PSM1346: Aspergillus fumigatus identification with 2D-LC Parent Study: PSE152 Organism: Aspergillus fumigatus Description In glucose minimal medium at 37°C with 0-hour, 4-hour, 6-hour, 8-hour culture. Statistics Proteins: 518 Peptides: 2223 Spectra: 2217 Dates Release: February 8, 2011 Deposit: February 4, 2011 Metadata update: February 7, 2011 Data update: February 7, 2011 Contact Organization: J. Craig Venter Institute Email: shuang@jcvi.org Address: 9704 Medical Center Drive Rockville, MD 20850 Preparation Growth: Asperfillus fumigatus was routinely grown in glucose minimal medium (GMM) containing a final concentration of 1% glucose at 37°C. Treatment: The frozen conidia pellet was ground to a fine powder using a mortar and pestle and then cell walls were incubated with 30mM NaOH at 4°C for 17 hours with gentle shaking. Extract: With the established mild alkali method for extraction of cell wall proteins, a class of proteins linked to glucans and cell walls were released. Digestion: Trypsin digestion using the FASP protocol (PMID: 19377485) Separation: For protein extracts: centrifugation at 16,100 x g to isolate solubilized proteins; for peptide digests: one-dimensional C18 reversed-phase LC using linear aqueous acetonitrile gradients. Characteristics Strain: Strain 293 Instrumentation Platform: ESI-ION-TRAP Manufacturer: Thermo Electron, San Jose, CA Ion source: Nano electrospray. Analyzer: Ion trap. Detector: Radial ejection and dual detector Setup: The Agilent 1100 nanoLC system (Agilent, Palo Alto, CA) was connected to a linear ion trap mass spectrometer (LTQ-IT mass spectrometer, Thermo-Finnigan, San Jose, CA); Peptide separation for LC-MS/MS was performed using a PicoTip microcapillary RP column (BetaBasic C18, 75mm X 11cm, New Objective, Woburn, MA) at a flow rate of 350 nL/min. LC method: 7 minute gradient - 3%-43%, Buffer A- 0.1% Formic Acid, Buffer B- 90% MeCN, 0.1% Formic Acid, loading buffer – 2% MeCN, 0.1% Formic Acid. MS method: top 5 ions are analyzed – 1st scan event is MS1 to obtain top 5 ions in spectra, remaining scan events (2-6) are MS2 to fragment selected ions. Exclusion list employed for selected ions for 100s. Processing Methods Software: Mascot (version 2.2, Matrix Science, London, UK) Parameters: DB: Aspergillus fumigatus Af293 protein database, enzymatic cleavage: trypsin, variable modification: oxidation (M), monoisotopic mass, max one missed cleavage, peptide tolerance = +/- 1.4 Da, MS/MS tolerance = +/- 0.5 Da, MS/MS, charge state = 1+, 2+, and 3+. Quantification: Not applied. Search Engines: Mascot Modifications Variable: Oxidation (35)