Humic Substances iTRAQ LC-MS/MS
iTRAQ analysis of Arabidopsis Thaliana roots treated with different Humic Susbstances.
Sample Processing Protocol
Roots were ground in liquid nitrogen and proteins precipitated with Acetone/TCA. Protein pellets were washed with cold acetone and suspended in 6M urea, 50 mM triethylammonium bicarbonate pH 8.5, 2% CHAPS and 1% PVPP. Proteins were quantified. Equal amounts of proteins from each sample were loaded onto a SDS-PAGE 4-12% and the electophoretic run was stopped after all protein extracts entered the running gel. Three biological replicates were prepared. Gel bands were cut and proteins were reduced (DTT) alkylated (iodoacetamide) and digested in-gel with trypsin. Peptides were extracted from the gel and subjected to iTRAQ labelling with a tag-swapping strategy. Samples were pooled and SCX fractionation was performed using a step-wise elution with KCl. Samples were desalted with C18 cartrdiges and analyzed by LC-MS/MS using a nano-HPLC Ultimate 3000 (Dionex- Thermo) interfaced to a LTQ-OrbitrapXL MS (Thermo). The instrument worked with a Top 3 DDA method: 1 full MS scan at 30000 resolution followed by MS/MS spectra on the top 3 ions both with CID and HCD fragmentation.
Data Processing Protocol
Raw data files were analyzed with Proteome Discoverer 1.4 (Thermo) interfaced to a Mascot server (2.2.4). Spectra were searched against the ARATH section of the Uniprot Database with a MudPIT protocol. Enzyme specificity was set to trypsin with up to 2 missed cleavages. Peptide and fragment tolerace was set to 10 ppm and 0.6 Da respectively. carbamidomethylation of cysteines and 4-plex iTRAQ modification at peptide N-term and K residues were set as fixed modifications. Met oxidation was set as variable modification. Percolator was used to assess FDR and data were filtered to keep only proteins identified with at least 2 unique peptides with q-value < 0.05. Proteins were merged inot protein families according to the principle of maximum parsimony.
Roomi S, Masi A, Conselvan GB, Trevisan S, Quaggiotti S, Pivato M, Arrigoni G, Yasmin T, Carletti P. Protein Profiling of Arabidopsis Roots Treated With Humic Substances: Insights Into the Metabolic and Interactome Networks. Front Plant Sci. 2018 9:1812 PubMed: 30619394