Computational protocol: Data for the characterization of the HSP70 family during osmotic stress in banana, a non-model crop

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[…] Musa HSP70 nucleotide and protein sequences were obtained from GreenPhyl and the Banana Genome Hub . Since many HSP70 genes were incorrectly predicted, all HSP70 predicted from the Acuminata A genome sequences were manually curated as well as the cytoplasmic and luminal HSP70 sequences predicted from the Balbisiana B genome. The manually curated B genome cytoplasmic and luminal protein sequences can be found in . The manually curated A genome protein sequences are available at the Banana Genome Hub. Cytoplasmic HSP70 from rice and Arabidopsis thaliana were retrieved from Greenphyl with the accessions as described by Jung et al. . Alignments of protein sequences were created using the ClustalX 2.1 software . An alignment of the main cytoplasmic HSP70 isoforms identified later in this manuscript can be found in . Phylogenetic trees were constructed via ClustalX 2.1 using the neighbor-joining algorithm with 1000 replicate bootstrap tests. Trees were visualized with njplot . The phylogenetic relationship between all curated cytoplasmic HSP70 protein sequences of the Musa A genome (GSMUA_Achr) and the cytoplasmic HSP70s of rice (LOC_Os) and Arabidopsis (AtHsp) can be found in . [...] Meristem and root proteins were extracted and analyzed using the phenol extraction/ammonium acetate precipitation protocol reported by Carpentier et al. . 50 µg of proteins was labeled with Cy2, Cy3 and Cy5 (GE Healthcare) for a total of 150 µg protein per gel, separated on gel and scanned according to Carpentier et al. . Data were analyzed using the DeCyder software version 7.0 (GE Healthcare). Statistical analysis of the standardized abundance of spots was performed in DeCyder. Statistical analysis of the raw spot intensities was performed using ANOVA in STATISTICA software 10 on the log of the peak height of the internal standard samples exported from DeCyder. Dynamic abundance profiles of the root HSP70 spots 1, 2, 3, 4, 5 and 6 after 0, 1, 4 and 14 days of stress (n=3) can be found in .For protein identification, gel pieces were extracted based on the protocol of Shevchenko et al. for the in-gel reduction, alkylation and destaining of the proteins. The destaining step was performed twice after which the gel pieces were covered with 3 µL of 0.1 µg/µL trypsin and 47 µL trypsin buffer (25 µM ammonium carbonate, 10% acetonitrile (ACN)). Digestion was performed overnight at 37 °C. Peptides were extracted by adding 100 µL 5% ACN in 0.1% FA, vortexing, centrifuging and sonicating for 5 min after which the supernatant is removed to a new eppendorf tube. The whole peptide extraction process is repeated twice with 50 µL 10% ACN in 0.1% FA the first time and 50 µL 95% ACN and 5% FA the last time. The accumulated supernatant was then dried in a vacuum centrifuge and stored at −20 °C. Before analysis, the samples were resuspended in 0.1% FA and 5% ACN, desalted using C18 Zip Tips (Millipore) and eluted in 10 µl Milli-Q water with 0.1% FA and 60% ACN, dried in a vacuum centrifuge and resuspended in 0.1% FA and 5% ACN.The HPLC-MS/MS analysis was performed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, USA). The samples (5 µL) were injected and separated on an Ultimate 3000 HPLC system (Dionex, Thermo Scientific) equipped with a C18 PepMap100 precolumn (5 µm, 300 µm×5 mm, Thermo Scientific) and an EasySpray C18 column (3 µm, 75 µm×15 cm, Thermo Scientific) using a gradient of 5–20% ACN in 0.1% FA in 10 min followed by a gradient of 10–35% ACN in 0.1% FA in 4 min and then a final gradient from 35% to 95% ACN in 0.1% FA in 2.5 min. The flow-rate was set at 250 µL/min. The Q Exactive was operated in positive ion mode with a nanospray voltage of 1.5 kV and a source temperature of 250 °C. ProteoMAss LTQ/FT-Hybrid ESI Pos. Mode CalMix (MSCAL5-1EA SUPELCO, Sigma-Aldrich) was used as an external calibrant and the lock mass 445.12003 as an internal calibrant. The instrument was operated in data-dependent acquisition (DDA) mode with a survey MS scan at a resolution of 70,000 (fwhm at m/z 200) for the mass range of m/z 350–1800 for precursor ions, followed by MS/MS scans of the top 10 most intense peaks with +2, +3 and +4 charged ions above a threshold ion count of 16,000 at 35,000 resolution using normalized collision energy (NCE) of 28 eV with an isolation window of 3.0 m/z and dynamic exclusion of 10 s. All data were acquired with Xcalibur 2.2 software (Thermo Scientific). For identification, all raw data were converted into mgf files using Progenesis v4.1 (Nonlinear Dynamics, UK). The spectra were searched using Mascot (version 2.2.04) against our in-house Musa database (76,220 sequences) containing all the protein sequences of the published A and B genome plus contaminant sequences (trypsin and keratin). Redundancy was eliminated from the database using the program cdhit . If both A and B isoforms were identical, the B genome isoform was eliminated. The original HSP70 protein sequences were removed and replaced by the manually curated HSP70 sequences. Search parameters were set at: tryptic digestion, one miscleavage allowed, 10 ppm precursor mass tolerance and 0.02 Da for fragment ion tolerance with a fixed modification of cysteine carbamidomethylation and a variable modification of methionine oxidation.The advantage of an LC-separation is nicely illustrated by the separation of the peptides FSDSSVQSDIK (encoded by gene GSMUA_Achr7T15160) and YSDASVQSDIK (encoded by gene GSMUA_Achr10T00900). These two isoforms of the peptide have the same monoisotopic mass but have different retention times on the RP column because of their different hydrophobicity (approximately 19 and 17 min) (). This would have resulted in a chimeric spectrum using MALDI-TOF/TOF MS but produces separate spectra using LC-MS/MS.An isoform was retained as positively identified in a spot if at least one tryptic specific peptide was found with an ion score higher than the Mascot identification score. Cytoscape v3.0 software was used to visualize tryptic specific peptides . contains a list of all identified HSP70 paralogs and allelic variants per spot based on this Mascot analysis. To quantify the different protein species in each spot, Mascot emPAI was exported and the ion intensity of the proteotypic peptide for each peptide was analyzed in Progenesis v4.1. Moreover, for all isoforms positively identified in at least one spot, we searched the unidentified MS/MS spectra in each spot in which they were not identified by performing a manual SRM approach. The ion intensity for a MS/MS spectrum was added to the quantification when the peptide fragment mass corresponded to the proteotypic peptide and a specific signature m/z was identified in the MS/MS spectrum. Several spots in a trail contain multiple proteins even on a 24 cm 3 pI zoom strip, as already been indicated by Schmidt et al. . Although all the spots look well separated on the 2-DE gels they consist of several proteins caused by neighbor spots. The isoelectric focusing of one particular isoform is not restricted to one physical location in the gel and each isoform has its highest abundance at a particular isoelectric point (). provides an overview of the ion intensity of the proteotypic peptide of each identified paralog and/or allelic variant in all spots. […]

Pipeline specifications

Software tools Clustal W, NJplot, Statistica
Databases The Banana Genome Hub
Applications Miscellaneous, Phylogenetics
Organisms Musa acuminata
Diseases Multiple Sclerosis