Mar 29, 2024
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
- Joanna Bons1,
- J P Rose1,
- M A Watson1,
- B Schilling1
- 1Buck Institute for Research on Aging
Protocol Citation: Joanna Bons, J P Rose, M A Watson, B Schilling 2024. 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. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld83bnv5b/v1
Manuscript citation:
dx.doi.org/10.17504/protocols.io.e6nvw1px7lmk/v
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 25, 2024
Last Modified: April 08, 2024
Protocol Integer ID: 97359
Keywords: Human, Ovary, Secretome, SASP, Senescence, Doxorubicin, LC-MS/MS, DIA, Orbitrap Eclipse, Mass Spectrometry, Conditioned Media
Funders Acknowledgement:
NIH Grant
Grant ID: 5U54AG075932-02
NIH Grant
Grant ID: 5U54AG075932-03
Disclaimer
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.
Abstract
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.
Conditioned Media Concentration with Amicon Ultra Centrifugal Filters
Conditioned Media Concentration with Amicon Ultra Centrifugal Filters
Protein Digestion with S-trap Spin Columns using Conditioned Concentrated Media
Protein Digestion with S-trap Spin Columns using Conditioned Concentrated Media
Proteolytic Peptide Desalting with C18 HLB Cartridges
Proteolytic Peptide Desalting with C18 HLB Cartridges
LC-MS/MS Acquisition by DIA on an Orbitrap Eclipse Tribrid Mass Spectrometer
LC-MS/MS Acquisition by DIA on an Orbitrap Eclipse Tribrid Mass Spectrometer
DIA Data Processing using Spectronaut/directDIA (Biognosys): Secretome Analysis
DIA Data Processing using Spectronaut/directDIA (Biognosys): Secretome Analysis