Phosphoproteomics combined with quantitative 14-3-3-affinity capture identifies SIRT1 and RAI as new regulators of cytosolic dsRNA-induced innate immune response
In this study, we have used global phosphoproteomic analysis to define phosphorylation-dependent signaling events regulated during RLR stimulation in human keratinocytes. In addition, we used quantitative 14-3-3-affinity capture to identify major target molecules of 14-3-3 regulation.
Sample Processing Protocol
HaCaT cells were transfected with and without polyI:C for 4 h, which after the cells were lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the protein contents were measured using the Bio-Rad DC™ protein assay. The proteins in cell lysate (10 mg per sample) were reduced, alkylated, and digested in-solution with trypsin. The undigested proteins and cell debris were removed by centrifugation and the samples were desalted with Sep-Pak Vac RP C18 cartridges. The peptides were fractionated by strong cation exchange chromatography (SCX) with a HPLC unit, an AKTApurifier instrument. The peptides were separated on a 200 x 4.6 mm, 5 μm, 200 Å PolySULFOETHYL A™ column (PolyLC) by applying a gradient run. The A buffer contained 10 mM KH2PO4, 20 % ACN, with a pH < 3. The gradient was set to 0-50 % buffer B (buffer A + 0.4 M KCl) in 25 min, followed by 50-100 % buffer B in 15 min. The flow rate was 1 ml/min and 1 ml fractions were collected by an autosampler. The fractions containing the phosphopeptides were collected and pooled. The samples were acidified with formic acid and desalted. The phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich) according to the manufacturer’s instruction. The enriched phosphopeptides were analyzed by nanoLC-MS/MS. The LC-MS/MS analyses were done with an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization. The samples were loaded on a ProteCol C18 trap column (SGE) and separated on a PepMap C18 analytical column (15 cm x 75 μm, 5 μm, 100 Å) (LC Packings/Dionex) at 200 nl/min with a linear gradient of 0-40 % acetonitrile in 120 min. The MS data was acquired with Analyst QS 2.0 software. Information-dependent acquisition method consisted of a 0.5 s TOF-MS survey scan of m/z 400-1400. From every survey scan two most abundant ions with charge states +2 to +4 were selected for product ion scans, and each selected target ion was dynamically excluded for 60 s. Smart IDA was activated with automatic collision energy and automatic MS/MS accumulation. Three independent biological experiments were performed.
Data Processing Protocol
The phosphopeptide identification was performed through ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) using an in-house Mascot database search engine version 4.0 (Matrix Science) and the ProteinPilot algorithm Paragon. The data were searched against human canonical sequences in the Swiss-Prot database (version 08132012). The search criteria for Paragon were: cysteine alkylation with iodoacetamide, trypsin digestion, biological modifications and phosphorylation emphasis allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). The search criteria for Mascot were: cysteine alkylation with iodoacetamide, trypsin digestion, methionine oxidation and serine/threonine/tyrosine phosphorylation allowed, identification threshold p < 0.05, and 1 missed cleavage allowed.
Ohman T, Soderholm S, Hintsanen P, Valimaki E, Lietzen N, MacKintosh C, Aittokallio T, Matikainen S, Nyman TA. Phosphoproteomics Combined with Quantitative 14-3-3-affinity Capture Identifies SIRT1 and RAI as Novel Regulators of Cytosolic dsRNA Recognition Pathway. Mol Cell Proteomics. 2014 Jul 5. pii: mcp.M114.038968 PubMed: 24997996