1200 series nano lc

The peptide samples prepared with the FASP method were analysed using a 1200 Series nano LC connected to a 6410 QQQ mass spectrometer via a chipcube interface (Agilent Technologies, UK). Peptide samples (1 µg in 0.1% formic acid) were injected for LC-MS/MS analysis with a 10 minute gradient from 3% (v/v) acetonitrile, 0.1% (v/v) formic acid to 100% (v/v) acetonitrile, 0.1% formic acid using a Large Capacity Chip (160 nl) 150 mm C-18 (Agilent Technologies, UK).

The desalted peptide samples were analysed using a 1200 Series nano LC connected via a chipcube interface to a 6520 Q-ToF mass spectrometer (Agilent Technologies, UK). In brief, peptide samples (1 µg in 0.1% formic acid) were injected for LC-MS/MS analysis with a 120-min gradient from 3% (vol/vol) acetonitrile-0.1% (vol/vol) formic acid to 40% (vol/vol) acetonitrile-0.1% formic acid and from 3% (vol/vol) acetonitrile-0.1% (vol/vol) formic acid to 75% (vol/vol) acetonitrile-0.1% formic acid by using a high-capacity (160-nl) 150-mm C18 chip (Agilent Technologies, UK). The mass spectrometer was operated with mass ranges of 250 to 2,000 m/z for MS and 50 to 2,000 m/z for MS/MS at a constant flow rate of 500 nl/min. Acquisition rates were 3 spectra/s for MS and 5.01 spectra/s for MS/MS, and the mass reference was 922.009798 m/z. Mass accuracy was better than 2-ppm for MS and 5-ppm for MS/MS, and mass resolution was up to 20,000. Proteins were identified by Mascot (Version 2.3, Matrix, UK) from the NCBI non-redundant (nr) database (Release date: 28-03-2011) as described previously [23] (link). Identified proteins were quantified by spectral counting, using Scaffold (Version 2.6, Proteome Software, USA). The software was used to identify differentially expressed proteins using the t test with P value of less than 0.05 after normalization.