Discovery proteomics by mass spectrometry comparing secreted proteins from young and old mouse lung mesenchymal stromal cells

Lung mesenchymal stromal cells (L-MSCs) were isolated from the young ( 3 months) and old (22-24 months) mice (N = 4 each) following collagenase digestion of the lungs and anchorage-dependent growth. Culture media was collected from young and old L-MSCs (10^6 cells) grown in culture for 24 hrs in serum-free condition. Culture media was then centrifuged to remove any cellular debris. Five (5) ml of supernatants were concentrated using 3kDa Molecular weight cut-off filters, and 10 micrograms of proteins were run as multiple MW 1D PAGE fractions using a standard GeLC approach with a nano-HPLC in-line with a Velos Pro Orbitrap MS. Individual data files were searched using Sequest, the results were then combined for each sample, followed by grouping, filtering, and quantifying by normalized spectral counts.