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Cell population-resolved proteomics of the developing lung

ABSTRACT: Cells from human lungs with age ranging between 0 and 8 years-old, were dissociated using a protocol previously described (DOI: 10.1152/ajplung.00041.2018). After dissociation, the cell population mix (PMX) was sorted into the four main populations composing the lung. Briefly, after removing erythrocytes (CD235a-), the different cell populations were sequentially isolated: mixed Immune cells (MIC: CD235a-/CD45+), Endothelial cells (ENDO: CD235a-/CD45-/PECAM1+), Epithelial cells (EPI: CD235a-/CD45-/PECAM-/EPCAM+), and Mesenchymal cells (MES: CD235a-/ CD45-/PECAM-/EPCAM-). Proteins, lipids, and polar metabolites were extracted using the MPLEx protocol. The protein fraction was reduced, alkylated, and digested with trypsin for 3 hours at 37 C (trypsin:protein ration 1:50). Sample desalting used C18 SPE cleanup (Discovery C18, 1 mL, 50 mg, Sulpelco). For each sorted population and for the PMX, 500 ng of peptides were analyzed by LC-MS/MS using a Waters nanoEquity UPLC system interfaced with a Q Exactive Plus Orbitrap mass spectrometer. A 180 min gradient was employed and a top 12 DDA strategy was employed. Raw mass spectrometry data were analyzed with MaxQuant (v1.6.0.16) using the default parameters, except with a matching window of 1.5 min for Match Between Runs. MaxLFQ was used for quantification.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Geremy Clair

PROVIDER: MSV000094189 | MassIVE | Tue Feb 27 19:24:00 GMT 2024

REPOSITORIES: MassIVE

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