膜蛋白质组学研究中样品制备与净化方法的发展与应用
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摘要
膜蛋白质在许多生命活动过程中发挥着重要的作用,包括细胞与环境之间的物质和能量交换以及信号转导等。然而,目前对于膜蛋白的研究仍然面临很大的挑战,这主要是因为膜蛋白大多具有低丰度和强疏水性的特点,从而给这些蛋白质的溶解、提取和酶解带来困难。为了解决这些问题,通常会在膜片的水平上对膜蛋白质进行富集,并且通过加入一些高浓度的添加剂(如去垢剂和有机溶剂等)来促进膜蛋白质的抽提和溶解。SDS是最有效、最经典的去垢剂。但是,由于SDS能够降低蛋白酶的活力,影响酶解产物的色谱分离和抑制质谱分析时的肽段的解离,所以,必须设法在酶解等后续实验步骤之前对样品进行净化处理,以除去样品中的SDS及其他干扰物质。传统的样品净化策略主要有沉淀法、透析、离子交换以及凝胶过滤等,尽管这些方法都能在一定程度上除去样品中的小分子干扰物质,但它们都有一个共同的缺陷,即在样品净化过程中往往导致明显的蛋白的损失,此外,这些方法还在蛋白质鉴定效果和实验成本等方面存在一定的局限性。在本研究中,我们针对现有方法的缺陷展开研究,发展了基于凝胶电泳和预冷丙酮沉淀的样品制备和净化方法并将其应用于膜蛋白质组学研究。
在基于凝胶电泳的方法研究中,我们采用了一种特殊设计的梯度凝胶电泳(GGE)系统,包括一个琼脂糖上样层,若干个不同浓度的聚丙烯酰胺凝胶分级分离层和一个高浓度(40%)的聚丙烯酰胺凝胶封闭层。采用富集的大鼠肝脏膜蛋白作为样品的实验结果表明,通过电泳驱动的方式,GGE系统能够去除样品和凝胶中85%以上的SDS。此外,通过这种电泳驱动的方式不仅能够对含有高浓度SDS的蛋白质样品进行有效的净化,而且能同时浓缩样品和对样品进行分级分离而降低样品复杂程度,因而有利于后续的蛋白质酶解和LC-MS/MS分析,大幅度提高蛋白质的鉴定效率。与文献报道的同类样品净化方法管胶法和三明治凝胶电泳法相比,GGE策略鉴定的膜蛋白质总数分别提高了213和222,尤其是那些具有极端性质(如分子量较高或较低、丰度低或疏水性强)的蛋白质的鉴定效果得到明显的提高。
在基于预冷丙酮沉淀的样品制备和净化策略研究中,我们优化了预冷丙酮沉淀法去除蛋白质样品中SDS及其他小分子干扰物质的实验条件,比较了沉淀蛋白质复溶和溶液酶解的不同策略,建立起一种基于溶液的蛋白质样品制备和净化的系统方法。该方法综合利用了SDS对于生物膜的强裂解能力和有效溶解疏水性膜蛋白的能力、丙酮沉淀法高效净化样品的能力以及NH4HCO3溶液提供的适合胰蛋白酶有效作用的适宜环境。实验结果表明,利用优化的预冷丙酮沉淀法能使蛋白质样品中的SDS的含量降低至0.01%以下,并使蛋白质的回收率保持在90%以上；预冷丙酮沉淀的蛋白质在NH4HCO3溶液中能被胰蛋白酶有效酶解。与文献报道的其他方法相比,此方法更有利于蛋白质的酶解和鉴定,它所鉴定到的总蛋白和膜蛋白的数量分别平均提高了26.6%和25.8%。此外,通过对所鉴定到的蛋白质的理化性质进行的比较分析表明,这种优化的系统方法对于各种类型的蛋白质包括高疏水性和多跨膜的蛋白质的鉴定具有普适性,在蛋白质化学和蛋白质组学研究领域具有重要的应用价值。
Membrane proteins play important role in many molecular processes, including the material and energy exchange between the cell and its environment, cell-cell interactions, and signal transduction, etc. However, the research on the membrane proteins still faces a great chanllenge at present due to the fact that most of membrane proteins are of low abundance and high hydrophobicity, which makes their solubilization, extraction and enzymolysis difficult.To address these problems, enrichment of membrane proteins at the level of membranes is usually employed and some additives such as detergents and organic solvents are introduced at high concentrations for improving the solubilization and extraction of them. SDS is the most efficient and classic detergent. Nevertheless, because SDS can reduce the activity of proteolytic enzymes, interfere with the chromatographic separation of the digests and suppress the ionization of the peptides during mass spectrometric analysis, the protein samples need to be cleaned up to remove SDS and other interfering substances prior to the subsequent emzymolysis and other expremental procedures. Conventional sample cleanup strategies mainly including precipitation, dislysis, ion exchange, gel filtration and so on. Although these strategies can eliminate the interfering substances with small molecular weight from the sample, they have a common limitation that often leads to a significant protein loss during the sample cleanup. Additionally, these methods have certain limitations in protein identification efficiency and experimental cost, etc.
