Peptide Analysis by LC-MS/MS

Peptide mixtures were analyzed by LC-MS MS using the LC/MSD Trap XCT Ultra (Agilent Technologies, CA, USA) equipped with a 1100 HPLC system and a chip cube (Agilent Technologies, CA, USA) as previously described [24 (link)]. Briefly, after loading, peptide mixture (8 μl in 0,2 % HCOOH) was pre-concentrated, washed at 4 μl/min in 40 nl enrichment column (Agilent Technologies chip) and separated on a RP-C18 column (75 μm × 43 mm) at a flow rate of 200 nl/min with a linear gradient of eluent B (2 % formic acid in acetonitryl) in eluent A (2 % formic acid) from 5 to 60 % in 60 min. Peptides were analyzed using data-dependent acquisition of MS scan (400–2000 m/z) followed by MS/MS scans of the three most abundant ions. Dynamic exclusion was used to acquire a more complete survey of the peptides. A permanent exclusion list of the most frequent peptide contaminants was included in the acquisition as previously described [25 (link)].