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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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Project description:Proteome analyses of human induced pluripotent cells (iPSC) were carried out by liquid chromatography–tandem mass spectrometry systems using meter-scale monolithic silica-C18 capillary columns without pre-fractionation. Tryptic peptides from five different iPSC lysates and three different fibroblast cell (FBC) lysates (4 μg each) were directly injected onto a 200 cm long, 100 μm i.d. monolithicsilica-C18 column and an 8-h gradient was applied at 500 nL/min at less than 20 MPa. Consequently, 98,977 non-redundant tryptic peptides from 9,510 proteins (corresponding to 8,712 genes), including low abundant protein groups such as 329 protein kinases, were successfully identified from triplicate measurements within 10 days. The obtained proteome profiles of 8 cells were successfully categorized into two groups, iPSC and FBC, by hierarchical cluster analysis. Further quantitative analysis based on an exponentially-modified protein abundance index approach combined with UniProt keyword enrichment analysis reveals that the iPSC group contains more ‘transcription regulation’ proteins and the FBS group contains more ‘transport’ related proteins. This simplified “one-shot” proteomics approach with long monolithic columns enables to accomplish the rapid, deep, sensitive and reproducible proteome analysis. Sample pretreatment was carried out according to the PTS protocol with some modifications shown below. Proteins were extracted from the pellets with 12 mM SDC, 12 mM SLS, and 50 mM ammonium bicarbonate, reduced with 10 mM dithiothreitol at room temperature for 30 min; and alkylated with 55 mM iodoacetamide in the dark at room temperature for 30 min. The protein mixture was 5-fold diluted with 50 mM ammonium bicarbonate and Lys-C/trypsin digestion was performed. An equal volume of ethyl acetate was added to the eluent solution, and the mixture was acidified with 0.5% trifluoroacetic acid (final concentration). The mixture was shaken for 1 min and centrifuged at 15,700 x g for 2 min, and then the aqueous phase was collected. Tryptic peptides were desalted with reversed phase-StageTips. Raw data files were searched against the IPI human database v3.87 (91,464 sequences) using AB SCIEX ProteinPilot v4.0 with the set parameter of “Instrument: TripleTOF 5600”and “Digestion: trypsin”. A precursor mass tolerance of 0.05 Da and a fragment ion mass tolerance of 0.1 Da were employed. Carbamidomethylation of cysteine, oxidation of methionine, phosphorylation of serine, threonine and tyrosine, deamidation of asparagine, glutamine, N-terminal pyro-glutamic acid of glutamine or glutamic acid and protein N-terminal acetylation were only accepted, and peptides were rejected if the peptide confidence was below 95%, the delta mass was over 0.05 Da, the charge state was more than 5 and the number of missed cleavage was more than 2. For protein identification, peptides were grouped into ‘protein group’ based on the rules previously established. Then, at least two confidently identified peptides per protein were used for protein identification. In addition, single peptides with higher confidence (p<0.01) were allowed for protein identification.

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Cell population-resolved proteomics of the developing lung

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