br Microorganism cultivation and secretome extraction The fu

Microorganism cultivation and secretome extraction The fungal isolates belonging to Aspergillus sp. isolated, identified and labeled as A. fumigatus LF9 was used in this study [3]. DNA sequence of this isolate is available in GenBank under accession no. JF815073. A. fumigatus LF9 was cultivated at 50°C at 100rpm in medium composed of ammonium sulfate 3.1gL−1, sodium chloride 1.5gL−1, dipotassium phosphate 1.2gL−1, monopotassium phosphate 0.9gL−1, magnesium sulfate 0.3gL−1 and 5.0gL−1 different carbon sources such as glucose, cellulose, xylan and starch. Mycelium was harvested in mid exponential phase (4 days) and supernatant was collected by centrifugation at 7500×g at 4°C (Beckman Coulter, Brea, CA, USA) for 7min. The experimental design included three flaks for each carbon source used. The supernatant henceforth called secretome was further filtered through 0.25μm filters. Proteins were precipitated using ice cold acetone for overnight and protein content was estimated by Bradford method [4]. The strains belonging to species A. fumigatus are pathogenic and hence biosafety guidelines were strictly followed while working with this fungal isolate.
Protein separation, protein digestion, and peptide extraction Proteins from each test condition were separated on 10% SDS-PAGE at 100V, and protein bands were visualized by staining with Coomassie Brilliant blue G-250. For proteomic analysis, proteins from all four conditions were digested separately by using our optimized protocol [2,5,6]. Concisely, 200µg proteins from each condition were loaded on SDS-PAGE and run at 100V for 30–40min and concentrated in separating gel. Each sample lane was sliced separately, cut into small pieces (approximately 1mm2) washed with 75% acetonitrile (ACN) containing triethylammonium bicarbonate buffer (TEAB, 25mM) and then distained using TEAB (25mM) alone and TEAB with 50% ACN. Then gel pieces were reduced with Tris 2-carboxyethyl phosphine hydrochloride (5mM) and alkylated with methyl methanethiosulfonate (10mM). The gel pieces were washed twice with TEAB to remove excess reducing and alkylating agent and dehydrated using 100% ACN. The gel pieces were subjected to sequencing grade modified trypsin (Promega, Madison, WI) histone deacetylase inhibitors at 37°C for overnight. The peptides were extracted using 50% ACN plus 5% acetic acid. The extracted peptides were concentrated using concentrator (Eppendrof AG, Hamburg, Germany) for iTRAQ labeling.
iTRAQ labeling and LC–MS/MS analysis iTRAQ labeling of peptides was performed using 4-plex iTRAQ reagent multiplex kit (Applied Biosystems, Foster City, CA) following manufacturer׳s protocol. The labeling was 113: A. fumigatus LF9 glucose (control); 114: A. fumigatus LF9 cellulose; 115: A. fumigatus LF9 xylan; 116: A. fumigatus LF9 starch. Thus, peptides from each test condition were labeled with respective isobaric tags, incubated for 2h at room temperature (20±2°C) and reaction was stopped by adding 100µL water. The labeled peptides were combined together and vacuum-centrifuged to dryness. The labeled samples were acidified with 0.1% trifluoroacetic acid and de-salted using Sep-Pak C18 cartridges and then HPLC fractionated. The iTRAQ labeled peptides were dissolved in buffer A (10mM ammonium acetate, 85% acetonitrile, 0.1% formic acid) and fractionated using ERLIC column (200×4.6mm, 5μm particle size, 300Å pore size) by HPLC system (Shimadzu, Japan) at flow rate of 1.0mLmin−1. The eluted 60 fractions were collected using automated fraction collector, combined to 20 fractions and vacuum dried before LC–MS/MS analysis. The labeled vacuum dried peptides were reconstituted in 3% ACN with 0.1% formic acid and analyzed by QStar Elite mass spectrometer (Applied Biosystems/MDS Sciex) coupled with online microflow HPLC system (Shimadzu). The labeled peptides were separated on a home-packed nanobored C18 column with a picofrit nanospray tip (New Objectives, Wubrun, MA) coupled to the LC–MS/MS system at a constant flow rate of 300nLmin−1. Analysis was carried out in positive ion mode using Analyst QS 2.0 software (Applied Biosystems) and data were acquired with a selected mass range of 300–1600 m/z. Peptides with charge +2 and above were selected for MS/MS. The three most abundantly peptides above a five-count threshold were selected for MS/MS, and dynamically excluded for 30s with 30mDa mass tolerance. Smart information-dependent acquisition (IDA) was activated with automatic collision energy and automatic MS/MS accumulation. The fragment intensity multiplier was set to 20 and maximum accumulation time was 2s. Data acquisition was performed with Analyst QS 2.0 software (Applied Biosystems/MDS SCIEX).