In the present study, we have developed gel electrophoresis- and pre-cooled acetone precipitaion-base methods for sample preparation and cleanup to address the limitations in the existing methods, and applied them to the analyses of a membrane proteome.
In the study of gel electrophoresis-based method, we introduced a specially-designed gradient gel electrophoresis (GGE) system that was composed of an agarose layer for sample loading, several polyacrylamide fractionation layers with different concentrations and a polyacrylamide sealing layer with high concentration (40%). The experimental results using a rat liver membrane-enriched preparation showed that GGE system could eliminate moren than 85% of SDS in the loaded sample and gel by electrophoretic driving. Besides, this electrophoresis-based method not only could efficiently cleanup the the SDS-containing sample, but also could simultaneously concentrate the sample and fractionate the sample to decrease its complexity, thereby improving the efficiency of protein identification by a big margin. Compared with tube-gel digestion and three-layer sandwich method, two representative gel-based sample cleanup methods reported in literature, the total numbers of identified membrane proteins by GGE strategy were increased by 213 and 222, respectively, especially the identification efficiency of those with extreme properties (such as very high or low-molecular weight, low abundance or high hydrophobicity) being improved obviously.
In the study of acetone precipitation-based method for sample preparation and cleanup, we optimized the experimental conditions for the removal of SDS and other interfering substances with small molecular weight from protein sample by pre-cooled acetone precipitation, compared different strategies for the re-dissolvation and in-solution enzymolysis of the precipitated proteins, and established a solution-based systematic method for protein sample preparation and cleanup. The method comprehensively utilized the strong ability of SDS to lyse the membranes and solibilize hydrophobic membrane proteins, the high efficiency of pre-cooled acetone precipitation to clean up the sample, and the optimum environment provided by NH4HCO3 solution for efficient trypsinolysis. Experimental results showed that, using the optimized pre-cooled acetone precipitation method, the SDS content in a sample could be reduced to below 0.01%, and at the same time the protein recovery rate was retained to above 90%. The precipitated proteins were demonstrated to be efficiently digested by trypsin in NH4HCO3 solution. Compared to the other strategies reported in literature, the method gave rise to more efficient protein digestion and identification, thereby increasing the numbers of identified total proteins and membrane proteins on average by 26.6% and 25.8%, respectively. Furthermore, the comparative analysis of physicochemical characteristics of the identified proteins also demonstrated that the optimized systematic method had a broad practicability in the identification of various proteins, including highly hydrophobic and/or multiple transmembrane proteins, and had important potential in protein chemistry and proteomics.
在基于凝胶电泳的方法研究中,我们采用了一种特殊设计的梯度凝胶电泳(GGE)系统,包括一个琼脂糖上样层,若干个不同浓度的聚丙烯酰胺凝胶分级分离层和一个高浓度(40%)的聚丙烯酰胺凝胶封闭层。采用富集的大鼠肝脏膜蛋白作为样品的实验结果表明,通过电泳驱动的方式,GGE系统能够去除样品和凝胶中85%以上的SDS。此外,通过这种电泳驱动的方式不仅能够对含有高浓度SDS的蛋白质样品进行有效的净化,而且能同时浓缩样品和对样品进行分级分离而降低样品复杂程度,因而有利于后续的蛋白质酶解和LC-MS/MS分析,大幅度提高蛋白质的鉴定效率。与文献报道的同类样品净化方法管胶法和三明治凝胶电泳法相比,GGE策略鉴定的膜蛋白质总数分别提高了213和222,尤其是那些具有极端性质(如分子量较高或较低、丰度低或疏水性强)的蛋白质的鉴定效果得到明显的提高。
在基于预冷丙酮沉淀的样品制备和净化策略研究中,我们优化了预冷丙酮沉淀法去除蛋白质样品中SDS及其他小分子干扰物质的实验条件,比较了沉淀蛋白质复溶和溶液酶解的不同策略,建立起一种基于溶液的蛋白质样品制备和净化的系统方法。该方法综合利用了SDS对于生物膜的强裂解能力和有效溶解疏水性膜蛋白的能力、丙酮沉淀法高效净化样品的能力以及NH4HCO3溶液提供的适合胰蛋白酶有效作用的适宜环境。实验结果表明,利用优化的预冷丙酮沉淀法能使蛋白质样品中的SDS的含量降低至0.01%以下,并使蛋白质的回收率保持在90%以上；预冷丙酮沉淀的蛋白质在NH4HCO3溶液中能被胰蛋白酶有效酶解。与文献报道的其他方法相比,此方法更有利于蛋白质的酶解和鉴定,它所鉴定到的总蛋白和膜蛋白的数量分别平均提高了26.6%和25.8%。此外,通过对所鉴定到的蛋白质的理化性质进行的比较分析表明,这种优化的系统方法对于各种类型的蛋白质包括高疏水性和多跨膜的蛋白质的鉴定具有普适性,在蛋白质化学和蛋白质组学研究领域具有重要的应用价值。
