Protein identification using nano LC-ESI-MS/MS was performed by Proteome Factory. The MS system consisted of an Agilent 1100 nanoLC system (Agilent, Waldbronn, Germany), PicoTip electrospray emitter (New Objective, Woburn, MA, USA) and an Orbitrap XL or LTQ-FT Ultra mass spectrometer (ThermoFisher, Bremen, Germany).
Protein spots were in-gel digested by trypsin (Promega, Mannheim, Germany) and applied to nanoLC-ESI-MS/MS. Peptides were trapped and desalted on the enrichment column (Zorbax SB C18, 0.3mm × 5 mm, Agilent) for five min using 2.5% acetonitrile/0.5% formic acid as eluent, then peptides were separated on a Zorbax 300 SB C18, 75 µm × 150 mm column (Agilent) using an acetonitrile/0.1% formic acid gradient from 5% to 35% acetonitril within 40 min. MS/MS spectra were recorded data-dependently by the mass spectrometer according to manufacturer’s recommendations.
Proteins were identified using MS/MS ion search of the Mascot search engine (Matrix Science, London, UK) and swissprot/uniprot database (Available online:http://www.uniprot.org/ ). Ion charge in search parameters for ions from ESI-MS/MS data acquisition were set to “1+, 2+, or 3+” according to the instrument’s and method’s common charge state distribution.
Protein spots were in-gel digested by trypsin (Promega, Mannheim, Germany) and applied to nanoLC-ESI-MS/MS. Peptides were trapped and desalted on the enrichment column (Zorbax SB C18, 0.3mm × 5 mm, Agilent) for five min using 2.5% acetonitrile/0.5% formic acid as eluent, then peptides were separated on a Zorbax 300 SB C18, 75 µm × 150 mm column (Agilent) using an acetonitrile/0.1% formic acid gradient from 5% to 35% acetonitril within 40 min. MS/MS spectra were recorded data-dependently by the mass spectrometer according to manufacturer’s recommendations.
Proteins were identified using MS/MS ion search of the Mascot search engine (Matrix Science, London, UK) and swissprot/uniprot database (Available online: