Protein expression under heat wave conditions

Protein ratios from quantitative proteomics of leaf samples collected after a four-day heat wave (42 degree) and control (28 degree) treatment for Eucalyptus grandis. Protein identity and quantity were determined using Proteome Discoverer V1.3 software (Thermo Scientific, United States) run from a local MASCOT server (Matrix Science, London, U.K.). The E. grandis protein sequences available through UniProt (http://www.uniprot.org, n = 44,150 proteins; October 2016) were used as a reference search database. The following parameters were used for peptide to spectrum matching in MASCOT: MS tolerance, ±10 ppm; enzyme, trypsin with one missed cleavage; fragment mass error, 0.1 Da; static modifications, carbamidomethylation of cysteine and 10-plex TMT tags on lysine residues and peptide N-termini; variable modifications, oxidation of methionine and deamidation of asparagine and glutamine. Spectra were also searched against a reversed-sequenced decoy database to determine false discovery rates (FDR) and filtered at a maximum FDR cut-off of 1% at the protein level. Only peptides below the MASCOT significance threshold filter of p = 0.05 were included in the final dataset, and proteins with at least two unique peptides were regarded as confident identifications. Relative quantification of proteins was achieved by pairwise comparison of TMT intensities, using the ratio of the labels for each of the treatment replicates (numerator) versus the labels of their corresponding pooled control reference (denominator).