Project description:P. savastanoi pv. phaseolicola isolate R5 was cultured in 10 mL Luria broth with 400 uM genistein or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform. Replicate R5 cultures of each treatment were centrifuged at 5,000 x g for 10 min., washed once in 1 mL phosphate buffered saline, centrifuged at 5,000 x g for 10 min., resuspended in 1 mL of 1:1:1:1 acetone/acetonitrile/methanol/water and 1,2500 pmol prednisone, and pulverized in the bead mill as before. Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without genistein). The milled extracts were centrifuged for 20 minutes at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 uL 50% methanol/0.1% formic acid. Fifteen uL of each sample was pooled to create a quality control (QC) sample. Five uL of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Three subsequent injections of the QC were performed to generate MS2 spectra. After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank 1, QC 1, sample replicate set 1, Blank 2, QC 2, sample replicate set 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. The samples also were analyzed in negative ion mode at -2,500 V but with the same other settings.

Project description:R5 was cultured in 10 mL Luria broth with 1.0 mM resveratrol or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform until the cultures without resveratrol reached an average optical density (O.D.) of 0.78 (mid-log phase growth) at 600 nm. Protein was prepared by centrifuging the cultures at 5,000 x g, washing the cells two times in 5 mL phosphate buffered saline, resuspending the cells in 1 mL 100 mM Tris pH 8.5\1 mM EDTA\10 mM DTT\0.5% n-dodecyl beta-D-maltoside\Proteinase Inhibitor cOmplete [1 tablet/10 mL, Roche Diagnostics, Indianapolis, IN], and pulverizing the cells with 0.5 mm glass beads with a Qiagen PowerLyzer 24 bead mill (Qiagen, Hilden, Germany) 10 times at 5,000 rpm for 20 seconds (cooled on ice for 2 minutes between cycles). The extracts were centrifuged at 10,000 x g for 5 minutes, and the supernatants sent to the Thermo Fisher Scientific Center for Multiplexed Proteomics at the Harvard Medical School. Protein (~300 ug) was precipitated from the supernatants with methanol/chloroform, reduced in Tris(2-carboxyethyl)phosphine, carboxyamidomethylated with iodoacetamide, and digested sequentially with LysC and trypsin using methods previously described (Isasa et al., 2015). Peptides (~120 ug) were labeled with Tandem Mass Tags (TMTpro; Thermo Fisher Scientific) per the manufacturer instructions. Tags 126, 127N, and 127C were assigned to control replicates, and 128N, 128C and 129N were assigned to resveratrol-treated replicates. Samples were analyzed to determine tag label incorporation percentage (>95%) and to estimate quantitative ratios between samples. On the basis of the quantitative ratios, labeled samples were mixed together and fractionated by high-pH reverse phase HPLC (Isasa et al., 2015). Twelve concatenated fractions were analyzed. Peptides from each fraction were separated using a gradient of 3 to 22% of 90% acetonitrile\0.1% formic acid over 150 minutes and were electrosprayed at 2.6 kV into an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) operating in data-dependent mode with positive polarity. Quadrupole isolation was enabled, and survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 350-1500 m/z. The automatic gain control (AGC) target was set to 1,000,000 and the maximum injection time was set to 50 milliseconds (msec). The most abundant precursor ions (minimum intensity threshold 5,000) were fragmented by collision-induced dissociation (35% energy) and fragment ions were detected in the linear ion trap (AGC 15,000, 50 msec maximum injection). Analyzed precursors were dynamically excluded for 180 seconds. Multiple MS2 fragment ions were captured using isolation waveforms with multiple frequency notches and fragmented by high energy collision-induced dissociation (50% normalized collision energy). MS3 spectra were acquired in the Orbitrap (AGC 150,000; maximum injection time 200 msec, 50,000 resolution scanning from 100-1200 m/z).

Project description:R5 was cultured in 10 mL Luria broth with 1.0 mM resveratrol or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform until the cultures reached an average O.D. of 1.0 at 600 nm. Five replicate cultures were used for metabolomics experimentation. The samples were centrifuged at 5,000 x g for 10 min., washed once in 1 mL phosphate buffered saline, centrifuged at 5,000 x g for 10 min., resuspended in 1 mL of 1:1:1:1 acetone/acetonitrile/methanol/water and 1,2500 pmol prednisone, and pulverized in the bead mill. Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without resveratrol). The milled extracts were centrifuged for 20 minutes at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 ul 50% methanol/0.1% formic acid. Fifteen ul of each sample was pooled to create a QC sample. Five ul of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Four subsequent injections of the QC were performed to generate MS2 spectra. After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank1, QC1, sample replicates 1, Blank2, QC2, sample replicates 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%.

