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Human plasma BOS iTRAQ

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Sophie Paczesny

PROVIDER: MSV000079203 | MassIVE | Thu Jul 30 14:20:00 BST 2015

REPOSITORIES: MassIVE

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Project description:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.

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Human plasma BOS iTRAQ

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