Dataset Information

Platelet Activation Results in Modulation of miRNA, mRNA, and Protein Expression Profiles: New Insights into the Pathophysiology of Platelet Activation

ABSTRACT: Leucocytes-decontaminated platelet-rich plasma (PRP) from 15 healthy volunteers was collected and activated with collagen, or Thrombin Activator Receptor Peptide (TRAP). Total protein extracts (75 microg protein/sample), coming from two biological replicates, were precipitated using 2-D Clean Up Kit (Amersham Bioscience). Total protein extracts were precipitated and dissolved into 20 microl of iTRAQ dissolution buffer. Protein alkylation, trypsin digestion and labeling of the resulting peptides were performed according to manufacturer’s instructions (Applied Biosystems) followed by strong cation exchange fractionation and LC-MS/MS analysis of the peptides as previously described (Lietzén et al, PLoS Pathogens). Protein identification and relative quantitation were performed with Paragon search algorithm using ProteinPilot 4 interface (Applied Biosystems). Database searches were performed against human protein sequences in UniProt database (version 120813 with 20231 human entries). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Paragon Unused ProtScore > 1.3). Additionally, automatic bias correction was used to correct for uneven protein loading.

INSTRUMENT(S): QSTAR

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tuula Nyman

PROVIDER: PXD000621 | Pride | 2015-04-27

REPOSITORIES: Pride

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Publications

Activating stimuli induce platelet microRNA modulation and proteome reorganisation.

Cimmino Giovanni G Tarallo Roberta R Nassa Giovanni G De Filippo Maria Rosaria MR Giurato Giorgio G Ravo Maria M Rizzo Francesca F Conte Stefano S Pellegrino Grazia G Cirillo Plinio P Calabro Paolo P Öhman Tiina T Nyman Tuula A TA Weisz Alessandro A Golino Paolo P

Thrombosis and haemostasis 20150423 1

Platelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet t ...[more]

PMID: 25903651

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Project description:Human keratinocytes, HaCaT cells were transfected with the dsRNA-analogue polyinosinic-polycytidylic acid (poly I:C) (Sigma-Aldrich) using Lipofectamine 2000 reagent (Invitrogen) for intracellular delivery. The cells were transfected with 7 ug/ml poly I:C for 1 h or left untreated. The cells were collected in PBS with a cell scraper and washed once with PBS before they 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 (Sigma-Aldrich). The cell lysates were centrifuged 2,264 x g for 15 min at 4°C, divided into smaller fractions and centrifuged again 11,686 x g for 15 min at 4°C . The supernatant was collected and the protein content was measured with Bio-Rad DC protein assay (Bio-Rad). For the samples, 8 mg of protein was used. The proteins were precipitated with 10 % TCA/acetone at -20°C overnight. Samples were centrifuged 10,733 x g for 20 min at 4°C and the supernatant was removed. The precipitation was washed once with acetone and dissolved in urea buffer (8 M urea, 400 mM NH4CO3, 20 mM DL-dithiotheitol, 1 mM EDTA, pH 8.5). The proteins were reduced, alkylated, and enzymatically digested in-solution with lysyl endopeptidase (Wako Chemicals) and trypsin (Promega). The peptides were fractionated by strong cation exchange chromatography. Phosphopeptide enrichment was performed with immobilized-metal affinity chromatography (IMAC) using PHOS-Select Iron Affinity Gel (Sigma Aldrich). The enriched phosphopeptides were analyzed by nanoLC-MS/MS using an Ultimate 3000 nano-LC (Dionex) coupled to a QSTAR Elite hybrid quadrupole TOF-MS (Applied Biosystems/MDS Sciex) with nano-ESI ionization.23,24 The LC-MS/MS data were submitted through the ProteinPilot 4.0 interface (Applied Biosystems/MDS Sciex) to an in-house Mascot database search engine version 4.0 (Matrix Science), and to the ProteinPilot algorithm Paragon. The data were searched against the human canonical sequences in the Swiss-Prot database (decoy version 08132012 for the Mascot searches and version 04012013 with 539,829 sequences for the Paragon searches). The Mascot search criteria were: fixed modifications: carbamidomethyl of cysteine, variable modifications: oxidation (M), phospho (ST), phospho (Y), acetylation (K), 1 missed cleavage allowed, enzyme: trypsin. The Paragon search criteria were: modifications: cystein alkylation with iodoacetamide, ID focus: biological modifications, phosphorylation emphasis, identification threshold: 95 % confidence (Unused ProtScore (Conf) > 1.3), Thorough search (ID), enzyme: trypsin.

Project description:Before stimulation, the cells were washed and the culture media was changed to RPMI-1640. After stimulation, cell culture media were collected, concentrated, and the proteins were precipitated with 2-D Clean-Up Kit (GE Healthcare) followed by protein alkylation, trypsin digestion and iTRAQ labelling of the resulting peptides according to manufacturer´s instructions (Applied Biosystems). Control sample was labeled with 114, LPS-stimulated with 115, Curdlan-stimulated with 116, and GBY-stimulated with 117 isobaric tag. After labelling the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing labelled peptides was analyzed twice with nano-liquid chromatography-tandem mass spectrometry using Ultimate 3000 nano-liquid chromatograph (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems / MDS Sciex) with nano-ESI ionization as previously described (Lietzén et al., 2011). MS data were acquired automatically using Analyst QS 2.0 software. Protein identification and relative quantitation were performed with ParagonTM search algorithm (Shilov et al., 2007) using ProteinPilot 2.0 interface (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. Database searching was done against UniProt human database (version 2008-01-28 with 20330 human sequences) and ´decoy` database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). Importantly, automatic bias correction was not used in quantitation. The false discovery rates were calculated as previously described (Elias and Gygi, 2007) and were 1.4% and 2% for the two biological replicates.

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Dataset Information

Platelet Activation Results in Modulation of miRNA, mRNA, and Protein Expression Profiles: New Insights into the Pathophysiology of Platelet Activation

Publications

Activating stimuli induce platelet microRNA modulation and proteome reorganisation.

Similar Datasets

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