کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1225215 1494742 2016 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment
چکیده انگلیسی


• We developed a novel method for identifying GPI-APs using TiO2-based affinity purification.
• A total of 73 ω-sites derived from 49 GPI-APs were identified.
• GPI-AP protein species with different ω-sites were observed.

Glycosylphosphatidylinositol anchored proteins (GPI-APs) in the outer leaflet of the membrane microdomains, commonly referred to as lipid rafts, play important roles in many biological processes such as signal transduction, cell adhesion, protein trafficking, and antigen presentation. From a topological viewpoint, elucidating the presence and localization of GPI-anchor modification sites (ω-sites) is important for the study of the biophysical properties and anchoring mechanisms of these proteins. However, very few reports have actually identified ω-sites of GPI-APs. To enable large-scale site-specific analysis of GPI anchoring, we developed a method for identification of ω-sites by mass spectrometry by combining titanium dioxide-based affinity purification and hydrogen fluoride treatment. This method was able to identify ~ 3-fold more GPI-APs than our previous method: the new technique identified a total of 73 ω-sites derived from 49 GPI-APs. In 13 of the 49 GPI-APs identified, the GPI-anchor attached to multiple amino acids in the C-terminal site, yielding a variety of different protein species. This method allows us to simultaneously identify many GPI-AP protein species with different ω-sites. We also demonstrated the C-terminal GPI anchor attachment signal peptide, based on information about the GPI anchor binding sites of 49 GPI-APs. Thus, our results provide evidence for new insight into the GPI-anchored proteome and the role of GPI anchoring.Biological significanceGPI-anchored proteins (GPI-APs) are localized to the outer leaflet of the plasma membranes. Because the GPI anchor is a complex structure, the identification of GPI-anchored peptides by mass spectrometry has always been considered difficult. To improve the feasibility of large-scale site-specific analysis of GPI anchoring, we developed a method for identification of GPI-anchored peptides by combining titanium dioxide–based affinity purification with hydrogen fluoride treatment. Using this novel technique, we identified a total of 73 ω-sites derived from 49 GPI-APs. These data may help us to develop a comprehensive understanding of the GPI-anchored proteome and the role of GPI anchoring. Moreover, this method could be used to discover GPI-APs as candidate biomarkers.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Proteomics - Volume 139, 29 April 2016, Pages 77–83
نویسندگان
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