Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides

摘要

Numerous studies have recently focused on the identification of specific glycan biomarkers; given the important roles that protein linked glycans play, for example, during development and disease progression. The identification of protein glycobiomarkers, which are part of a very complex proteome, has involved the use of fractionation techniques such as lectin affinity chromatography. In this study, the glycoproteomic characterization of pluripotent murine embryonic stem cells (ES) and from ES cells that were differentiated into embroid bodies (EB) was performed using immobilized Concanavalin A (ConA). This procedure allowed the isolation of glycopeptides that express biantennary and hybrid N-linked structures (ConA2 fraction) as well as high mannose glycans (ConA3 fraction), that were abundant in both ES and EB stages. A total of 293 unique N-linked glycopeptide sequences (from 180 glycoproteins) were identified in the combined data sets from ES and EB cells. Of these glycopeptides, a total of 119 sequences were identified exclusively in only one of the lectin bound fractions, (24 in the ES-ConA2, 15 in the ES-ConA3, 16 in the EB-ConA2 and 64 in the EB-ConA3). Results from this study allowed the identification of individual N-glycosylation sites of proteins that express specific glycan types. The absence of some of these lectin bound glycopeptides in a cell stage suggested that they were derived from proteins that were either expressed exclusively on a defined developmental stage, or were expressed in both cell stages but carried the lectin bound oligosaccharides in only one of them. Therefore, these lectin bound glycopeptides can be considered as stage specific glycobiomarkers.