Hemicellulose

Culture protein lysates were prepared by French press of glucose and hemicellulose cultures in mid-log phase and from cellulose cultures after 2 weeks of growth. Supernatant proteins were isolated by three rounds of centrifugation (5k, 15 min) with transfer of the supernatant to a fresh tube, followed by 0.1 μ filtration. Proteins were precipitated with ¼ volume of 100% (w/w) trichloroacetic acid (TCA) and incubated for 60 min at 37°C in 1% SDS, 0.2 M NaOH, and 10 mM DTT. Cysteines were alkylated with 30 mM iodoacetamide at room temperature in the dark for 60 min. Proteins were again TCA precipitated and resuspended in 50 mM Tris–HCl (pH 7.6), 1 M urea and digested overnight at 37°C with sequencing grade trypsin (Promega, WI, USA) in a ∼50:1 substrate-to-enzyme ratio.
Peptide solutions were acidified with trifluoroacetic acid (TFA) to a final concentration of 0.5% and 500 μg of peptides were subjected to C18 reversed-phase extraction using a 100 mg Sep-Pak cartridge (Waters, MA). Following sample loading and a 4 ml wash step with 0.1% TFA, the cartridge was washed with 2 ml of 0.2 M citric acid/sodium phosphate buffer (pH 5.5) and peptides were labeled through ReDi with 8 ml of 0.8% formaldehyde (D₂ labeled or in normal isotopic distribution, Sigma, MO) and 0.12 M sodium cyanoborohydride (Sigma) or sodium cyanoborodeuteride (CDN Isotopes, Canada) for 8 min. After washing with 0.1% TFA and 0.5% acetic acid AcOH, labeled peptides were eluted with 40% acetonitrile (ACN), 0.5% AcOH and 80% ACN, 0.5% AcOH. Light and heavy peptides were mixed and separated into 16 fractions by immobilized pH gradient isoelectric focusing (IPG-IEF) on a 18 cm pH 3–10 IPG strip using a IPGphor apparatus (Chick et al, 2008 (link)) (GE Healthcare, WI). Peptides were desalted using C₁₈-StageTips (Rappsilber et al, 2003 (link)) and analyzed by microcapillary lLC-MS/MS using a hybrid dual pressure linear ion trap/Orbitrap mass spectrometer (LTQ Orbitrap Velos, Thermo Scientific, Bremen, Germany) essentially as described (Haas et al, 2006 (link)). Peptides were dissolved in 5% formic acid (FA) and 5% ACN and ∼1 μg was separated on a 100 μm × 20 cm C₁₈-reversed phase (Magic C18AQ, Michrom Bioresources) column applying a gradient from 10% ACN to 32% ACN in 0.125% FA over a 75 or 100 min gradient at a flow rate of ∼300 nl min⁻¹. The mass spectrometer was operated in a data-dependent mode with a full MS scan (resolution, 60 000) acquired in the Orbitrap analyzer followed by linear ion trap MS/MS spectra on the 20 most abundant ions detected in the full MS spectrum. Automatic gain control (AGC) targets were 3 × 10⁶ for the full MS and 2000 for MS/MS, maximum ion accumulation times were set to 1000 ms (MS) and 150 ms (MS/MS). Fragmented peptide precursor ions were excluded from further selection for MS/MS for 20–60 s (see RAW data). The RAW data files have been deposited in Proteome Commons (http://www.proteomecommons.org) and can be downloaded using the following hash key:

Peptides were identified from MS/MS spectra using SEQUEST and were filtered to a 1% MS/MS spectra assignment false discovery rate using a target-decoy sequence database (Elias and Gygi, 2010 (link)) of the 3926 C. phytofermentans (NCBI NC_010001.faa) proteins, common contaminants such as trypsin and human keratins, and a decoy component of the reversed sequences of these proteins. Absolute protein abundances within each treatment were estimated from MS/MS spectral counts using APEX, a machine learning-supported analysis based on spectral counting (see APEX section of Supplementary Information). Extracted ion current peak areas (MS1 peaks) of light and heavy versions of the same peptide were compared to determine their relative concentration differences using VISTA (Bakalarski et al, 2008 (link)). Protein expression changes were calculated as the median peptide concentration change, here called the median MPA ratio. Significant differential expression was assessed using aggregate Z-scores for all peptides in a protein (see ReDi section of Supp Info). Peptide pairs were quantified only when their average signal-to-noise ratio was above five and proteins for which exclusively light or heavy peptides were detected are reported only if their identification was based on at least two unique peptides. After this additional filtering, the protein false discovery rate for each experiment was lower than 5%. Expression measurements are from individual cultures (APEX) or culture comparisons (ReDi). Replicate experiments were run to establish high reproducibility between biological replicates for APEX (Supplementary Figure S10) and for ReDi labeling of biological duplicates (Figure 3D), treatment comparisons (Figure 3E), and differentially labeled samples mixed at various ratios (Supplementary Figure S11).

Myceliophthora thermophila was cultured in 1× VMM supplemented with 2% Avicel, 2% xylan, or AX to induce the production of cellulases or hemicellulases. The protein concentration in the supernatant was determined using the Bio-Rad Protein Assay kit (absorbance of 595 nm) (Bio-Rad, Hercules, CA, USA). The hemicellulose/cellulose hydrolysis reaction system included the condensed culture (50 μg protein), 2% substrate (Avicel or xylan), and 1 μg/mL sodium azide in 1 mL 1× VMM. The samples were incubated at 45 °C for 24 h with shaking at 150 rpm and then centrifuged (14,000 rpm for 15 min at 4 °C). The supernatants were filtered through a 0.22 μm membrane and analyzed using an HPLC system.