 
Phosphoprotein Characterization
Sites of protein phosphorylation can be identified by analyzing [32P]-labeled tryptic peptides isolated from [32P]-proteins, using MALDI-MS mapping techniques, and/or FT-ICR MS analysis. With non-radiolabeled phosphoproteins, we have most often used affinity purification of phosphopeptides (TiO2, IMAC) and MALDI-MS/MS analysis of the putative phosphopeptides. This method provides you with detection of phosphopeptides, the peptide amino acid sequence and identification of the phosphorylation site.
Other types of post-translational modifications with small molecular weight change often can be identified using similar approaches of MALDI-MS mapping and FT-ICR analysis.
Glycoprotein Characterization
It’s a tough task to study the glycan chains compared with the other modifications of protein because of their complexity in structure, their hard-to-ionization character and lack of analysis databases. Modification to the glycan sides (e.g., permethylation) can improve their detection by MS, but this treatment needs a large amount of glycoproteins (at least 10ug) and its mass spectrum analysis completely depends on time-consuming manual interpretation. In our lab, we have developed a novel method to detect the glycosylation sites and structures of glycoproteins with sialic acids. This method has a much better sensitivity (ten times) greater than the traditional methods. We can complete the glycosylation analysis of those protein spots with sialic acids separated by 2-DE at an amount of less than 1ug/spot. In the past we successfully identified the glycan chains and glycosylation sites of human transfferin, haptoglobin and antitrypsin from 2-DE gels. Our lab is the first one in the world that can simultaneously analyze protein glycosylation sites and the structures of glycoproteins with sialic acids at 2-DE level. Additionally, we offer a traditional method by using specific glycosidases to digest saccharides from glycan chains before MS analysis.
From LUMC
|
Default Home
