MassIVE MSV000079203

Description

We compared 4 pools of plasma in the same iTRAQ proteomic experiment. The pool 1 contained plasma from twelve patients with strict BOS criteria and with plasma sample available at onset of the clinical symptoms (named BOS), pool 2 contained plasma from sixteen patients with pulmonary infections (infection), pool 3 contained plasma from fifteen patients with chronic GVHD without pulmonary involvement (CGVHD no lung), and pool 4 contained plasma from fifteen patients after transplant with no chronic complications (at similar time point as BOS samples) (no complications). Each pool contained 25 μl of plasma. The four pooled plasmas were then individually immunodepleted of the twenty common hyper-abundant proteins with a ProteoPrep®20 plasma immunodepletion kit (Sigma-Aldrich) according to manufacturer?s procedure. The cysteine residues were alkylated and all samples were trypsinized. Each pool was labeled with a different tag allowing for differential quantification. The samples were labeled in the following order: 1) BOS with 114, 2) Infection with 115, 3) cGVHD with 116, and 4) No complication with 117. The four pooled plasma samples were dissolved in buffer A (7mM potassium phosphate, 30% acetonitrile, pH 2.65) and combined right before fractionation with a SCX column (Zorbax 300-SCX 5µm, 2.1 x 150 mm, Agilent). The fractions were collected every minute at 200 µL/min flow rate from 1% solvent B (7mM potassium phosphate, 500 mM KCl, 30% acetonitrile, pH 2.65) to 60% over 40 minutes (1% B for 7 minutes, 6 to 15% B for 18 minutes, 15 to 34% B for 10 minutes, and 34 to 60% B for 5 minutes) as well as during column washing at 98% solvent B for 10 minutes. These fractions were consolidated into 11 fractions using the UV trace to distribute the peptide quantities to be similar. LC-MS/MS analysis was performed with an Easy-nLC 1000 (Thermo Scientific) coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific). The peptide sample was diluted in 30 µL of 2% acetonitrile and 0.1% formic acid in water and 8-4 µL was loaded onto the column in triplicates and separated using a two-mobile-phase system consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A 90 minute gradient from 7% to 35% B at a flow rate of 400 nL/min was used for chromatographic separations. The mass spectrometer was operated in a data-dependent MS/MS mode over the m/z range of 400-1800. The mass resolution was set at 60,000. For each cycle, the 10 most abundant ions from the scan were selected for MS/MS analysis using 40% normalized HCD collision energy and analyzed with an orbitrap with the resolution set to 15000. [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: iTRAQ quantification BOS

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