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 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.
Institute of Biotechnology in Animal Production, Interuniversity Department for Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.|Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria.
Hans
H
Yu
Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany.
Judith
J
Reiser
Institute of Biotechnology in Animal Production, Interuniversity Department for Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.|Reproduction Centre Wieselburg, University of Veterinary Medicine Vienna, Vienna, Austria.
Urban
U
Besenfelder
VetCore Facility for Research, University of Veterinary Medicine Vienna, Vienna, Austria.
Ebrahim
E
Razzazi-Fazeli
Analytical Chemistry, Department of Chemistry-Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Jonas
J
Bergquist
Institute of Biotechnology in Animal Production, Interuniversity Department for Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.|Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria.
Gottfried
G
Brem
Analytical Chemistry, Department of Chemistry-Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Konstantin
K
Artemenko
Institute of Biotechnology in Animal Production, Interuniversity Department for Agrobiotechnology (IFA Tulln), University of Natural Resources and Applied Life Sciences Vienna, Tulln, Austria.|Institute of Animal Breeding and Genetics, Department for Biomedical Sciences, University of Veterinary Medicine Vienna, Vienna, Austria.
Corina
C
Mayrhofer
D000818:Animals; D001826:Body Fluids / Q000737:chemistry*; D003208:Concanavalin A / Q000378:metabolism; D005187:Fallopian Tubes / Q000737:chemistry; D005260:Female; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D008297:Male; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014909:Wheat Germ Agglutinins / Q000378:metabolism; D057188:Workflow
D006023:Glycoproteins; D020543:Proteome; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A
Concanavalin A; Glycoproteins; Oviductal fluid; Technology; Wheat germ agglutinin