Targeted glycoprotein enrichment and identification in stromal cell secretomes using azido sugar metabolic labeling

Abstract

Purpose: Effectively identifying the proteins present in the cellular secretome is complicated due to the presence of cellular protein leakage and serum protein supplements in culture media. A metabolic labeling and click chemistry capture method is described that facilitates the detection of lower abundance glycoproteins in the secretome, even in the presence of serum.

Experimental design: Two stromal cell lines were incubated with tetraacetylated sugar-azide analogs for 48 h in serum-free and low-serum conditions. Sugar-azide labeled glycoproteins were covalently linked to alkyne-beads, followed by on-bead trypsin digestion and MS/MS. The resulting glycoproteins were compared between media conditions, cell lines, and azide-sugar labels.

Results: Alkyne-bead capture of sugar-azide modified glycoproteins in stromal cell culture media significantly improved the detection of lower abundance secreted glycoproteins compared to standard serum-free secretome preparations. Over 100 secreted glycoproteins were detected in each stromal cell line and significantly enriched relative to a standard secretome preparation.

Conclusion and clinical relevance: Sugar-azide metabolic labeling is an effective way to enrich for secreted glycoproteins present in cell line secretomes, even in culture media supplemented with serum. The method has utility for identifying secreted stromal proteins associated with cancer progression and the epithelial-to-mesenchymal transition.

Figure 1

Secretome analysis using sugarazide metabolic labels. (A) Summary of the experimental workflow. (B) Conditioned media from WPMY1 cells grown 48 h with and without tetraacetylated N-azidoacetyl-d-mannosamine (ManNAcAz) were collected and reacted with biotin-alkyne. A portion of the ManNAcAz proteins were also treated with protein N-glycanase. Proteins from each condition were loaded in replicate and separated on the same SDS-gel. One half of the gel was silver stained (lanes 1–3), and the other half was blotted to nitrocellulose for detection with an IR800 streptavidin antibody (lanes 4–6). The samples treated with protein N-glycanase are in lanes 3 and 6. (C) Venn diagram comparison of the secretome proteins detected in the serum-free WPMY1 cells with no ManNAcAz, serum-free WPMY1 media from cells treated with ManNAcAz, and 1% serum WPMY1 media from cells treated with ManNAcAz.

Figure 2

Venn diagram comparisons of WPMY1and HS5 stromal cell secretomes following ManNAcAz and GalNAcAz treatments with expressed prostatic secretion proteomes. The cumulative secretome glycoproteins from both cell lines and both sugarazide labels listed in Supporting Information Table 1 were combined for comparison of shared expression with the proteomes from clinical prostatic fluids, expressed prostatic secretions (EPS) in urine [16], and direct EPS obtained at the time of prostatectomy [17]. Keratins were excluded from the comparison.