C18 macro spincolumns

Samples previously purified with C18 Macro Spin Columns (Harvard Apparatus) were fractionated using off-gel electrophoresis (OGE) separation using an Agilent 3100 off-gel fractionator as previously described [66 (link)]. Samples were then desalted and purified using MicroSpins C18 columns, dried in a speed-vacuum and stored at −20 °C until analysis. An LTQ Orbitrap Q-exactive Plus mass spectrometer (Thermo Fisher, Waltham, MA, USA) coupled with nano-HPLC was used to analyzed the OGE fractions [42 (link)]. Peptides were reconstituted using 5% ACN and 0.1% FA; were trapped on a 2 cm × 75 µm ID, 3 µm pre-column; and separated on a 50 cm × 75 µm ID, PepMap C18, 2 µm easy-spray column (Thermo Scientific, Waltham, MA, USA). The analytical separation was run for 60 min using a gradient of H2O/FA (99.9%/0.1%; solvent A) and CH3CN/FA (99.9%/0.1%; solvent B) at a flow rate of 300 nL/min. For MS survey scans, the OT resolution, the ion population, the number of precursor ions and the collision energy used have been previously described [39 (link)].

TMT‐labelled phosphorylated peptides were combined at equal amounts for each channel and subjected to HPRP. Labelled peptides were solubilized in 20 mM ammonium formate (pH 8.0) and separated on a Gemini C18 column (250 × 3 mm, 3 μm C18 110 Å pore size; Phenomenex). Using a DGP‐3600BM pump system equipped with a SRD‐3600 degasser (Thermo Fisher), a 40 min gradient from 1 to 90% acetonitrile (flow rate of 0.25 ml/min) separated the peptide mixtures into a total of 40 fractions. The 40 fractions were merged into 12 samples, acidified with 1% (v/v) TFA (pH ~ 2), desalted via C18 Macro SpinColumns (Harvard Apparatus), dried under vacuum centrifugation and resuspended in 2% (v/v) ACN/0.1% (v/v) TFA for LC‐MS/MS analysis.

Phosphopeptide enrichment was similar to previously described (Larsen et al, 2005; Trost et al, 2009, 2012; Lai et al, 2015). Tryptic peptides (5 mg per TMT channel) were resuspended in 1 ml of 2 M lactic acid/50% (v/v) acetonitrile and centrifuged 15,000 × g for 20 min. Supernatants were placed in an Eppendorf LoBind Tube containing 24 mg of titanium dioxide beads (GL Sciences) and vortexed for 1 h at room temperature. Beads were washed twice with 2 M lactic acid/50% (v/v) acetonitrile and once with 0.1% (v/v) TFA in 50% (v/v) acetonitrile. Phosphopeptides were eluted twice with 150 μl of 60 mM ammonium hydroxide (pH > 10.5), then acidified with 40 μl of 20% (v/v) formic acid and desalted via C18 Macro SpinColumns (Harvard Apparatus).

A. niger fungi were obtained from domestic cat hair samples and cultured on an agar medium. D-Glucosamine hydrochloride was obtained from MP Biomedicals (Santa Ana, CA). Chitin polymer (100% acetylated) and N-acetyl-D-glucosamine (> 98.0%) were obtained from TCI (Portland, OR). Chitin polymer (20–30% deacetylated) was obtained from Alfa Aesar. ACS-grade acetone and Optima LC-MS grade formic acid were obtained from Fisher Chemical (Waltham, MA). Hydrochloric acid was obtained from Ward’s Science (Rochester, NY). LC-MS grade water, LC-MS grade acetonitrile, low molecular weight chitosan (96% deacetylated), and ammonium acetate (> 98.0%) were obtained were obtained from EMD Millipore (Burlington, MA). 0.2-μm Captiva Econofilters were obtained from Agilent (Palo Alto, CA). C18 Macro spin columns were obtained from Harvard Apparatus (Holliston, MA).

Samples (40 mg) were resuspended in 800 μL of lysis buffer (0.25% sodium deoxycholate, 1 mM EDTA, 0.5% Igepal in PBS pH 7.4, and protease inhibitor (Roche)), the internal standard was added and the samples were homogenized with a tissue lyser (TissueLyser II, QIAGEN) for 30 min at 30 Hz. After homogenization, lysates were put on rotation at 4°C for 2 h, centrifuged at 21,000 g for 30 min. The supernatants were recovered and incubated with previously blocked protein A magnetic beads (Merck) for 2 h in rotation at 4°C. Beads were recovered and washed with lysis buffer, buffer A (150 mM NaCl, 20 mM Tris in water, pH 7.5) and buffer B (400 mM NaCl, 20 mM Tris in water, pH 7.5). Proteins were eluted with glycine (0.1 M, pH 3) and incubated shaking for 30 min. Cysteines were reduced and carbamylated with TCEP·HCl and IAA respectively. Proteins were finally digested overnight with trypsin at 37°C. Tryptic peptides were purified on C₁₈ Macrospin columns (Harvard Apparatus) according to the manufacturer’s instructions and eluates were dried at room temperature with a vacuum centrifuge (Eppendorf). Dried samples were finally resuspended in a solution of 3% MeCN and 0.1% FA (25 μL). Samples (6 µL) were then injected in the nanoLC-MS system.