Naslov
Expanding the Human GalNAc-type O-glycoproteome through SimpleCell Technology

Autori
Crnko, Sandra

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, diplomski rad, diplomski

Fakultet
Odjel za biotehnologiju

Mjesto
Rijeka

Datum
22.09

Godina
2015

Stranica
54

Mentor
Peter-Katalinić, Jasna ; Kraljević Pavelić, Sandra

Ključne riječi
O-Glycoproteomics; GalNAc; SimpleCells; Mass Spectrometry

Sažetak
In O-glycoproteomics the analysis of O-glycan structures is well established while localization of O-glycans on proteins offers considerable challenges. A significant breakthrough in the discovery of localization of O-glycosites has been recently made by developing a strategy based on mass spectrometry applied to genetically engineered cells, so-called "SimpleCells" (SC). In SC virtually all GalNAc-type (mucin-type) O-glycans are truncated to GalNAc (Tn) or NeuAc-GalNAc (SiaTn), which then carry the site information through a straightforward shotgun high resolution mass spectrometry facilitated by ETD-MS2 (Orbitrap MS, Thermo) fragmentation preserving intact the glycan-peptide linkage. In screening of 12xSC cell lines around 600 O-GalNAc glycoproteins and nearly 2000 O-glycosites have been reported. This discovery was made predominantly using trypsin as a proteolytic enzyme leaving a large area for potential O-glycosylation unexplored. To expand structural information on human O-glycoproteome 12x human SC cell lines were repeatedly screened by the same MS approach upon extended proteolysis with additional proteolytic enzymes orthogonal to trypsin, such as chymotrypsin. Taking advantage of the high speed of data processing by Sequest HT algorithm (Thermo) new data were sequentially processed with the full and semi-specific enzymatic digestion. In addition, previously published data were reprocessed with semi-specific digestion as well. As a result the total number of O-glycoproteins in a data base (DB) has been increased to a nearly to 3, 000 and the number of glycosites to 10, 000.

Izvorni jezik
Engleski

Znanstvena područja
Biotehnologija

POVEZANOST RADA