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Contribution of primary human fibroblasts and endothelial cells to the hallmarks of inflammation as determined by proteome profiling - cytoplasmic proteins of untreated endothelial cells
While the most important players of inflammation have been well described, a systematic analysis of the proteins fulfilling the effector functionalities during inflammation has not yet been undertaken. Here we present a systematic proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-beta and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying an FDR of less than 0.01 at both peptide and protein level, a total of 8235 protein groups assembled from 163858 peptides was identified. Comparative proteome analysis allowed us to determine proteins regulated in each kind of cells during inflammation. Remarkably, cells were working on similar inflammation-related tasks, however, by regulating different proteins. Thus, we were able to determine cell type-specific inflammatory signatures, apparently resulting from cell type-specific regulatory mechanisms. Hallmarks of inflammation emerged from these findings, representing commonly and cell type-specific responsibilities of cells during inflammation.
Sample Processing Protocol
Primary human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Walkersville Inc., USA). HUVEC were cultured in endothelial basal medium supplemented with the EGM-2 SingleQuot Kit (both EBM, Lonza Walkersville Inc., USA), 10% FCS and 100U/ml penicillin/streptomycin (both ATCC, USA) at 37°C and 5% CO2. Experiments were performed up to passage 7, in 75cm2-culture flasks, using approximately 5x106 cells per flask. Cell numbers, as well as cell viability which was consistently better than 98%, were determined using a MOXI cell counter (ORFLO, USA). Cells were incubated for 24h, and then they were washed with PBS and further cultured for 6 hours in 6 ml of serum-free medium. Seven biological replicates were prepared. To obtain the cytoplasmic fraction, cells were lysed in isotonic lysis buffer supplemented with protease inhibitors and mechanical shear stress. By centrifugation at 2300g and 4°C for 5min the cytoplasmic proteins were separated from the nuclei and precipitated overnight with ice-cold ethanol at -20°C. After precipitation, samples were dissolved in sample buffer and the protein concentrations were determined by means of Bradford assay (Bio-Rad-Laboratories, Germany). For digestion of cytoplasmic proteins, a variation of the FASP protocol was used. 3kD MWCO filters were rinsed with LC-MS grade water. 20µg of a protein sample obtained each from five of the seven biological replicates was concentrated onto the pre-washed filter by centrifugation at 15000g for 15min to get a final sample volume of 10-20µl. For reduction, 200µl of DTT solution (5mg/ml dissolved in 8M guanidinium hydrochloride in 50mM ammonium bicarbonate buffer (ABC buffer), pH 8) were added and incubation was performed at 56°C for 30min. After centrifugation at 14000g for 10min, a washing step with ABC buffer was performed. For alkylation, 200µl of IAA solution (10mg/ml in 8M guanidinium hydrochloride in 50mM ABC buffer) were added and incubation was performed in the dark for 45min. After centrifugation at 14000g for 10min, proteins on top of the filter were washed with ABC buffer. Afterwards, filters (with a maximum of 50µl sample volume) were placed in a new Eppendorf tube and 100µl ABC buffer as well as 10µl trypsin solution (0.1µg/µl) were added and incubation was performed at 37°C for 18h. After trypsin digestion, peptide samples were cleaned up with C-18 spin columns (Pierce, Thermo Scientific). Therefore, columns were pre-washed two times with 500µl ACN and equilibrated with 200µl 5% ACN, 0.5% trifluoroacetic acid (TFA) by centrifugation at 1500g for 1min. The peptide samples were acidified to a final concentration of 1% TFA and transferred from the MWCO filters to spin columns. After