellulose acetate membrane with an inner diameter of It is 100-400μm, the molecular weight cut-off is 3000-8000Da, and the effective length is 2cm-4cm.4.按照权利要求3所述的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的膜接口(A1)的连接毛细管(1)内径与外径要与醋酸纤维素膜内径相匹配，以保证连接毛细管(1)可以穿入膜中。4. The method for protein sample enrichment and capillary electrophoresis analysis according to claim 3, characterized in that: the inner diameter and outer diameter of the connecting capillary (1) of the membrane interface (A1) should be the same as that of cellulose acetate Match the inner diameter of the plain membrane to ensure that the connecting capillary (1) can penetrate into the membrane.5.按照权利要求1所述的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的联用系统中的聚四氟乙烯管(A2)的内径接近连接毛细管(1)的外径，联用系统中的聚四氟乙烯管(A2)与连接毛细管(1)为密封连接。5. The method for protein sample enrichment and capillary electrophoresis analysis combined according to claim 1, characterized in that: the inner diameter of the polytetrafluoroethylene tube (A2) in the described combined system is close to the connecting capillary ( 1), the polytetrafluoroethylene tube (A2) in the combined system is in a sealed connection with the connecting capillary (1).6.按照权利要求1所述一种用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述硅橡胶(A3)可以用毛细管聚合物整体柱代替。6. A method for protein sample enrichment combined with capillary electrophoresis analysis according to claim 1, characterized in that: the silicone rubber (A3) can be replaced by a capillary polymer monolithic column.7.按照权利要求1所述一种用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述硅橡胶(A3)可以用毛细管硅胶整体柱代替。7. A method for protein sample enrichment combined with capillary electrophoresis analysis according to claim 1, characterized in that: the silicone rubber (A3) can be replaced by a capillary silica gel monolithic column.8.按照权利要求1所述一种用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述硅橡胶(A3)可以用毛细管填充柱代替。8. A method for protein sample enrichment combined with capillary electrophoresis analysis according to claim 1, characterized in that: the silicone rubber (A3) can be replaced by a capillary packed column.9.按照权利要求1所述的用于蛋白质样品富集与毛细管电泳分析联用的方法，其特征在于：所述的分离毛细管的内径为50或者75μm。9. The method for combining protein sample enrichment and capillary electrophoresis analysis according to claim 1, characterized in that: the inner diameter of the separation capillary is 50 or 75 μm.

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