Proteomic datasets

The extracellular proteome was extracted as described by Vincent et al. [44], using a modified tricholoacetic acid (TCA)/acetone protein precipitation procedure. Briefly, proteins from the extracellular culture filtrate were precipitated, collected by centrifugation and washed with 100% acetone. The protein pellet was subsequently air-dried at room temperature and suspended in 20 mM Tris pH 7. Residual TCA was progressively removed by dialysis of the suspension using D-Tube Dialyzer Maxi, MWCO 3.5 kDa (Novagen, Darmstadt, Germany) in several changes of 20 mM Tris pH 7 at 4°C for 48 hrs. Solubilised proteins were retained and stored at -80°C until further manipulation.

The intracellular proteome was extracted as previously described by the authors of this study [41]. Briefly, intracellular proteins from mechanically ground freeze-dried mycelia were solubilised in 20 mM Tris-Cl pH 7 and de-salted using a PD10 chromatography column (GE Healthcare, Little Chalfont, UK). Solubilised proteins were retained and stored at -80°C until required.

To facilitate cell wall/membrane (CWM) proteome extraction, freeze-dried fungal mycelia were ground with a mortar and pestle and washed three times with 20 mM Tris-Cl pH 7 to release and remove soluble intracellular proteins. The pellet was then washed three times with 0.1 M Na₂CO₃ to further remove soluble and peripherally-attached proteins. The pellet was then resuspended in 20 mM Tris-Cl pH 7 and subjected to 3 cycles of slow freeze and thaw to further break up the cellular material. Membrane-bound proteins were extracted using two methods: Extraction Procedure 1 (EP1)– 100 mg of membrane enriched pellet was extracted with 2% (w/v) SDS, 100 mM EDTA and 50 mM DTT in 100 mM Tris/HCl (pH7.8) by vortexing and boiling for 5 min followed by 5 min on ice (based on methods presented in Meijer et al., 2006 and Feiz et al., 2006); Extraction Procedure 2 (EP2)– 100 mg of membrane enriched pellet was extracted with 2% (w/v) SDS, 7 M urea, 2 M thiourea and 50 mM DTT in 125 mM triethylammonium bicarbonate (TEAB, pH 8.5) by vortexing and sonication for 15 min in an ice-cold sonication bath followed by resting for 30 min on ice. Vortexing and sonication steps were repeated. Subsequent sample processing for suspensions derived from ‘Extraction Procedures’ EP1 and EP2 were identical. Suspensions were centrifuged at 16,000 x g for 5 min (4°C) and the supernatants removed. Pellets were washed twice with either 100 mM Tris/HCl (pH 7.8) for EP1 or 100 mM TEAB (pH 8.5) for EP2. Respective supernatants were pooled, centrifuged at 20,000 x g for 15 min (4°C) and collected for further processing. Proteins were precipitated from supernatants by the addition 100% TCA to a final concentration of 20% (v/v) and incubated on ice for 30 min. Protein precipitates were harvested by centrifugation at 20,000 x g for 10 min (4°C). Pellets were washed twice with 90% (v/v) acetone and centrifuged each time as before. Protein pellets were briefly dried under a gentle stream of nitrogen and used immediately. The final pellets were re-suspended in 45 μL of EP2 extraction buffer (without DTT) and 5 μL of 1 M TEAB (pH 8.5) by repeated vortexing and incubating the tubes for 10 min in an ice cold sonication bath. Samples were centrifuged at 20,000 x g for 10 min (4°C) and supernatants collected for further processing. Protein concentration of all samples was determined using the 2D-Quant kit (GE Healthcare) according to the manufacturer’s ‘Standard procedure’ protocol. CWM proteins were digested without prior fractionation.

