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Post-translational modification of the polypeptide and protein signal in mass spectrometry sensitization study

Author: ChengZhiHui
Tutor: CaoShengLi；LiYanMei
School: Capital Normal University
Course: Physical and chemical
Keywords: Phosphorylated proteins. β-elimination / Michael- addition MALDI-TOF MS
CLC: O629.73
Type: Master's thesis
Year: 2009
Downloads: 18
Quote: 0
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Abstract

The post-translational modification of the protein is a regulator of protein localization, function, the normal physiological mechanism of the flip. A large number of post-translational modifications have been reported, in which protein phosphorylation is the most common, the most important post-translational modification way. Protein phosphorylation and dephosphorylation regulate almost the entire process of life activities, including cell proliferation, development, apoptosis, and differentiation, as well as signal transduction, etc.. Order to be able to better study these physiological processes at the molecular level, to know the state of protein phosphorylation is essential, so the development of more selective and more sensitive determination of protein phosphorylation sites is very important. Mass spectrometry because of its high sensitivity, high accuracy, and easy to operate and does not require radioactive labeling advantages become protein structure analysis method. However, due to the following reasons, the extensive application of a conventional phosphorylation of protein characterization MS persists constraints. One is suppressed due to the negative charge of the phosphate group with the ionization efficiency of the peptides in mass spectrometry in the positive ion mode of the mass spectrometry of the characteristic peaks of the signal is very low and difficult to detect. In addition, in protein digests mixing, with respect to non-phosphorylated polypeptide, polypeptide phosphorylation chemical content is very low, Mass Spectrometry phosphopeptides signal easily be the MS signal coverage of the non-phosphorylated peptide. Phosphorylated serine, threonine, under alkaline conditions can occur β-elimination, and further Michael addition reaction, the introduction efficiency of the ionized or no effect can be enhanced to a group of the MS signal is the current research in this area hot spots. Introduced some of the nucleophilic addition of a mercapto group and an amino group-containing reagent with these molecules via β-elimination / Michael Addition substituted with the negatively charged phosphate groups significantly enhanced, so that the ionization efficiency of the peptides in mass spectrometry, mass spectrometry Fig. in signal may also be significantly enhanced. And sequentially after addition of these molecules and polypeptides of the sensitizing effect of contrast, eliminating the reaction may be under the action of barium hydroxide completely, and the effect of addition depends on the addition reagent and polypeptide sequences. The experimental results showed that the ethyl mercaptan with respect to to eliminate after addition of the results of various phosphorylated peptide phosphorylated peptides in mass spectrometry have been a significant sensitization. Also with these molecules glycosylated polypeptide eliminate the addition reaction, after addition of the polypeptide in the mass spectrum of the signal is also significantly enhanced. In summary, through the development of protein chemistry modification techniques, using mainly \phosphorylated peptide signal re-modification of the method can be detected easily, is no longer non-phosphorylated peptide the MS signal coverage, which will help phosphorylation sites identified by mass spectrometry.

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