GeLC-MS/MS analysis of hen egg vitelline membrane from freshly-laid eggs and eggs stored for 28 days at 4°C and 20°C

ABSTRACT: The hen egg vitelline membrane is a protein layer separating the yolk content from the egg white. The integrity of egg vitelline membrane is important for both the shell egg and the egg product industries. However, its physical properties are altered during prolonged storage, depending on storage temperature. The aim of this work was to identify proteomic changes of the hen egg vitelline membrane during 28-day storage at +4°C and +20°C. Vitelline membrane samples were collected from freshly-laid eggs (n=24) and 28-day-old eggs stored at 4°C (n=24) and 20°C (n=24). They were freeze-dried and solubilized using a buffer containing SDS and beta-mercaptoethanol. For proteomic analyses, equal protein amounts of pooled samples (n=4 individual samples per pool, n=6 pools per condition) were loaded on a 10% SDS-polyacrylamide separating gel. Proteins were included and concentrated in the gel after a brief migration, and stained with Coomassie Blue. Each protein band (containing the whole protein sample) was cut into small pieces and processed for mass spectrometry analysis. Following in gel digestion with trypsin, peptides were extracted and analyzed by nanoflow liquid chromatography tandem mass spectrometry (Nano LC-MS/MS) in technical triplicate. Experiments were performed on a dual linear ion trap Fourier Transform Mass Spectrometer LTQ Orbitrap Velos Pro (Thermo Fisher Scientific) coupled to an Ultimate 3000 RSLC Ultra High Pressure Liquid Chromatographer (Thermo Fisher Scientific). Data were acquired using Xcalibur version 3.0.63 software (Thermo Fisher Scientific). MS/MS ion searches were performed using Mascot search engine (Matrix Science) via Proteome Discoverer software (ThermoFisher Scientific) against NCBIprot_gallus gallus database. Peptide and protein identifications were validated using the Peptide Prophet and Protein Prophet algorithms with a probability of 95% for proteins and 99% for peptides. Protein identifications were accepted if they contained at least two identified peptides. The search parameters included trypsin as a protease with two allowed missed cleavages and carbamidomethylcysteine, methionine oxidation and acetylation of N-term protein as variable modifications. The tolerance of the ions was set to 5 ppm for parent and 0.8 Da for fragment ion matches. After applying a protein identification filter requiring a minimum of two unique peptides, a 99% peptide identification probability, and a 95% protein identification probability, the data were normalized and quantified using the eXtracted Ion Chromatogram (XIC) method in Scaffold Quant (Proteome Software).

INSTRUMENT(S):

ORGANISM(S): Gallus Gallus (chicken)

TISSUE(S): Egg, Acellular Membrane

SUBMITTER: Valerie LABAS  

LAB HEAD: GUYOT Nicolas

PROVIDER: PXD066675 | Pride | 2026-03-16

REPOSITORIES: Pride