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Exploring the oviductal fluid proteome by a lectin‐based affinity approach
Authors:Hans Yu  Judith Reiser  Urban Besenfelder  Ebrahim Razzazi‐Fazeli  Jonas Bergquist  Gottfried Brem  Konstantin Artemenko  Corina Mayrhofer
Institution:1. Institute of Biotechnology in Animal Production, Interuniversity Department for Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria;2. Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria;3. Institute of Molecular Animal Breeding and Biotechnology, Ludwig‐Maximilian University, Munich, Germany;4. Reproduction Centre Wieselburg, University of Veterinary Medicine Vienna, Vienna, Austria;5. VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria;6. Analytical Chemistry, Department of Chemistry—Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden
Abstract: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.
Keywords:Concanavalin A  Glycoproteins  Oviductal fluid  Technology  Wheat germ agglutinin
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