Project : PXD000574

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Dectin-1/Syk pathway activates autophagy-dependent unconventional protein secretion from human macrophages

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.

Sample Processing Protocol

See details in reference PMID : 24808366

Data Processing Protocol

See details in reference PMID : 24808366

Contact

Tuula Nyman, Institute of Biotechnology

Submission Date

08/11/2013

Tissue

Not available

Instrument

QSTAR

Software

Not available

Experiment Type

Bottom-up proteomics

Publication

    Ohman T, Teirilä L, Lahesmaa-Korpinen AM, Cypryk W, Veckman V, Saijo S, Wolff H, Hautaniemi S, Nyman TA, Matikainen S. Dectin-1 Pathway Activates Robust Autophagy-Dependent Unconventional Protein Secretion in Human Macrophages. J Immunol. 2014 May 7 PubMed(s) : 24808366

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