Intracellular and extracellular proteins were separated into 24 fractions using isoelectric point-based fractionation of proteins (Agilent 3100 Offgel fractionator) with liquid-phase recovery followed by digestion and LC-MS analysis of peptides. Offgel separations were performed using high resolution separation kits (pH range 3–10, 24 cm IPG gel strips; Agilent) and approx. 1 mg of protein per strip loading as described previously [45]. The pH of 100 μL aliquots of recovered Offgel fractions was adjusted by adding 10 μL of 1 M TEAB (pH 8.5). CWM sample aliquots of 80 μg protein were diluted with 10 μL of 1 M TEAB (pH 8.5) and the volume adjusted to 110 μL with EP2 extraction buffer (without DTT).

Proteins were reduced with 5 μL of 0.5 M tris(2-carboxyethyl)phosphine (TCEP, 22 mM final conc.) for 2.5 hours at 4°C and then alkylated with 16 μL of 1 M iodoacetamide (122 mM final conc.) in the dark at 22°C for 2 hours. Reducing agent TCEP was dissolved in 100 mM TEAB (pH 8.5) and neutralised with 10 M sodium hydroxide solution to pH 8. Sample proteins were co-precipitated with 1 μg of modified trypsin (Roche, sequencing grade) by adding 10 volumes of methanol as follows: One microliter of trypsin was added to the side of the Eppendorf tube and quickly flushed into the sample solution with 1.3 mL of 100% methanol at -20°C. Tubes were incubated overnight at -20°C. Protein precipitates were harvested by centrifugation at 20,000 x g for 15 minutes (4°C). Pellets were washed twice, once with 1 mL of 90% (v/v) methanol at -20°C and finally with 1 mL of 100% methanol at -20°C and centrifuged each time as before. Protein pellets were briefly dried under a gentle stream of nitrogen and continued immediately. The final pellets were re-suspended in 40 μL of 100 mM TEAB buffer (pH 8.5) containing 5% acetonitrile by repeated vortexing and incubating the tubes for 1 min in the sonication bath. Samples were incubated at 37°C for 2 hours followed by the addition of further 1 μg of modified trypsin and 6 hours (Offgel fractions) and 14 hours (CWM proteome) digestion at 37°C. Protein digests were stored at -80°C until analysis.

For LC-MS/MS analysis, trypsin-digested samples were centrifuged for 5 min at 20,000 x g and an aliquot of 5 μL (Offgel fractions) or 3 μL (CWM proteome) was diluted to a volume of 20 μL with 6.5% formic acid prior to injection. Tryptic peptides were separated on a Prominence nano HPLC system (Shimadzu, Kyoto, Japan) and data collected on a Hybrid LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Mobile phases for chromatographic peptide separation were as follows: Eluent A was milliQ water containing 0.1% formic acid and eluent B was 80% acetonitrile / 20% milliQ water (v/v) containing 0.1% formic acid.

Acidified Offgel fractions were loaded onto a reversed-phase trap column (Dionex Acclaim PepMap μ-Precolumn C18, 0.3 mm x 5 mm) at 30 μL/min in 100% eluent A for 3.5 minutes and subsequently separated on a reversed phase capillary column (Vydac Everest C18 5μm 300 Å, 150 μm x150 mm, Alltech) at 45°C and a flow rate of 1 μL/min. Separation was performed with gradients of 2–30% B over 60 and 90 minutes (depending on sample complexity), followed by a 95% B wash step, resulting in a total run time of 110 and 140 minutes, respectively. Data was either acquired on an LTQ Orbitrap Velos as outlined below or an LTQ Orbitrap XL as described in Hastie et al. [45] with the following modifications to data acquisition: target value of 1 x 10³ for ion trap MS/MS scans; dynamic exclusion set to 70 s; ion selection threshold 1000 counts.

