Computational protocol: Proteome quantification of cotton xylem sap suggests the mechanisms of potassium-deficiency-induced changes in plant resistance to environmental stresses

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Protocol publication

[…] Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the EASY-nLC 1000 system (Thermo Finnigan) with a trap column (EASY column SC001 traps; 150 μm × 20 mm (RP-C18)) and analysis column (EASY column SC200; 150 μm × 100 mm (RP-C18)). Each sample was auto-sampled into the trap column and separated by the analysis column at a flow rate of 400 nl/min. The analysis column was balanced with 100% mobile phase A (water solution with 0.1% formic acid and 2% acetonitrile). Peptides were eluted with a linear gradient from 0–45% mobile phase B (water solution with 0.1% formic acid and 84% acetonitrile) for 100 min and 45–100% B for 8 min and maintained at 100% for 12 min. The eluate was electro-sprayed into a Q-Exactive Orbitrap Mass (Thermo Finnigan) for 120 min. The Q-Exactive was operated with one full precursor scan scope (m/z 300–1800) (MS1 scan) and in a HCD top 10 mode (MS2 scan). The resolution was 70,000 for the full scan and 17,500 for the fragments (both specified at an m/z of 200). The exclusion time was 90 sec. Raw files were processed using MaxQuant version 1.3.9.3 (http://www.maxquant.org/) with the iBAQ and match between runs (match time window 2 min) options. For protein identification, the MS/MS spectra were automatically searched by MaxQuant against the target/reverse UniProt Eudicotyledons database (FASTA-formatted protein sequence database). The identified proteins were further statistically and bioinformatically analyzed using Perseus version 1.3.04. The fixed modification was carbamidomethyl (C). The variable modifications were oxidation (M) and acetyl (protein N-term). The initial mass tolerances for the full scans were 6 ppm and 20 ppm for MS/MS. Two missed cleavages were allowed. The peptide and protein false discovery rates (FDR) were both set to 0.01. [...] The gene ontology (GO) annotations in terms of cellular components, molecular functions and biological processes for the identified proteins were obtained from http://www.uniprot.org. The theoretical molecular weights (MWs) and isoelectric points (pIs) of the proteins were collected from http://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/. The proteins were predicted for secretion with a signal peptide using SignalP (www.cbs.dtu.dk/services/SignalP/) and without a signal peptide using SecretomeP (www.cbs.dtu.dk/services/SecretomeP). In addition, the non-secreted proteins that were predicted by SignalP and SecretomeP were predicted by TargetP (www.cbs.dtu.dk/services/TargetP) for their locations. [...] To obtain comprehensive transcription profile of proteins listed in for K-deficient cotton root, we use the Illumina Hiseq2000 to perform high-throughput RNA-seq of K-deficient root and K-efficient root. In total, 8.99 Gb of raw RNA-seq data were generated (BGI-Tech., China).RNA-seq reads were mapped to the cotton genotype TM-1 genome using Tophat (Version 2.0.8). To measure the gene expression level in LK and NK root tissues, we calculated the expression of each gene using FPKM (Fragments per Kilobase of exon model per Million mapped reads) with Cufflinks (Version2.1.1) (http://cufflinks.cbcb.umd.edu/).We analyzed the gene (corresponding to proteins listed in ) expression changes of K-deficient cotton root, compared with K-efficient root, and present a heatmap for the coordination of gene transcription and protein expression by using software MultiExperiment Viewer (MeV). […]

Pipeline specifications

Software tools MaxQuant, Perseus, EMBOSS, SignalP, SecretomeP, TargetP
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Gossypium hirsutum
Chemicals Potassium