Comparative proteome analysis in different media using three Xanthomonas species
Purpose -Comparison of proteome incubated in different media (rich and minimal media) -Three Xanthomonas species were used (Xanthomonas oryzae pv. oryaze, X. campestris pv. vesicatoria, and X. axonopodis pv. glycines. -Shotgun proteomic was used
Sample Processing Protocol
Bacterial strains and growth conditions Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A, X. campestris pv. vesicatoria (Xcv) strain 85-10, and X. axonopodis pv. glycines (Xag) strain 8ra were used in this study [1-3]. For Xoo, peptone sucrose (PS) medium (peptone 10 gL-1, sucrose 10 gL-1, L-glutamic acids 1 gL-1) and XOM2 were used as rich and minimal media, respectively. For Xcv and Xag, tryptic soy (TS) medium (Tryptic Soy Broth Soybean-Casein Digested 30 gL-1). tryptic soy (TS) medium (Tryptic Soy Broth Soybean-Casein Digested 30g/L). In minimal media, XOM2 was used for XOO and XVM2 was used for XCV and Xag. Protein extraction and Peptide preparation Bacteria cells grown to 0.6 at OD600 were harvested by centrifugation (15 min, 7,300 x g, 28 oC). The 0.5 g (wet weight) of cells were resuspended in 1 ml of 50mM Tris-HCl (pH7.8) and centrifuged again (10 min, 7,300 x g, 28 oC). This washing process was repeated two times. After removing the supernatants, the pellets were resuspended in 1ml of lysis buffer (6 M Guanidine-HCl, 10mM DTT, 50 mM Tris-HCl, pH 7.8). Resuspended cells were disrupted by Ultrasonic Processor (Cole Parmer) ten times (10 s on / 60 s off cycle) in ice, and centrifuged (20 min, 8,000 x g, 4 oC). After collecting supernatants, concentration of proteins was quantified by BCA protein assay kit (ThermoFisher) and 1,000 μg of samples were used for the next step. Samples were incubated for 1 hr at 60℃, alkylated by treatment of 100mM iodoacetamide in the dark chamber at room temperature for 1 hr, and then incubated again with 20 mM DDT (final concentration) at room temperature for 1hr. For protein precipitation, 0.3 volumes of ice-cold trichloroacetic acid were added into the samples, and incubated at 4oC for 12 hrs. After centrifugation (30 min, 15,700 x g, 4 oC), pellets were resuspended in 1ml of acetone (Sigma), and centrifuged again (30 min, 15,700 x g, 4 oC). This process was repeated three times. The pellets were dried in clean bench and dissolved in the start volume of 50mM ammonium bicarbonate (pH 7.8). For digestion of proteins, after 5 μg of trypsin (Promega) were treated in 200 μg of extracted proteins, the mixture were incubated for 24 hrs at 37 oC. Trypsin-treated samples were acidified with 0.4% trifluoroacetic acid (TFA, Sigma), and loaded into the Sep-Pak Vac 1cc tC18 cartridges (Waters) for cleaning samples. After washing the cartridges by 1 ml of 0.1% TFA, samples were eluted with 1 ml of elution buffers (0.1% TFA, 50% acetonitrile). Eluted samples were dried by Speed Vac concentrator (Vision), dissolved in 30 μl of 0.4% acetic acid, and quantified the protein by BCA protein assay kit (ThermoFisher). Samples were diluted to 1 μg/μl for mass spectrometry analysis.
Data Processing Protocol
Mass spectrometry analysis Tryptic peptide mixtures from each sample (2 μl, ~ 2 μg) were analyzed by split free nano LC (EASY-nLC II, ThermoFisher) connected to LTQ Velos Pro instrument (ThermoFisher) via a nanospray ion mode. Sample digested were separated by a column packed in-house with 7.5 cm of MAGIG C18AQ 200A (5 μm) material (Michrom). Peptides were eluted over a 420 min gradient at a flow rate of 300 nL/min by a water/ACN gradient (Solvent A, Water with 0.1% Formic Acid; Solvent B, 100% Acetonitrile with 0.1% Formic Acid) consisting of 5 min 7% B, 380 min gradient to 35% B, and 10 min to 80% B with final hold at 7% B for 25 min. Full MS spectra were acquired in 6 data dependent MS/MS scans over a mass range of m/z 300–2000. Dynamic exclusion was allowed with a repeat count of 1, repeat duration of 0.5 min, and exclusion duration of 3.0 min, with charge state selection enable to preferentially select 2+ and 3+ ions. Peptides were transferred with a 1.8 kV spray voltage and a desolvation capillary temperature of 200 oC. Up to the 6 most intense ions in each full MS scan were consecutively collected for fragmentation and examined in centroid mode within the linear ion trap part of the instrument. Three biological replicates were carried out. Protein/Peptide identification and quantification Thermo Proteome Discoverer 1.3(ver. 188.8.131.529) with SEQUEST search algorithm was used for interpretation of acquired MS/MS spectra. Spectra were searched against the Xcv strain 85-10 database. Trypsin was fixed as an enzyme, and two missed cleavage was allowed. All accepted Xcv peptides had a false discovery rate (FDR) of 0.01 with reversed database searches, and precursor mass accuracies of 100 ppm. In addition, probability scores of all peptides were >20.Integrated decoy database search were carried out. The oxidation of methionine was fixed as a possible modification. Proteins matched at least two unique peptides were considered as present in the sample. And then, proteins identified were re-imported into scaffold 4 proteome software for visualization of proteomics data and comparison between biological samples. For comparative analyses, each protein were normalized with total peptide spectra matches (PSM). The average of normalized PSMs was calculated per protein and used as a comparison value to identify differently expressed proteins between xanthomonomas spp. in rich-media and minimal-media, respectively.
Park HJ, Bae N, Park H, Kim DW, Han SW. Comparative proteomic analysis of three Xanthomonas spp. cultured in minimal and rich media. Proteomics. 2017 PubMed: 29044975