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

Abstract

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.

Figure 1. MALDI-TOF spectrum of permethylated N-linked oligosaccharides obtained from glycoprotein extracts of ES(A) and EB(B) cells

Glycoproteins were obtained from ES and EB cell pellets (ca 1×10⁷ cells) after delipidation and N-linked oligosaccharides were released with PNGase F, permethylated and analyzed by MALDI-TOF/MS GlcNAc, Gal, Man, NeuNAc, Fuc.

Figure 2. Venn diagram with the distribution of the glycopeptide sequences identified in the ConA bound fractions from ES and EB cells

A total of three cell pellets from each ES or EB cell stage were subjected to lectin affinity fractionation and glycoproteomic analysis and a total of 12 samples were obtained, which represented the following lectin bound fractions (3 samples for each fraction): ES-ConA2, ES ConA3, EB ConA2, and EB ConA3. ConA2 represents the glycopeptides fraction eluted with 10 mM α-methyl-glucopyranoside (which contains biantennary and hybrid N-glycans) and ConA3 the glycopeptides eluted with 100 mM α-methyl-mannopyranoside which contain high mannose N-glycans.

Figure 3. Distribution of the proteins identified in the present study according to cell stage or Con A bound fraction

The proteins that were identified in the lectin bound fractions were classified in three groups according the cell stage in which they were present (ES only, EB only, or ES and EB or the lectin bound fraction (ConA2 only, ConA3 only, or ConA2 and ConA3). Arrows denote the number of proteins identified in the indicated fractions.

Figure 4. Predicted subcellular localization of the proteins identified in the ConA bound fractions from ES or EB stages

(A) subcellular distribution of all 180 ConA bound proteins identified in ES or EB stages. (B) subcellular distribution of the 116 proteins that were shared by both ES and EB stages. (C) subcellular distribution of the 17 proteins identified only in the ES stage. (D) subcellular distribution of the 47 proteins identified only in the EB stage