Northern elephant seal blubber proteome (repeated ACTH challenge)

Juvenile northern elephant seals received once-daily injections of synthetic adrenocorticotropic hormone (ACTH) for four consecutive days. Blubber samples were collected immediately prior to and four hours after the first and fourth ACTH administrations. Proteins were isolated from the inner, metabolically active half (closest to muscle) of each blubber sample from two study animals for which transcriptomes have been published. Blubber was homogenized in Qiazol and proteins were precipitated from the organic phase after phase extraction with chloroform. Denatured, trypsinized, and alkylated peptides were labeled with iTRAQ 8plex and fractionated off-line using a high pH reverse-phase peptide fractionation kit. Fractions were pooled in pairs and injected onto a C18 LC column. Mass spectrometry analysis was performed on a Thermo Orbitrap Fusion Tribrid mass spectrometer in DDA mode with HCD collision. MS/MS data analysis (SEQUEST search against SwissProt UniProt database) and iTRAQ-labeled peptide quantification at the MS2 level were conducted using Proteome Discoverer v2.2.