4.5. MALDI-TOF/TOF MS/MS and Database Search

Images of the stained gels were captured with a scanner (UMAX Powerlook 2100 XL; UMAX, Taiwan, China). Spot detection, matching, and background subtraction were performed using the ImageMaster 2D Platinum software (version 6.0; Amersham Biosciences, Uppsala, Sweden), followed by manual editing. All the spots detected in each gel were matched with the corresponding spots from the reference gels. To exclude the likely differences introduced by sample loading or gel staining/destaining, the normalized relative percent volume values (% volume) of the protein spots from three replicates were used for further statistical analysis. Selected spots were digested with gold grade trypsin, and then analyzed by a MALDI-TOF/TOF tandem mass spectrometer ABI4800 proteomics analyzer (Applied Biosystems, Framingham, MN, USA). For protein identification, the acquired MS/MS data were uploaded on the Protein Pilot software (Applied Biosystems, Framingham, MN, USA) and compared against P. trichocarpa genome (V3.0) database (https://phytozome.jgi.doe.gov/pz/portal.html). Proteins identified with a Mowse score ≥60 (p < 0.05) were reported. To annotate the identified proteins, the Gene Ontology (GO) was used to classify the proteins into three main classes, biological process (BP), molecular function (MF), and cellular component (CC). In addition, the enriched GO terms were slimmed in REVIGO web server [23].