Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors
Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors
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DOI：
10.5772/30198
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发表时间：
2012-02
期刊：
影响因子：
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通讯作者：
H. Yamaguchi;M. Miyazaki;H. Maeda
中科院分区：
文献类型：
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作者：
H. Yamaguchi;M. Miyazaki;H. Maeda
蛋白质组学是对蛋白质，特别是其结构和功能的大规模研究。其中最重要的一点是发展高效、快速的方法来鉴定靶蛋白。肽质量图和基于串联质谱（MS/MS）的肽序列分析是当前蛋白质组学研究中蛋白质鉴定的关键方法。蛋白质通常被消化成肽，随后通过MS进行分析。因此，通过序列特异性蛋白酶的蛋白水解是与MS整合的蛋白质组学分析中正测序的关键步骤（Aebersold & Mann，2003）。通过蛋白酶进行溶液中消化的常规方法是耗时的程序（在37 ° C下过夜）。底物/蛋白酶比率必须保持较高（通常> 50），以防止蛋白酶及其自动消化产物对样品的过度污染。但这会导致消化相对缓慢。此外，通过MS分析获得可靠的肽图谱和有意义的序列数据不仅需要分离消化的肽，还需要严格限定的蛋白水解条件（Domon & Aebersold，2006; Witze等人，2007年）。此外，包括修饰肽如磷酸肽的消化片段的电离效率取决于肽大小或肽序列，其与通过MS分析的靶蛋白的序列覆盖度直接相关。此外，从溶液中消化的肽回收高度依赖于靶蛋白的结构性质，因为具有刚性结构（例如通过二硫键）的蛋白倾向于抵抗完全消化。事实上，用于蛋白水解的样品的典型制备包括变性、二硫键还原和烷基化程序，导致构象稳定性降低。很明显，不充分的序列覆盖可能会损害蛋白质组表征的准确性。因此，重要的是开发新的消化方法以实现用于基于MS的肽图谱的高效蛋白水解（Park & Russell，2000; Slysz等人，2006年）。微反应器是用于在微通道中处理小体积样品以进行化学或酶促反应的合适的反应系统。酶固定
Proteomics is the large-scale study of proteins, particularly their stuructures and functions. One of the most important point is to develop efficient and rapid approachehes to identify the target proteins. Peptide mass mapping and tandem mass spectrometry (MS/MS)-based peptide sequencing are key methods in current protein identification for proteomic studies. Proteins are usually digested into peptides that are subsequently analyzed by MS. Therefore, proteolysis by sequence-specific proteases is the key step for positive sequencing in proteomic analysis integrated with MS (Aebersold & Mann, 2003). The conventional method of in-solution digestion by proteases is a time-consuming procedure (overnight at 37 C). The substrate/protease ratio must be kept high (generally > 50) in order to prevent excessive sample contamination by the protease and its auto-digested products. But this leads to a relatively slow digestion. In addition, obtaining reliable peptide maps and meaningful sequence data by MS analysis requires not only the separation of the digested peptides but also strictly defined proteolysis conditions (Domon & Aebersold, 2006; Witze et al., 2007). In addition, the ionization efficiency of the digested fragments including a modified peptide such as a phosphopeptide is dependent on peptide-size or peptidesequence, which directly correlates with sequence coverage of the target protein by MS analysis. Furthermore, peptide recovery from in-solution digestion is highly dependent on the structural properties of the target proteins because proteins with rigid structures, e.g. by disulfide bonds tend to be resistant to complete digestion. In fact, the typical preparation of a sample for proteolysis includes denaturation, reduction of disulfide bonds, and alkylation procedures lead to a decrease the conformational stability. It is obvious that insufficient sequence coverage could compromise the accuracy of proteome characterization. Therefore, it is important to develop novel digestion methods to achieve a highly efficient proteolysis for MS-based peptide mapping (Park & Russell, 2000; Slysz et al., 2006). A microreactor is a suitable reaction system for handling small-volume samples in a microchannel to perform chemical or enzymatic reactions. Enzyme-immobilized