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 and Wheat Germ Agglutinin as ligands to fractionate and enrich glycoproteins from oviductal fluid (OF), which is a source of molecules involved in fertilization. First, the selectivity was corroborated by a gel-based approach using glycoprotein staining and enzymatic deglycosylation. Nanoliquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS) further allowed the reliable identification of 134 nonbound 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 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.
Animals Body Fluids/chemistry* Concanavalin A/metabolism Fallopian Tubes/chemistry Female Glycoproteins/analysis* Glycoproteins/metabolism Male Proteome/analysis* Proteome/metabolism Rabbits Reproducibility of Results Spectrometry, Mass, Electrospray Ionization/methods Wheat Germ Agglutinins/metabolism Workflow