Acclaim pepmap 100 c18 hplc column 0.3 5 mm with 5 μm particles

Samples were analyzed on a TSQ Altis (Thermo Scientific) coupled to an UltiMate 3000 (Thermo Scientific), and an easy spray analytical column (ES802A, 25 cm, 75 mm ID PepMap RLSC, C18, 100 A˚, 2 mm particle size column (Thermo Scientific)). First, samples were reconstituted in 2% LC–MS grade formic acid, containing the heavy labeled peptides. Samples were loaded on a trap column (Acclaim™ PepMap™ 100C18 HPLC Column 0.3 × 5mm with 5 μm particles (Thermo Scientific)) with 2.2% Buffer A (0.1% FA) for 3 min, and subsequently separated using 0-32% buffer B (99.9%ACN, 0.1%FA) in 35 min at 300 nL/min. Followed by a 20 min column wash with 80% buffer B at 300 nL/min, and 10 min column equilibration at 2.2% B. The TSQ Altis spray voltage was set at 1.9 kV and fragmented at 1.5 mTorr in the second quadrupole. The first quadrupole was set at 0.7 da FWHM, and the third quadrupole at 1.2 da FWHM. All transitions were measured with an optimized collision energy without scheduling and a cycle time of 1.5 s.