Membrane proteins play important role in many molecular processes, including the material and energy exchange between the cell and its environment, cell-cell interactions, and signal transduction, etc. However, the research on the membrane proteins still faces a great chanllenge at present due to the fact that most of membrane proteins are of low abundance and high hydrophobicity, which makes their solubilization, extraction and enzymolysis difficult.To address these problems, enrichment of membrane proteins at the level of membranes is usually employed and some additives such as detergents and organic solvents are introduced at high concentrations for improving the solubilization and extraction of them. SDS is the most efficient and classic detergent. Nevertheless, because SDS can reduce the activity of proteolytic enzymes, interfere with the chromatographic separation of the digests and suppress the ionization of the peptides during mass spectrometric analysis, the protein samples need to be cleaned up to remove SDS and other interfering substances prior to the subsequent emzymolysis and other expremental procedures. Conventional sample cleanup strategies mainly including precipitation, dislysis, ion exchange, gel filtration and so on. Although these strategies can eliminate the interfering substances with small molecular weight from the sample, they have a common limitation that often leads to a significant protein loss during the sample cleanup. Additionally, these methods have certain limitations in protein identification efficiency and experimental cost, etc.
In the present study, we have developed gel electrophoresis- and pre-cooled acetone precipitaion-base methods for sample preparation and cleanup to address the limitations in the existing methods, and applied them to the analyses of a membrane proteome.
In the study of gel electrophoresis-based method, we introduced a specially-designed gradient gel electrophoresis (GGE) system that was composed of an agarose layer for sample loading, several polyacrylamide fractionation layers with different concentrations and a polyacrylamide sealing layer with high concentration (40%). The experimental results using a rat liver membrane-enriched preparation showed that GGE system could eliminate moren than 85% of SDS in the loaded sample and gel by electrophoretic driving. Besides, this electrophoresis-based method not only could efficiently cleanup the the SDS-containing sample, but also could simultaneously concentrate the sample and fractionate the sample to decrease its complexity, thereby improving the efficiency of protein identification by a big margin. Compared with tube-gel digestion and three-layer sandwich method, two representative gel-based sample cleanup methods reported in literature, the total numbers of identified membrane proteins by GGE strategy were increased by 213 and 222, respectively, especially the identification efficiency of those with extreme properties (such as very high or low-molecular weight, low abundance or high hydrophobicity) being improved obviously.
In the study of acetone precipitation-based method for sample preparation and cleanup, we optimized the experimental conditions for the removal of SDS and other interfering substances with small molecular weight from protein sample by pre-cooled acetone precipitation, compared different strategies for the re-dissolvation and in-solution enzymolysis of the precipitated proteins, and established a solution-based systematic method for protein sample preparation and cleanup. The method comprehensively utilized the strong ability of SDS to lyse the membranes and solibilize hydrophobic membrane proteins, the high efficiency of pre-cooled acetone precipitation to clean up the sample, and the optimum environment provided by NH4HCO3 solution for efficient trypsinolysis. Experimental results showed that, using the optimized pre-cooled acetone precipitation method, the SDS content in a sample could be reduced to below 0.01%, and at the same time the protein recovery rate was retained to above 90%. The precipitated proteins were demonstrated to be efficiently digested by trypsin in NH4HCO3 solution. Compared to the other strategies reported in literature, the method gave rise to more efficient protein digestion and identification, thereby increasing the numbers of identified total proteins and membrane proteins on average by 26.6% and 25.8%, respectively. Furthermore, the comparative analysis of physicochemical characteristics of the identified proteins also demonstrated that the optimized systematic method had a broad practicability in the identification of various proteins, including highly hydrophobic and/or multiple transmembrane proteins, and had important potential in protein chemistry and proteomics.
引文
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