Proteomic Analysis of Human Ovarian Cortex and Medulla Secretome Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer

The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.

Ovaries from human donors were cut into 3-5 mm sections. These sections were further processed into 500 μm slices containing cortex and medulla. The slices were then processed into pieces (1 mm x 1 mm x 500 µm), and cortex and medulla pieces were cultured separately as explants in static cultures.

Explants were cultured and treated with either DMSO vehicle control or 0.1 µg/mL doxorubicin to induce

senescence. After 10 days, cortex and medulla explants were thoroughly washed with serum-free basal media and transferred to a clean plate with pre-equilibrated serum-free basal media and inserts. The conditioned media were collected after 24 hours for secretome proteomics profiling.

The concentrated conditioned media was subjected to tryptic digestion using S-trap Spin columns. The reconstituted peptide elution was desalted with C18 hydrophilic-lipophilic balance (HLB) cartridges. The final reconstituted peptides were diluted with 2% ACN and 0.1% FA. Proteolytic peptide measurement was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid mass spectrometer for peptide/protein identification and quantification.