Acidified CWM digests were loaded onto a reversed-phase trap column (ReproSil-Pur C18-AQ 3μm, 0.3 mm x 10 mm; Dr. Maisch, Ammerbuch-Entringen, Germany) and washed for 3.5 minutes at 30 μL/min using 100% eluent A. Peptide mixtures were subsequently back flushed onto a capillary column (150 μm x 150 mm) packed in-house with reversed-phase beads (ReproSil 100 C18 3μm; Dr. Maisch) and separated at a flow rate of 1 μL/min. Peptides were separated at 55°C using a sequence of linear gradients: to 5% B over 3.5 minutes; to 35% B over 166.5 minutes; to 45% B over 10 minutes; to 95% B over 10 minutes and then holding the column at 95% B for 10 minutes. Data was acquired on an LTQ Orbitrap Velos Pro as described below.

Column-separated peptides were electrosprayed into the LTQ Orbitrap Velos and LTQ Orbitrap Velos Pro through a Nanospray Flex Ion Source (Thermo Fisher Scientific) using 30 μm inner diameter uncoated silica emitter (New Objective). Spray voltage was 1.5 kV with no sheath, sweep or auxiliary gases used. The heated capillary temperature was set to 250°C and 285°C for the LTQ Orbitrap Velos and LTQ Orbitrap Velos Pro, respectively. An S-lens value of 50 to 55% was used.

The LTQ Orbitrap Velos (OT Velos) and LTQ Orbitrap Velos Pro (OT Velos Pro) were controlled using Xcalibur 2.2 software (Thermo Fisher Scientific) and operated in data-dependent acquisition mode to automatically switch between Orbitrap-full scan MS and ion trap- MS/MS acquisition. Full scan MS spectra (OT Velos: m/z 300–2000; OT Velos Pro: m/z 380–1700) were acquired in the Orbitrap mass analyser with a resolving power set to 30,000 (OT Velos) and 60,000 (OT Velos Pro) at 400 m/z after accumulation to a target value of 1 x 10⁶ in the linear ion trap. The top 20 (OT Velos) and 15 (OT Velos Pro) most intense ions with charge states ≥ +2 were sequentially isolated with a target value of 5,000 and fragmented using collision-induced dissociation (CID) in the linear ion trap. ‘Rapid’ scan mode was selected for the ion trap- MS/MS acquisition in the OT Velos Pro. Fragmentation conditions were set as follows: 35% normalized collision energy; activation q of 0.25; 10 ms activation time; ion selection threshold 1000 (OT Velos) and 5000 (OT Velos Pro) counts. Maximum ion injection times were 200 ms for survey full scans and 50 ms for MS/MS scans. Dynamic exclusion was set to 70 s and 90 s for OT Velos and OT Velos Pro runs, respectively. Lock mass of m/z 445.12 was applied with an abundance was set at 0%.

Mass spectra were then searched using the Tide search engine [46] implemented in the Crux toolkit [47] with specifications as follows: spectra mapped against: 6-frame translations of both the new and the old genome assemblies and the set of predicted protein sequences from both the new and the old annotations. The search parameters used were: variable modifications, oxidation (M); and deamidation (NQ); fixed modification, carbamidomethyl (C); peptide tolerance, 20 ppm; MS/MS tolerance: ±0.8 Da; Digestion enzyme: trypsin; maximum missed cleavages: 1. Peptide-spectrum matches were refined using Percolator [48], again as implemented in the Crux toolkit.

For 1D-LC MALDI MS/MS analysis of the SN15 extracellular proteome, SN15 trypsin-digested peptides were resuspended in 20 μl of 2% acetonitrile and 0.05% trifluoroacetic acid. Peptides were loaded onto a C18 PepMap100, 3 mm column (Dionex, CA, USA) through the Ultimate 3000 nano HPLC system (Dionex, CA, USA). Mass spectrometry analysis was carried out on a 4800 MALDI TOF/TOF Analyser as previously described [49]. These spectra were also searched using the Tide search engine [46] with specifications: variable modifications, oxidation (M); fixed modification, carbamidomethyl (C) and other parameters and post-processing as above.

Conflicts with existing annotations were identified where proteomic spectra searched against the six-frame translation of the genome mapped into intergenic regions, intronic annotations or coding regions in the wrong frame.