ABSTRACT: Herein, proteomic profiling was conducted on susceptible sugarbeet infected with BNYVV to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugarbeet leaf tissue infected with BNYVV (beet necrotic yellow vein virus), quantified, and analyzed by mass spectrometry. A proprietary sugarbeet variety, KWS-rzNI, susceptible to all forms of BNYVV, was provided by KWS (Einbeck, Germany). To enable detection of a broad range of proteins associated with compatible interactions, two sources of BNYVV were used; standard BNYVV pathotype A, originally collected from Spence Field at the USDA-ARS in Salinas, CA, and resistance-breaking BNYVV-IV originating from the field, Rockwood 158, Imperial County, CA. Pooled leaf tissue from each cup was ground in liquid nitrogen and lyophilized. Total protein was extracted from 100 mg (dry weight) of lyophilized leaf tissue of each sample using the Plant Total Protein Extraction Kit (Sigma, St. Louis, MO) according to manufacturer’s recommendations. Protease inhibitors were added to liquid protein extracts (Pierce, Rockford, IL), and samples were stored at -80oC prior to analysis. Protein concentrations were determined via Bradford Assay [10] (Thermo Scientific, Rockford, IL) and 30 g of each sample underwent in-solution proteolytic digestion as previously described [11]. Briefly, samples were solubilized in 8 M urea, 0.2% Protease Max (Promega, Madision, WI), then reduced with dithiothreitol, alkylated with iodoacetamide, and digested with 1% Protease Max and trypsin at 37oC for 3 hours. Samples were dried in a Speed Vac® vacuum centrifuge, desalted using Pierce PepClean C18 spin columns (Pierce, Rockford, IL), dried and resuspended in 30 ull 3% ACN, 0.1% formic acid. All solvents, water, and acid were LC-MS/MS grade from Sigma (St. Louis, MO). 1 l of each sample (three replicates from each soil type) was analyzed in duplicate injections via LC-MS/MS . Peptides were purified and concentrated using an on-line enrichment column (Thermo Scientific 5m, 100 m ID x 2 cm C18 columns). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Thermo Scientific EASYnano-LC, 3m, 75 m ID x 100 mm C18 column) using a 90 minute linear gradient from 10%-30% buffer B (100% ACN, 0.1% formic acid) at a flow rate of 400 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific Orbitrap Velos Pro) and spectra collected over a m/z range of 400-2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific) with a S/N threshold of 1.5 and 1 scan/group. MS/MS spectra were searched against a Uniprot Amaranthacae database (3,645 entries, version 03/06/13) concatenated to a reverse database and the Uniprot Mus musculus database (50,665 entries, version 03/06/13) using the Mascot database search engine (Matrix Science, version 2.3.02) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research). Due to the limited size of the Amaranthaceae database, the Mus musculus sequences were added to ensure adequate database size for statistical scoring and calculation of false discovery rates. The following search parameters were used in Mascot: monoisotopic mass, parent mass tolerance of 20 ppm, fragment ion mass tolerance of 0.8 Da , complete tryptic digestion allowing two missed cleavages, variable modification of methionine oxidation, and a fixed modification of cysteine carbamidomethylation. SEQUEST search parameters were the same except for a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 0.0120 Da. Peptide identifications from both of the search engines were combined using probabilistic protein identification algorithms in Scaffold 4 [13, 14] (Version 4.0.3, Proteome Software, Portland, OR) with protein clustering enabled. All data files were then combined using the “mudpit” option in Scaffold 4 generating a composite listing for all proteins identified. Thresholds were set to 99% protein probability, 95% peptide probability, and a 2 unique peptide minimum was required. The peptide FDR was 0% after manual validation of all proteins identified by 2 unique peptides [15]. Criteria for manual validation included the following: 1) a minimum of at least 5 theoretical y or b ions in consecutive order that are peaks greater than 5% of the maximum intensity; 2) an absence of prominent unassigned peaks greater than 5% of the maximum intensity; and 3) indicative residue specific fragmentation, such as intense ions N-terminal to proline and immediately C-terminal to aspartate and glutamate.
Carolyn Broccardo 