Deutzmann Rainer
Institute for Biochemistry, University of Regensburg, Germany.
Methods Mol Med. 2004;94:269-97. doi: 10.1385/1-59259-679-7:269.
The primary structure of proteins is nowadays determined by DNA sequencing, and a variety of genomes are already known. Nevertheless, protein sequencing/identification is still indispensable to analyze the proteins expressed in a cell, to identify specific proteins, and to determine posttranslational modifications. Proteins of interest are typically available in low microgram amounts or even less. The separation method of choice is gel electrophoresis, followed by blotting to PVDF membrane for N-terminal sequencing or by in-gel digestion to generate peptides that can be separated by HPLC. Structural analysis can be done by Edman degradation or mass spectrometry (MS). Edman degradation is the older method based on successive removal of N-terminal amino acids by chemical methods. Sequencing of a peptide requires many hours, the sensitivity is in the range of 2-5 pmol of a purified peptide. Nevertheless, Edman degradation is still the workhorse in the lab for routine work such as identification of blotted proteins. It is also the method of choice for sequencing unknown proteins/ peptides and modified peptides. MS has routinely been used with peptides in the range of 100 fmol or even less. In contrast to Edman degradation, complex mixtures such as tryptic digests can be analyzed, making HPLC separation of peptides unnecessary. MS is a very fast method that can be automated. It is the method of choice for sensitive analysis and large-scale applications (proteomics). Two different ionization methods are commonly used to generate peptide/protein ions for MS analysis. These are MALDI (matrix assisted laser desorption and ionization) and ESI (electrospray ionization). They can be combined with a variety of mass analyzers (TOF, quadrupole, ion trap). Proteins are either identified by searching databases with the masses of proteolytic peptides (peptide mass fingerprinting) or using fragmentation data (raw MS/MS spectra or sequence tags). This approach requires that the protein is known and listed in the database. De novo sequencing by MS of peptides is possible, but very time consuming and not a routine application, in contrast to Edman degradation. The aim of this chapter is to introduce to basic theory, practical applications and limitations of the various methods, to enable the non-expert scientist to decide which method is best suited for his project and which kind of sample preparation is necessary.
如今，蛋白质的一级结构是通过DNA测序来确定的，并且已经知晓了多种基因组。然而，蛋白质测序/鉴定对于分析细胞中表达的蛋白质、鉴定特定蛋白质以及确定翻译后修饰而言仍然不可或缺。目标蛋白质的量通常只有低微克级甚至更少。首选的分离方法是凝胶电泳，随后将其印迹到PVDF膜上进行N端测序，或者进行胶内消化以生成可通过HPLC分离的肽段。结构分析可通过埃德曼降解法或质谱法（MS）来完成。埃德曼降解法是一种较老的方法，基于通过化学方法连续去除N端氨基酸。对一个肽段进行测序需要数小时，灵敏度在纯化肽段2 - 5皮摩尔的范围内。尽管如此，埃德曼降解法在实验室中仍是常规工作（如鉴定印迹蛋白质）的主要方法。它也是对未知蛋白质/肽段和修饰肽段进行测序的首选方法。质谱法通常用于分析100飞摩尔甚至更少的肽段。与埃德曼降解法不同，它可以分析诸如胰蛋白酶消化产物等复杂混合物，无需对肽段进行HPLC分离。质谱法是一种非常快速且可自动化的方法。它是灵敏分析和大规模应用（蛋白质组学）的首选方法。通常使用两种不同的电离方法来产生用于质谱分析的肽/蛋白质离子。它们是基质辅助激光解吸电离（MALDI）和电喷雾电离（ESI）。它们可以与多种质量分析器（飞行时间质谱仪、四极杆质谱仪、离子阱质谱仪）联用。蛋白质要么通过用蛋白水解肽段的质量搜索数据库（肽质量指纹图谱）来鉴定，要么使用片段化数据（原始的MS/MS谱图或序列标签）来鉴定。这种方法要求该蛋白质是已知的且列在数据库中。通过质谱对肽段进行从头测序是可行的，但与埃德曼降解法相比，非常耗时且并非常规应用。本章的目的是介绍各种方法的基本理论、实际应用和局限性，以使非专业科学家能够决定哪种方法最适合其项目以及需要哪种样品制备方法。
