Exploring the oviductal fluid proteome by a lectin-based affinity approach

The analysis of glycoproteins in body fluids represents a central task in the study of vital processes. Herein we assessed the combined use of Concanavalin A (ConA) and Wheat Germ Agglutinin (WGA) as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF) that is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nano-liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further on allowed the reliable identification of 134 non-bound as well as 130 lectin bound OF proteins. Enrichment analysis revealed that 77% of the annotated proteins in the lectin bound fraction were known glycoproteins (p-value (FDR) = 1.45E-31). The low variance of the number of peptide spectrum matches (PSMs) for each protein within replicates indicated a consistent reproducibility of the whole workflow (median CV 17.3 % for technical replicates and 20.7% for biological replicates). Taken together, this study highlights the applicability of a lectin-based workflow for the comprehensive analysis of OF proteins and gives for the first time an insight into the broad glycoprotein content of OF. This article is protected by copyright. All rights reserved