Dataset Information

GNPS - Transient expression of NHP-Glc biosynthetic genes in tomato

ABSTRACT: Experimental Goal: To determine the metabolic profile of tomato leaves when transiently expressing NHP and NHP-Glc biosynthetic enzymes. Methods: Transient expression and SAR assays were performed as previously (Holmes et al., 2019. Science Signaling. 12, eaay3066). Briefly, combinations of Agrobacterium C58C1 pCH32 strains harboring combinations of GFP, FMO1, ALD1, and UGT76B1 were infiltrated into two proximal (bottom, local (L)) leaflets of the third and fourth compound leaves of 4-5-week old tomato plants for 48 h. For metabolic profiling, two proximal and three distal (D) leaflets of the third compound leaf were harvested, lyophilized to dryness, and homogenized using a ball mill (Retsch MM 400) at 25 Hz for 2 min. Samples were resuspended in 20 ul of 80:20 MeOH:H2O per mg dry tissue and incubated at 4C for 10 min. The liquid fraction of each sample was split for LC-MS and GC-MS analysis respectively. Samples for GC-MS analysis were further derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Holmes et al., 2019. Science Signaling. 12, eaay3066). NHP-Glc was measured using previously published methods on an Agilent 1260 HPLC coupled to an Agilent 6520 quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer (Chen et al., 2018. PNAS. E4920-E4929). SA and SA-Glc were measured using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6470 triple quadrupole (QQQ) mass spectrometer. A 1.8 um, 2.1 x 50 mm Zorbax RRHD Eclipse Plus C18 column was used for reverse phase chromatography with mobile phases of A [water with 0.1% formic acid (FA)] and B [acetonitrile (AcN) with 0.1% FA]. The following gradient was used for separation with a flow rate of 0.6 ml/min (percentages indicate percent buffer B): 0-0.2 min (5%), 0.2-4.2 min (5-95%), 4.2-5.2 min (95-100%). The MS was run in negative mode with the following parameters: gas temperature, 250C; gas flow rate, 12 l/min; nebulizer, 25 psig. SA was measured using monitored transitions with the following parameters: Precursor ion, 137.0239; product ions, 93 and 65.1; dwell, 150 ms; fragmentor voltage, 158 V; collision energy, 20 V and 32 V respectively, cell accelerator voltage, 4 V. SA-Glc was measured using monitored transitions with the following parameters: Precursor ion, 299.0767; product ions, 137 and 93; dwell, 150 ms; fragmentor voltage, 158 V; collision energy, 5 V and 20 V respectively, cell accelerator voltage, 4 V. TMS-derivatized samples were measured for Pip and NHP using published methods on an Agilent 7820A gas chromatograph coupled to an Agilent 5977B mass spectrometer (Holmes et al., 2019. Science Signaling. 12, eaay3066).

INSTRUMENT(S): 6520A Quadrupole Time-of-Flight LC/MS, 6470 Triple Quadrupole LC/MS (Agilent instrument model), 7820A/5977B GC/MS (Agilent instrument model)

ORGANISM(S): Solanum Lycopersicum (ncbitaxon:4081)

SUBMITTER: Elizabeth Sattely

PROVIDER: MSV000086056 | GNPS |

REPOSITORIES: GNPS

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Experimental Goal: To determine the metabolic profile of Arabidopsis WT, ugt76b1, and fmo1 leaves during systemic acquired resistance. Methods: Three lower leaves (leaf number 5-7) of 30-32-day-old Col-0, fmo1 and ugt76b1 Arabidopsis plants were infiltrated with 10 mM MgCl2 and a 5e6 cfu/ml suspension of Pst avrRpt2 in 10 mM MgCl2. Forty-eight h later, the three treated lower leaves (local, L) and three untreated upper leaves (distal, D) (leaf number 8-10) were harvested, pooled, respectively, then frozen in liquid nitrogen for metabolic profiling by LC-MS, and triple quadrupole (QQQ)-MS analysis. Plant tissue was harvested, lyophilized to dryness, and homogenized using a ball mill (Retsch MM 400) at 25 Hz for 2 min. Samples were resuspended in 20 ul of 80:20 MeOH:H2O per mg dry tissue and incubated at 4C for 10 min. NHP-Glc and DC-NHP were measured using previously published methods on an Agilent 1260 HPLC coupled to an Agilent 6520 quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer (Chen et al., 2018. PNAS. E4920-E4929). SA and SA-Glc were measured using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6470 triple quadrupole (QQQ) mass spectrometer. A 1.8 um, 2.1 x 50 mm Zorbax RRHD Eclipse Plus C18 column was used for reverse phase chromatography with mobile phases of A [water with 0.1% formic acid (FA)] and B [acetonitrile (AcN) with 0.1% FA]. The following gradient was used for separation with a flow rate of 0.6 ml/min (percentages indicate percent buffer B): 0-0.2 min (5%), 0.2-4.2 min (5-95%), 4.2-5.2 min (95-100%). The MS was run in negative mode with the following parameters: gas temperature, 250C; gas flow rate, 12 l/min; nebulizer, 25 psig. SA was measured using monitored transitions with the following parameters: Precursor ion, 137.0239; product ions, 93 and 65.1; dwell, 150 ms; fragmentor voltage, 158 V; collision energy, 20 V and 32 V respectively, cell accelerator voltage, 4 V. SA-Glc was measured using monitored transitions with the following parameters: Precursor ion, 299.0767; product ions, 137 and 93; dwell, 150 ms; fragmentor voltage, 158 V; collision energy, 5 V and 20 V respectively, cell accelerator voltage, 4 V.

