Ultimate 3000 nanoflow uhplc system

nLC–ESI–MS/MS analyses were performed on a Bruker amaZon ETD ion trap™ arranged in-line with an Ultimate 3000 nanoflow uHPLC system™ (Thermo Scientific, USA). Peptides were directly eluted over a 35 min LC gradient and a full scan mass spectrum acquired over m/z 300–1800, with the three most abundant ions being selected for isolation and activation with either CID and/or ETD (alternating). Peaklists from both the CID and ETD MS/MS spectra were extracted using Data Analysis software v. 4.0 (Bruker, Germany) and converted into mascot generic files (.mgf). .mgf files were searched against the concatenated SwissProt database (2011.05.03) and decoy using Mascot (v. 2.2.06), specifying H. sapiens for taxonomy. Parameters were set as follows: precursor mass tolerance at 0.4 Da; product ion tolerance at 0.6 Da; less than or equal to one missed cleavage; fixed modification: carbamidomethyl Cys; variable modifications: deamidation of Asn and Gln, oxidation of Met, phosphorylation of Ser, Thr and Tyr. Top-ranking peptide matches in Mascot were using ion score greater than or equal to 25 and an expectation value of less than or equal to 0.1. False-discovery rates at the protein and peptide level were assessed by searching against the decoy database (max. 1%). All CID and ETD tandem mass spectra of phosphorylated peptides were manually inspected.