Quantitative Proteomic Analysis of A549 Cells Infected with Human Respiratory Syncytial Virus

Munday, Diane C.; Emmott, Edward; Surtees, Rebecca; Lardeau, Charles-Hugues; Wu, Weining; Duprex, W. Paul; Dove, Brian K.; Barr, John N.; Hiscox, Julian A.

LC-MS/MS-LC-MS/MS analysis was performed by Dundee Cell Products Ltd. as described previously (37) and for completeness is reproduced here. Trypsin-digested peptides were separated using an Ultimate U3000 (Dionex Corp.) nanoflow LC system consisting of a solvent degasser, micro-and nanoflow pumps, flow control module, UV detector, and a thermostated autosampler. 10 l of sample (a total of 2 g) was loaded with a constant flow of 20 l/min onto a PepMap C 18 trap column (0.3-mm inner diameter ϫ 5 mm; Dionex Corp.). After trap enrichment, peptides were eluted off onto a PepMap C 18 nanocolumn (75 m ϫ 15 cm; Dionex Corp.) with a linear gradient of 5-35% solvent B (90% acetonitrile with 0.1% formic acid) over 65 min with a constant flow of 300 nl/min. The HPLC system was coupled to an Linear quadrupole trap Orbitrap XL (Thermo Fisher Scientific Inc.) via a nanoelectrospray ion source (Proxeon Biosystems). The spray voltage was set to 1.2 kV, and the temperature of the heated capillary was set to 200°C. Full-scan MS survey spectra (m/z 335-1800) in profile mode were acquired in the Orbitrap with a resolution of 60,000 after accumulation of 500,000 ions. The five most intense peptide ions from the preview scan in the Orbitrap were fragmented by collisioninduced dissociation (normalized collision energy, 35%; activation Q, 0.250; and activation time, 30 ms) in the LTQ after the accumulation of 10,000 ions. Maximal filling times were 1,000 ms for the full scans and 150 ms for the MS/MS scans. Precursor ion charge state screening was enabled, and all unassigned charge states as well as singly charged species were rejected. The dynamic exclusion list was restricted to a maximum of 500 entries with a maximum retention period of 90 s and a relative mass window of 10 ppm. The lock mass option was enabled for survey scans to improve mass accuracy (38) . The data were acquired using Xcalibur software.