Protein solution method for direct identification of Arabidopsis stem proteins

Identifying the intracellular and cell wall-ionically bound glycoside hydrolases (GHs) carbohydrate esterases (CEs) at the late growth stage of Arabidopsis stems is important for understanding the mechanisms regulating the cell wall integrity. The fact that whole plant stems are used as biomass feedstocks and the mixing of intracellular proteins with the cell wall proteome caused by an increasing proportion of broken cells of stems at the late growth stage pose a challenge in identifying these GHs and CEs. Here, we used a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by protein solution method for direct identification of stem proteins, and by SDS-PAGE separation and protein gel band method for improving the identification of stem proteins, using Nano-LC-MS/MS analysis. Totally, 75 and 236 stem proteins were identified by using these two methods, respectively, confirming the later method (i.e. protein gel method) identified three time more stem proteins. Among these proteins, based on cell wall protein databases and data mining analyses, 6 and 22 proteins were identified as cell wall proteins from the late growth stage Arabidopsis stems, by using these two methods respectively.