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Eicosadomics, proteomics and metabolomics of inflammatory stimulated human fibroblasts reveals specific functions related to chronic inflammation - nuclear proteins of dexamethasone-treated inflammatory stimulated cells
Fibroblasts have only recently been identified as important effector cells in inflammation. In this study, human dermal fibroblasts were inflammatory stimulated with interleukin-1beta and comprehensively analysed with respect to proteins, eicosanoids and metabolites. For eicosadomics, we have established a data-dependent shotgun analysis method capable of identifying inflammation-regulated lipids of yet unknown function. Several classical inflammatory agonists were found induced, including PGA2, PGB2, PGE2 and TXB2, but also modulators such as PGA3 and PGE3, while 8-HETE and several HODE family members remained unaffected. Using targeted metabolomics, several acylcarnithins, phosphatitylcholins and sphingomyelins were found significantly downregulated. Proteome profiling with orbitrap-MS demonstrated the strong induction of several chemokines, metalloproteinases and other effector molecules. Treatment of stimulated fibroblasts with dexamethasone almost completely abrogated the formation of all inflammation-induced eicosanoids and restored levels of acylcarnithins back to normal. As expected, the secretion of IL-6, MMP1, MMP3, CXCL2 and CXCL3 was strongly down-regulated. However, instead of counter-regulating, dexamethasone further enhanced consequences of inflammatory stimulation with respect to CXCL1, CXCL6, complement C3 as well as sphingomyelins. Shotgun secretome data were confirmed by targeted analysis with triple-quadrupol-MS. These molecules have been described to be involved in chronic inflammation. In peripheral blood mononuclear cells, actually dexamethasone successfully downregulated the formation of all detectable inflammation mediators. The present data suggest that successful pharmacological abrogation of the formation of lipid inflammatory mediators in fibroblasts may not suffice to suppress the release of several other powerful inflammatory mediators which we thus understand to be capable of establishing chronic inflammation states.
Sample Processing Protocol
Normal human dermal fibroblasts (NHDF), kindly provided by Verena Paulitschke from the General Hospital of Vienna, were cultured in RPMI 1640 (Life Technologies, UK) supplemented with 10% FCS and 100U/ml penicillin/streptomycin (both ATCC, USA) at 37°C and 5% CO2. Experiments were performed up to passage 22, in 25cm2-culture flasks, using approximately 1.5x106 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). For inflammatory activation, 10ng/ml IL1b were added to the cells and incubated at 37°C and 5% CO2. For dexamethasone treatment, 100ng/ml dexamethasone (Sigma-Aldrich) was added after 1h activation. After further 23h of cultivation, cells were grown for 6h in serum-free medium for secretome analysis and afterwards harvested. Two biological replicates were prepared. To obtain the nuclear protein extract, cells were lysed in isotonic lysis buffer (10 mM HEPES/NaOH, pH 7.4, 0.25 M sucrose, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100) supplemented with protease inhibitors (pepstatin, leupeptin and aprotinin, each at 1 μg/ml; 1 mM PMSF) and mechanical shear stress. By centrifugation at 2300g and 4°C for 5min the cytoplasmic proteins were separated from the nuclei. For gaining nuclear proteins, pellets were swelled up for 10min in extraction buffer (500 mM NaCl) and 1:10 diluted with NP-40 buffer for another 15min. To obtain the nuclear fraction, centrifugation at 2300g and 4°C for 5min was performed. The extracted proteins were then precipitated overnight with ice-cold ethanol at -20°C. After precipitation, samples were dissolved in sample buffer (7.5 M urea, 1.5 M thiourea, 4% CHAPS, 0.05% SDS, 100 mM DDT) and the protein concentrations were determined by means of Bradford assay (Bio-Rad-Laboratories, Germany). For digestion of nuclear proteins, a variation of the FASP protocol was used. 3kD MWCO filters (Pall Austria Filter GmbH) were rinsed with LC-MS grade water (Millipore GesmbH). 20µg of protein samples obtained each from two of the two 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. 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 and qualitative data analysis. 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.
Tahir A, Bileck A, Muqaku B, Niederstaetter L, Kreutz D, Mayer RL, Wolrab D, Meier SM, Slany A, Gerner C. Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation. Anal Chem. 2017 Feb 7;89(3):1945-1954 PubMed: 28208246
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||63454||no assay title provided (mzIdentML)||4281||24222||17567||29241||15834||
|2||63455||no assay title provided (mzIdentML)||4211||27214||18102||42731||19727||
|3||63452||no assay title provided (mzIdentML)||4377||26696||19091||34573||18169||
|4||63453||no assay title provided (mzIdentML)||4142||28966||19045||49322||21580||