Sciex 5500 qtrap triple quadrupole mass spectrometer

Samples were previously quantified by microBCA analysis (Pierce) and similar amounts (5 µg per sample) were individually dissolved in 8 M urea, 25 mM ammonium bicarbonate, reduced with DTT, and alkylated with iodoacetamide, according to a method described by López-Ferrer et al. (2004) (link). Digested samples were diluted with 0.2% trifluoroacetic acid in water and subjected to multiple reaction monitoring analysis using a 1D Plus nanoLC Ultra system (Eksigent, Dublin, CA, USA) interfaced to a Sciex 5500 QTRAP triple quadrupole mass spectrometer (Sciex, Framingham, MA, USA) equipped with a nano-electrospray ionization source and controlled by Analyst v.1.5.2. software (ABSciex). Trypsin-digested samples were loaded online on a C18 PepMap 300 µm internal diameter × 5 mm trapping column (5 µm, 100 Å, Thermo Scientific) and separated using a BioSphere C18 75 µm internal diameter × 150 mm capillary column (3 µm, 120 Å, Nanoseparations). A list of 84 transitions (usually 3–4 per peptide, with a preference toward higher-mass y series ions), corresponding to 21 unique peptides selected for 10 different proteins, was monitored. Skyline software determined automatically the collision energy values for the candidate peptides according to MacLean et al. (2010) (link).