Mass Spectrometry Molecular Weight Change of Ubiquitination Sites
Ubiquitination is a crucial post-translational modification that affects protein stability, activity, and function. This process involves the attachment of a 76-amino-acid ubiquitin molecule to target proteins through a cascade mediated by a trio of enzymes: E1, E2, and E3. Identifying ubiquitination sites is essential for understanding the molecular mechanisms involved. Mass spectrometry is typically employed for this purpose. During ubiquitination, the C-terminal glycine of ubiquitin forms an isopeptide bond with the lysine residue of the target protein, resulting in an increase of the protein's molecular weight by approximately 8.5 kDa, which can be detected by mass spectrometry.
In practice, due to enzymatic cleavage, ubiquitin is often extensively trimmed after tryptic digestion of ubiquitinated proteins. This leaves a single glycine residue on the lysine of the target protein, producing a molecular weight increase of approximately 114 Da, detectable with high-resolution mass spectrometry.
By comparing mass spectra from ubiquitinated and non-ubiquitinated proteins, researchers can identify ubiquitination sites. This analysis requires specialized software, such as MaxQuant, which is widely used for processing protein mass spectrometry data and can automatically detect mass changes indicative of ubiquitination. The comprehensive identification of ubiquitination sites is a detailed process, heavily reliant on advanced mass spectrometry and specialized data processing tools.
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