Project description:P. savastanoi pv. phaseolicola isolate R5 was cultured in 10 mL Luria broth with 400 uM genistein or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform. Replicate R5 cultures of each treatment were centrifuged at 5,000 x g for 10 min., washed once in 1 mL phosphate buffered saline, centrifuged at 5,000 x g for 10 min., resuspended in 1 mL of 1:1:1:1 acetone/acetonitrile/methanol/water and 1,2500 pmol prednisone, and pulverized in the bead mill as before. Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without genistein). The milled extracts were centrifuged for 20 minutes at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 uL 50% methanol/0.1% formic acid. Fifteen uL of each sample was pooled to create a quality control (QC) sample. Five uL of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Three subsequent injections of the QC were performed to generate MS2 spectra. After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank 1, QC 1, sample replicate set 1, Blank 2, QC 2, sample replicate set 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. The samples also were analyzed in negative ion mode at -2,500 V but with the same other settings.

Project description:R5 was cultured in 10 mL Luria broth with 1.0 mM resveratrol or without (but with 50 uL DMSO) at 28 C at 200 rpm on a shaking platform until the cultures reached an average O.D. of 1.0 at 600 nm. Five replicate cultures were used for metabolomics experimentation. The samples were centrifuged at 5,000 x g for 10 min., washed once in 1 mL phosphate buffered saline, centrifuged at 5,000 x g for 10 min., resuspended in 1 mL of 1:1:1:1 acetone/acetonitrile/methanol/water and 1,2500 pmol prednisone, and pulverized in the bead mill. Two matrix blank samples were prepared the same way starting with 10 mL Luria broth (without R5 and without resveratrol). The milled extracts were centrifuged for 20 minutes at 12,000 x g. The supernatants were transferred to fresh tubes and centrifuged again. The supernatants were transferred to glass vials and dried under vacuum. The residues were resuspended in 115 ul 50% methanol/0.1% formic acid. Fifteen ul of each sample was pooled to create a QC sample. Five ul of the samples were separated on a 150 x 2.1 mm Hypersil GOLD VANQUISH HPLC column with 1.9 um particles (Thermo Fisher Scientific) coupled to a Vanquish HPLC pump (Thermo Fisher Scientific) controlling a 10-minute linear gradient from 0% to 95% acetonitrile and 0.1% formic acid at a flow rate of 0.2 mL per minute. Eluent was electrosprayed at 3.5 kV positive polarity into an Exploris 240 mass spectrometer (Thermo Fisher Scientific) using an internal mass calibrant. Sheath gas was 35, auxiliary gas was 7, and sweep gas was 1 (arbitrary units). The ion transfer tube temperature was 325 C and the vaporizer temperature was 275 C. Advanced peak determination, mild trapping, and internal mass calibration was enabled. Default charge state was 1 and the expected peak width was 6 sec. AcquireX software was used to create a background ion exclusion list from the matrix blank sample and an inclusion ion list from the QC sample. Four subsequent injections of the QC were performed to generate MS2 spectra. After each QC injection, the resolved ions were appended to the exclusion list. Survey scans were recorded in the Orbitrap at 60,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%. Monoisotopic precursor selection was enabled, the minimum intensity was 5,000, and charge states were filtered to 1. Precursors selected within a 1.0 Da isolation window were fragmented by high energy collision-induced dissociation (30%, 50%, 70% normalized stepped collision energy), and fragment ions were resolved in the Orbitrap at 30,000 resolution. Subsequently, all test samples were analyzed alongside QC and matrix blank samples in the following order: Matrix blank1, QC1, sample replicates 1, Blank2, QC2, sample replicates 2, etc. Survey scans were recorded in the Orbitrap at 120,000 resolution over a mass range of 70-800 m/z. The RF lens was 70%.

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Pseudomonas savastanoi pv phaseolicola R5 treated with genistein (proteomics)

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