Large-scale Identification of N-Glycosylated Proteins of Mouse Tissues and Construction of a Glycoprotein Database, GlycoProtDB

Abstract

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H₂¹⁸O, IGOT) and finally LC-MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology.

Tissue-specific sequence and structural environments of lysine acetylation sites

“…Comprehensive studies of protein glycosylation (Kaji et al, 2012), phosphorylation (Lundby et al, 2012a) and acetylation (Lundby et al, 2012b) revealed thousands of differentially modified sites, opening up the possibility that PTM sites may possess substantially different sequence and spatial properties across tissues, depending on which particular enzyme catalyzes a particular modification event. The existence of compartment-specific sequence signatures for phosphorylation (Chen et al, 2014;van Wijk et al, 2014) and lysine acetylation (Choudhary et al, 2009;Kim et al, 2006;Lundby et al, 2012b;Shao et al, 2012) has already been firmly established.…”

Glycoprotein Disease Markers and Single Protein-omics

“…O-GalNAc glycoproteins are estimated to comprise 10% of proteins in the human proteome and 50% among those going through the secretory pathway (14). Two recent papers find, in various human tissues, ϳ2,500 N-glycosylated proteins each, but the sets have only partial overlap (11,15). It is likely that additional glycosylated proteins will be identified as detection methods improve.…”

Using Databases and Web Resources for Glycomics Research

“…GlycoProtDB (GlycoProtein Database) (39) GlycoProtDB is a database of N-glycoproteins that have been experimentally identified in C. elegans N2 and mouse tissues (strain C52BL/6J, male). GMDB (Glycan Mass Spectral DataBase)…”