Quantitative Proteomic Analysis of Hemp

50 μg of protein were labelled with Cy-dyes. Following the labelling, the samples were handled as described in [65 (link)]. The analysis of the gel images was performed with the DeCyder™ software (GE Healthcare, v. 7.0.8.53). Spots were considered as significantly different when detected on at least 75% of analysed gel images, protein abundance with a minimum fold change of 1.5 with a Student’s t-test p-value below 0.05 [Additional file 3]. Following MALDI analysis, the mass spectra of digested peptides were identified by carrying out a MASCOT database search against our in-house hemp transcriptome database (170,598 sequences; 64,508,806 residues) annotated using Blast2GO PRO version 3.0 against the A. thaliana non-redundant database and the NCBI Viridiplantae database, with the following parameters: fixed modifications: carbamidomethyl (C); variable modifications: dioxidation (W), oxidation (HW), oxidation (M), Trp → kynurenin (W); mass values: monoisotopic; peptide mass tolerance: ± 100 ppm; fragment mass tolerance: ± 0.5 Da and Max number of missed cleavages: 2. Individual ions scores greater than 42 indicate identity or extensive homology (p < 0.05), protein scores greater than 65 are significant (p < 0.05). A protein was identified with only one peptide if the individual ion score was higher than 84. Principal Component Analysis (PCA) was performed with the DeCyder™ software.