centrifugation at 1500g for 1min, the flow-through was re-loaded on the column to maximize peptide binding and again centrifuged. After a washing step with 5% ACN, 0.5% TFA, the peptides were eluted two times with 40µl 50% ACN, 0.1% TFA and once with 40µl 80% ACN, 0.1% TFA into a new Eppendorf tube. Finally the digested peptide samples in the flow-through were dried and stored at -20°C until further MS analyses. In parallel, 20µg of the cytoplasmic protein fraction each of two biological replicates was loaded on an SDS-PAGE. Proteins in the gels were stained by a MS-compatible silver staining. After fixation with 50% methanol/10% acetic acid, the gels were washed and sensitized with 0.02% Na2S2O3. Gels were then stained with 0.1% AgNO3 for 10min, rinsed and developed with 3% Na2CO3/0.05% formaldehyde. Afterwards, each protein band was cut into 4 slices and destained. Upon reduction with DTT and alkylation with IAA, the proteins were digested enzymatically overnight at 37°C using trypsin (Roche Diagnostics, Germany). The peptides were eluted, dried and stored at -20°C until LC-MS analysis. For LC-MS/MS analyses dried samples were reconstituted in 5µl 30% formic acid (FA) and diluted with 40µl mobile phase A (98% H2O, 2% ACN, 0.1% FA). 10µl of this solution were then injected into the Dionex Ultimate 3000 nano LC-system coupled to a QExactive orbitrap mass spectrometer equipped with a nanospray ion source (Thermo Fisher Scientific, Austria). All samples were analyzed in duplicates. As a pre-concentration step, peptides were loaded on a 2cm x75µm C18 Pepmap100 pre-column (Thermo Fisher Scientific, Austria) at a flow rate of 10µl/min using mobile phase A. Elution from the pre-column to a 50cm x75µm Pepmap100 analytical column (Thermo Fisher Scientific, Austria) and subsequent separation was achieved at a flow rate of 300nl/min using a gradient of 8% to 40% mobile phase B (80% ACN, 2% H2O, 0.1% FA) over 235min. For mass spectrometric detection, MS scans were performed in the range from m/z 400-1400 at a resolution of 70000 (at m/z =200). MS/MS scans of the 12 most abundant ions were achieved through HCD fragmentation at 30% normalized collision energy and analyzed in the orbitrap at a resolution of 17500 (at m/z =200).
Data Processing Protocol
Proteome Discoverer 1.4 (Thermo Fisher Scientific, Austria) running Mascot 2.4 (Matrix Science, UK) was used for protein identification. Protein identification was achieved searching against the SwissProt Database (version August 2014 with 20 194 entries) allowing a mass tolerance of 10ppm for MS spectra and 50mmu for MS/MS spectra as well as a maximum of 2 missed cleavages. Furthermore, search criteria included carbamidomethylation on cysteins as fixed modification and methionine oxidation as well as N-terminal protein acetylation as variable modifications.
Slany A, Bileck A, Kreutz D, Mayer RL, Muqaku B, Gerner C. Contribution of human fibroblasts and endothelial cells to the Hallmarks of Inflammation as determined by proteome profiling. Mol Cell Proteomics. 2016 Mar 29. pii: mcp.M116.058099 PubMed: 27025457
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||60695||no assay title provided (mzIdentML)||4149||31437||19641||38645||21854||
|2||60696||no assay title provided (mzIdentML)||3219||27716||13022||35639||17099||
|3||60697||no assay title provided (mzIdentML)||3444||23203||12357||33112||15403||
|4||60698||no assay title provided (mzIdentML)||2932||15576||8159||26564||9792||
|5||60699||no assay title provided (mzIdentML)||4151||31559||19692||39325||22029||
|6||60690||no assay title provided (mzIdentML)||3332||28331||13382||36316||17347||
|7||60691||no assay title provided (mzIdentML)||3495||23485||12583||33012||15451||
|8||60692||no assay title provided (mzIdentML)||3003||16160||8575||27140||10203||
|9||60693||no assay title provided (mzIdentML)||5382||44990||24789||97060||33411||
|10||60694||no assay title provided (mzIdentML)||5199||31252||21963||35524||21885||