Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells
Thrombin is the key serine protease of the coagulation cascade and a potent trigger of protease-activated receptor (PAR)1-mediated platelet aggregation. In recent years, PAR1 has become an appealing target for anticoagulant therapies. However, the inhibitors that have been developed so far increase bleeding risk in patients, likely because they interfere with endogenous PAR1 signaling in the endothelium. Due to its complexity, thrombin-induced signaling in endothelial cells has remained incompletely understood. Here, we have combined stable isotope amino acids in cell culture, affinity-based phospho-peptide enrichment and high resolution mass spectrometry and performed a time-resolved analysis of the thrombin-induced signaling in human primary endothelial cells. We identified 2224 thrombin-regulated phosphorylation sites, the majority of which has not been previously related to thrombin. Those sites were localized on proteins which are novel to thrombin signaling, but also on well-known players such as PAR1, Rho-associated kinase 2, phospholipase C, and proteins related to actin cytoskeleton, cell-cell junctions and Weibel-Palade body release. Our study provides a unique resource of phosphoproteins and phosphorylation sites which may generate novel insights into an intimate understanding of thrombin-mediated PAR signaling and the development of improved PAR1 antagonists that affect platelet but not endothelial cell function.
Sample Processing Protocol
Phosphoproteome. Equal amount of light, medium and heavy SILAC-labeled lysate were mixed together and processed as previously described16,17. Briefly, mixed proteins were reduced, alkylated and digested with trypsin using the FASP method27. Digested peptides were acidified to pH 2.5 with TFA and separated with strong cation exchange (SCX) chromatography (Resource S column, GE Healthcare) using 5 mM KH2PO4 pH 2.7, 30% acetonitrile (ACN) and 5 mM KH2PO4 pH 2.7, 30% ACN, 350 mM KCl buffers. SCX fractions were pooled into 6 according to UV absorbance and phosphopeptides enriched using TiO2 beads (GL sciences) in the presence of 2,5-dihydroxybenzoic acid (DHB)28. Phosphorylated peptides were eluted with 15% ammonium hydroxide, 40% ACN, loaded onto Empore-C18 StageTips, eluted with 80% ACN, 0.5% acetic acid, and stored at -80°C until MS analysis. Also the SCX flow through was collected and processed as described above performing 4 consecutive rounds of TiO2 enrichment, which were run separately at the MS. Secretome. The light, medium, heavy supernatants were combined and ultra-centrifuged at 4°C for 70 min at 100,000g. Proteins were isolated using silica-based resin, dissolved in 4x sample buffer (NuPAGE LDS loading buffer, Life Technologies) supplemented with 0.1 M DTT, incubated for 5 min at 95°C and separated on a 4%-12% NuPAGE Novex Bis-Tris gel (Life Technologies). Each gel lane was cut in seven slices and proteins in-gel digested with trypsin30. Peptides were loaded onto Empore-C18 StageTips29, eluted with 80% ACN, 0.5% acetic acid and stored at -80°C until MS analysis. Digested peptides were separated by nanoscale C18 reverse chromatography (Easy nLC, Thermo Scientific) coupled on line to a linear trap quadrupole (LTQ)-Orbitrap Elite mass spectrometer (Thermo Scientific) via a nanoelectrospray ion source (Nanospray Flex Ion Source, Thermo Scientific). Peptides were loaded on 20 cm fused silica emitter (New Objective) packed in-house with ReproSil-Pur C18-AQ, 1.9 μm resin (Dr Maisch GmbH) and eluted with 0-30% solvent (80% ACN, 0.5% acetic acid) over 90 min. Full scan MS spectra were acquired in the Orbitrap analyzer with a resolution of 120,000 at m/z 400, and a target value of 1,000,000 charges. The 10 most intense ions were selected for high collision dissociation fragmentation with a target value of 40,000 charges and acquired in the Orbitrap with resolution of 15,000 at m/z 400 Th. All data were acquired with Xcalibur software.
Data Processing Protocol
The RAW MS files were processed with the MaxQuant computational platform, 184.108.40.206. Proteins and peptides were identified using the Andromeda search engine by querying the human Uniprot database (release-2012 01, 81,213 entries). To search for precursor and fragment ions, an initial maximal mass deviation of 7 ppm and 20 ppm, respectively, was required. Trypsin with full enzyme specificity and only peptides with a minimum length of 7 amino acids were selected. A maximum of two missed cleavages was allowed. Carbamidomethylation (Cys) was set as fixed modification, while Oxidation (Met) and N-acetylation as variable modifications, as well as phospho(STY) for the phosphoproteome analysis. For protein, peptide and phosphorylation site identification, we required a maximum false discovery rate (FDR) of 1%. The relative quantification based on SILAC was performed by MaxQuant enabling the ‘re-quantify’ and ‘match between runs’ options.
van den Biggelaar M, Hernández-Fernaud JR, van den Eshof BL, Neilson LJ, Meijer AB, Mertens K, Zanivan S. Quantitative phosphoproteomics unveils temporal dynamics of thrombin signaling in human endothelial cells. Blood. 2014 Feb 5 PubMed: 24501219