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. 

Methods
Search Engine: Sequest 

  Version: 27, rev. 12

  Parent Tolerance: 0.015 Da (Monoisotopic)

  Variable Modifications: +16 on M (Oxidation), +57 on C (Carbamidomethyl)

  Database: the mjdb_mouse-20150516 database (24937 entries)

  Digestion Enzyme: Trypsin

  Max Missed Cleavages: 3

Scaffold: Version: Scaffold_4.8.7 

  Protein Grouping Strategy: Experiment-wide grouping with binary peptide-protein weights

  Peptide Thresholds: 80.0% minimum

  Protein Thresholds: 99.0% minimum and 2 peptides minimum

  Peptide FDR: 2.8% (Prophet)

  Protein FDR: 0.1% (Prophet)

  ID Source(s): FASTA:UniRef/NREF (UniProt)