4.1.2. Mass Spectrometry Examination

In the Laboratory of Mass Spectrometry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland, proteins were examined by liquid chromatography linked to a mass spectrometer. Using two technical replicates, tryptic peptide mixtures were evaluated by LC-ESI-MS/MS employing nanoflow HPLC and an LTQ-Orbitrap XL (Thermo Fisher Scientific, Waltham, MA, USA) as the mass analyzer. Trypsin was used to break down the proteins. The synthesized peptides were concentrated, desalted on an RP-C18 precolumn (LC Packings, Coventry, UK), and then separated by UltiMate

nano-HPLC (LC Packings, San Diego, CA, USA) with a linear acetonitrile gradient (from 10 to 30%) over 50 min. The column was linked directly to a nanospray ion source working in a data-dependent MS to MS/MS mode transition. Proteins were identified by tandem mass spectrometry (MS/MS) by acquiring fragmentation spectra of multiple-charged peptides in a manner dependent on information.
