Pseudomonas savastanoi pv phaseolicola R5 treated with salicylic acid
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ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Pseudomonas Savastanoi Pv. Phaseolicola R5
SUBMITTER: 
Bret Cooper
PROVIDER: MSV000087853 | MassIVE | Mon Jul 19 10:15:00 BST 2021
REPOSITORIES: MassIVE
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Project description:The Chromosome-centric Human Proteome Project (C-HPP) was recently initiated as an international collaborative effort. Our team adopted chromosome 9 (Chr 9) and performed a bioinformatics and proteogenomic analysis to catalog Chr 9-encoded proteins from normal tissues, lung cancer cell lines and lung cancer tissues. TQ orbitrap, Orbitrap full MS scans were acquired from m/z 350 to 1500 at a resolution of 15 000 (at m/z 400). Parent ions were fragmented using the LTQ (isolation width of 2 m/z units) , with a maximum injection time of 100 ms combined with an AGC value of 1 x104 using three fragmentation modes such as collision-induced dissociation (CID) alone, the reagent ion source emission current, reagent ion electron energy, and reagent ion source chemical ionization pressure were set to 35 mA, 70 V, and 26 psi, respectively. Database : UniProt database (rel. 2012-06, 86,875 entries). Search software : MASCOT software (version 2.2.04). Database search criteria were as follows: taxonomy Homo sapiens, carboxyamidomethylated (+57 Da) at cysteine residues for fixed modifications, oxidized at methionine (+16 Da) residues for variable modi?cations, two maximum allowed missed cleavage, 10 ppm MS tolerance. Only peptides resulting from trypsin digestion were considered. PeptideProphet and ProteinProphet were used to estimate the false discovery rate (FDR). We identified proteins using two or more unique peptides with an FDR < 1% at the protein level.
2014-03-18 | PXD000603 | Pride
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).
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Project description:Orbitrap Fusion (Thermo Fisher Scientific) LC-MS/MS analyses were performed on an Easy-nLC 1000 liquid chromatography system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion via a nano-electrospray ion source. Tryptic peptides were dissolved with a loading buffer (acetonitrile and 0.1% formic acid), and were eluted with a flow rate of 350 nL/min. Survey scans were acquired after an accumulation of 5×105 ions in the Orbitrap for m/z 300-1,400 using a resolution of 120,000 at m/z. The top speed data-dependent mode was selected for fragmentation in the cell at a normalized collision energy of 32%, and fragment ions were then transferred into the ion trap analyzer with the AGC target at 5×103 and maximum injection time at 35 ms. The dynamic exclusion of previously acquired precursor ions was enabled at 18 s. The Proteome Discoverer 1.4.1.14 was used for analysis of the protein spectrum. Oxidation (Methionine) and acetylation (Protein-N term) were chosen as variable modifications, cysteine carbamidomethylation was chosen as a fixed modification. Two missed cleavage sites for trypsin were allowed. The intensity-based absolute quantification (iBAQ)-based protein quantification were performed by an in-house software. The interaction of SNT-related differentially expressed proteins was investigated by STRING 11.0 (https://string-db.org). The differentially expressed protein interaction network (high reliability, interaction score > 0.4, PPI enrichment P-value < 1.0×10 -16) was selected for the analysis.
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