title: Simultaneous characterization of glyco- and phosphoproteomes of mouse brain membrane proteome with electrostatic repulsion hydrophilic interaction chromatography.
abstract: Characterization of glyco- and phosphoproteins as well as their modification sites poses many challenges, the greatest being loss of their signals during mass spectrometric detection due to substoichiometric amounts and the ion suppression effect caused by peptides of high abundance. We report here an optimized protocol using electrostatic repulsion hydrophilic interaction chromatography for the simultaneous enrichment of glyco- and phosphopeptides from mouse brain membrane protein digest. With this protocol, we successfully identified 544 unique glycoproteins and 922 glycosylation sites, which were significantly higher than those from the commonly used hydrazide chemistry method (192 glycoproteins and 345 glycosylation sites). Moreover, a total of 383 phosphoproteins and 915 phosphorylation sites were recovered from the sample, suggesting that this protocol has the potential to enrich both glycopeptides and phosphopeptides simultaneously. Of the total 995 glycosylation sites identified from both methods, 96% were considered new as they were either annotated as putative or not documented in the newly released Swiss-Prot database. Thus, this study could be of significant value in complementing the current glycoprotein database and provides a unique opportunity to study the complex interaction of two different post-translational modifications in health and disease without being affected by interexperimental variations.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Huoming
authors.initials: H
authors.lastname: Zhang
authors.forename: Tiannan
authors.initials: T
authors.lastname: Guo
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Arnab
authors.initials: A
authors.lastname: Datta
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Park
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Sai Kiang
authors.initials: SK
authors.lastname: Lim
authors.forename: James P
authors.initials: JP
authors.lastname: Tam
authors.forename: Siu Kwan
authors.initials: SK
authors.lastname: Sze
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002845:Chromatography / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008810:Mice, Inbred C57BL; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis*; D055672:Static Electricity; D053719:Tandem Mass Spectrometry
chemical_list: D006023:Glycoproteins; D010750:Phosphoproteins; D020543:Proteome