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Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells - Cytoplasmic Proteins of Untreated Cells
Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced up-regulation of proteins such as IL-1beta, IL-6, CXCL2 and GROα was confirmed, however with strong quantitative inter-individual differences. Furthermore, the inability of dexamethasone to down-regulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. While most consequences of dexamethasone were found compatible with the expected way of action, some unexpected but significant observations may relate with adverse effects.
Sample Processing Protocol
PBMCs were isolated from three different healthy volunteers with written consent of each donor and approval of the Ethics Committee of the Medical University of Vienna. 50ml of non-coagulated whole blood were collected in 6ml CPDA tubes (Greiner Bio-One GmbH, Austria) and immediately processed by diluting it 1:2 with RPMI1640 medium (Gibco, Life Technologies, Austria) supplemented with 100U/ml penicillin and 100µg/ml streptomycin (ATCC, LGC Standards GmbH, Germany). The diluted blood suspension was carefully overlaid on Ficoll Paque (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) and centrifuged at 500g for 20min at 24°C. The interphase was then collected and washed with PBS. The washed cell pellet was resuspendend in diluted autologous plasma and 4h incubated at 37°C and 5% CO2. Following 4h cultivation, cells were further grown for 3h in serum-free medium for secretome analysis and afterwards harvested. To obtain the cytoplasmic fraction, 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 and 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). Afterwards 20µg of each sample was loaded on 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 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.
Bileck A, Kreutz D, Muqaku B, Slany A, Gerner C. Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells. J Proteome Res. 2014 Oct 27 PubMed: 25347463
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||40969||no assay title provided (mzIdentML)||5746||431025||172862||321130||186862||
|2||40971||no assay title provided (mzIdentML)||3790||152468||72858||166000||88466||
|3||40970||no assay title provided (mzIdentML)||3776||88287||37115||131000||55102||
|4||40973||no assay title provided (mzIdentML)||2133||44145||16984||78010||32089||
|5||40968||no assay title provided (mzIdentML)||4682||335541||124933||303000||166441||
|6||40972||no assay title provided (mzIdentML)||1911||20044||11853||43165||14593||