Vacuum lyophilizer

A mixture of 1.5% CNF aqueous suspensions and lignin (10 wt% of dry CNF) was poured into an open tube (1.5 inches in length and 0.81 inches in diameter) and sealed with aluminum foil. The samples were flash frozen in liquid nitrogen for 1 min and lyophilized in a vacuum lyophilizer (Labconco, Inc., Kansas City, MO, USA) at −51 °C for 2 days. A columnar aerogel was obtained. The samples were oven heated at 160 °C for 2 h to promote lignin melting to form 3D networks. Finally, CNF-lignin aerogel was prepared.

Eluates were incubated in 10 mM dithiothreitol (DTT) for 45 min and subsequently alkylated with 30 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. IAA was quenched by DTT (20 mM final concentration). Samples were then cleaned up using four volumes of acetone for precipitation, and pellets were resuspended in 50 mM ammonium bicarbonate. Trypsin (Promega) was added to samples at a ratio of 1:50 (Trypsin–protein) and incubated overnight at 37 °C to digest protein. Peptides were acidified with fluoroacetic acid (FA) and desalted using a C18 desalting column equilibrated in 200 mL acetonitrile (ACN), then twice with 200 mL 60% ACN followed by 200 mL 0.1% FA. Samples were loaded onto the C18 column, washed three times with washing buffer (0.1% FA, 2% ACN), then eluted with 60% ACN. Eluents were collected and lyophilised in a vacuum lyophilizer (Labconco, Kansas City, MO, USA) before LC-MS/MS analysis.