Refrigerated vapor trap

A Thermo Scientific Savant Speedvac high capacity concentrator in combination with a Thermo Scientific Savant Refrigerated Vapor Trap was used for evaporation under vacuum of the samples during sample pretreatment. An ultrasonic cleaner from Branson was used for reconstitution and homogenization of the residue.
The HPLC-MS/MS system consisted of an Ultimate 3000 autosampler and pump with inline degasser, all of Dionex (Sunnyvale, CA, USA), connected to a degasser from LC Packings. The autosampler with a 100 μL sample loop was coupled to an AB Sciex API4000 mass spectrometer (Framingham, MA, USA) via a Shimadzu Prominence Column Oven (type CTO-20A) which was kept at 35 °C. Separation was performed with an Agilent column 2.1 × 100mm packed with material of Zorbax Extend 3.5 μm C-18 and kept at 35 °C using a CTO-20A column oven (Shimadzu, Kyoto, Japan). Instrument control and data acquisition was done using Analyst v1.6.2 and DCMS-link v2.12 software (Dionex).

Avicel (0.2%), phosphoric acid swollen cellulose (0.2%) and carboxy-methyl cellulose (0.05%) at 13.12nM SMECel6A (1 µg mL⁻¹) for 1 h at optimum conditions. To sample, the reaction was centrifuged at 4600 rpm for 1 min and 4 volumes of 100% 4 °C ethanol added to precipitate proteins and impurities. The hydrolysate was dehydrated in a refrigerated vapor trap (Thermo). Samples were re-suspended in the original volume taken and analysed with ion exchange as above. Apparent processivity (P_app) was determined semiquantitatively as the molar ratio of cellobiose/(glucose + cellotriose)^{49 (link),50 (link)}.