Dynamism analysis of posttranslational modifications by mass spectrometry.

质谱法是一种被广泛接受和可靠的蛋白质表征方法。该方法具有高通量、高精度、高灵敏度的测量优势，非常适合于鉴定多种蛋白质，如通过2D-PAGE和LC分离的蛋白质，以及分析在各种生物事件中起重要作用的翻译后修饰。利用积累的蛋白质/DNA序列数据库的优势，前者已经成为在细胞中表达的肽或蛋白质以及从体液或组织中分离的肽或蛋白质的总体分析的常规任务。后者，特别是对蛋白质中未知或多重修饰的分析，仍然是一项具有挑战性的任务，只能通过高精度质量测量和串联质谱等尖端的质谱技术来实现。为了建立高灵敏度的蛋白质修饰结构分析方法，我们进行了更多的质谱（MS）实验；1)利用电喷雾电离（ESI）或基质辅助激光解吸电离（MALDI）质谱法仔细研究了各种修饰的片段特征。在测量核组蛋白修饰肽的过程中，观察到一个新的片段，它产生的离子与传统的前体离子或序列离子相隔59 Da，这将有助于探测序列中修饰的氨基酸。2)研制了在ESI-MS或MS/MS上进行精确质量测量的装置，该装置由两个纳米esi探针组成，可以对分析物和标准样品进行有效的喷雾，并由两个高压电源供电。因此，在肽（1000 Da）的测量上达到了5ppm以内的精度，这对于在MS/MS中阐明未知修饰或片段离子特别有用。少

Mass spectrometry represents a well-accepted and reliable method for characterization of proteins. The method has great advantages in terms of high throughput, high accuracy, and high sensitivity in measurements, which is well suited for the identification of a wide variety of proteins, such as those separated by 2D-PAGE and LC, and for the analysis of posttranslational modifications that play important roles in various biological events. Taking advantages of accumulating protein/DNA sequence databases, the former has been a routine task for overall profiling of peptides or proteins expressed in a cell and those isolated from body fluid or tissue. The latter, especially, the analysis of unknown or multiple modifications in a protein, is still a challenging task and could only be achieved by the cutting-edge MS techniques such as high-accuracy mass measurement and tandem mass spectrometry.In order to establish the high-sensitivity structural analysis of protein modifications, the follow … More ing experiments based on mass spectrometry-(MS) have been executed; 1) Fragmentations characteristic for various kinds of modifications were examined carefully using electrospray-ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) MS. During the measurements of the modified peptides derived from nuclear histones, a novel fragmentation, which was observed to produce ions separated by 59 Da from the conventional precursor ion or sequence ions, would be useful for probing this modified amino acid in the sequence. 2) The device for accurate mass measurement, which would be powerful for structural elucidation of modifications, on ESI-MS or MS/MS was developed, which consists of two nano-ESI probes that allow for the efficient spray of analyte and standard samples, which are operated by two high-voltage electric power supplies. As a result, the accuracy within 5 ppm has been achieved on the measurements of peptides ( 1000 Da), which will be particularly useful for elucidating unknown modifications or fragment ions in MS/MS. Less