| Research Abstract |
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
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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
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