Project description:The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle samples by removing invalid and low signal-high-variance probes Human primary skeletal muscle cells were isolated from muscle biopsies from healthy adults as previously described (Crossland et al. 2016; Crossland et al. 2017). Myogenic cell enrichment used magnetic-activated cell sorting (MACS) and anti-CD56 microbeads (130-050-401; Miltenyi Biotec). Myogenic purity was assessed through measurement of fractional desmin positivity (Crossland et al. 2016; Crossland et al. 2017). Cells were washed in PBS and fixed in ice-cold 1:1 acetone/methanol, before being blocked in 5% (v/v) goat serum for 30 min at room temperature. Cells were subsequently incubated with rabbit anti-desmin monoclonal antibody (ab32362; Abcam) for 1h at room temperature, washed, and incubated with anti-rabbit TRITC-conjugated secondary antibody as previously detailed (Crossland et al. 2016; Crossland et al. 2017). Finally, cells were washed in PBS and mounted/DAPI-stained using FluoroshieldTM mounting medium with DAPI. Myoblasts at passage 5-6 were cultured on Collagen Type I-coated 6-well dishes in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Life Technologies) containing 20% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), 1% (v/v) antibiotic-antimycotic (AbAm) solution and 4mM L-glutamine (Life Technologies). Cells were maintained at 37oC with 5% CO2. Once cells reached ~95% confluency, differentiation was induced by switching the medium to DMEM/F-12 containing 2% (v/v) horse serum (Sigma-Aldrich), 4mM L-glutamine and 1% (v/v) AbAm solution (Life Technologies). Six days following the initiation of differentiation, a media change was carried out, using differentiation media supplemented with long R3 IGF-1 (10 ng/ml; Sigma-Aldrich), with or without 100 nM rapamycin (Sigma-Aldrich). DMSO (final concentration 0.01% (v/v)) was used as a vehicle control for rapamycin. Samples were collected at 0h baseline and following 4h and 24h treatment.

Project description:This study corresponds to a re-analysis and meta-analysis of the five transcriptomic studies related to spontaneous tolerance to renal allograft in human. It examines the transcriptome of peripheral blood samples from six distinct clinical groups: healthy volunteers (HV); tolerant patients (TOL); stable patients on minimal immunosuppression (MIS) or maintained on classical immunosuppressive therapy (STA); patients showing either signs of chronic rejection (CR) or acute rejection (AR). The initial studies are the following: -Study 1 from Braud C. et al. (J Cell Biochem., 2008) published under GEO accession number GSE47755 and comprising 250 unique samples (8 HV; 21 TOL; 190 STA; 31 CR). -Study 2 from Brouard S. et al. (Proc Natl Acad Sci U S A., 2007) published under GEO accession number GSE47683 and comprising 67 unique samples (8 HV; 12 TOL; 10 MIS; 12 STA; 11 CR; 14 AR). - Study 3 from Lozano JJ. et al. (Am J Transplant., 2011) published under GEO accession number GSE22707 and comprising 42 unique samples (6 HV; 12 TOL; 12 STA; 12 CR). - Study 4 from Newell KA. et al. (J Clin Invest., 2010) published under GEO accession number GSE22229 and comprising 58 unique samples (12 HV; 19 TOL; 27 STA). - Study 5 from Sagoo P. et al. (J Clin Invest. 2010) published under GEO accession number GSE14655 and comprising two distinct cohorts of patients. The IOT (“Indices of Tolerance”) cohort (5a) corresponds to a European group of patients and totalizes 74 unique samples (8 HV; 10 TOL; 11 MIS; 36 STA; 9 CR). The ITN (“Immune Tolerance in Transplantation”) cohort (5b) corresponds to an American group of patients and totalizes 105 unique samples (20 HV; 22 TOL; 11 MIS; 34 STA; 18 CR). The final data matrix corresponds to the combination of the data from the five independent studies and examines the expression of 1846 meta-genes (MG) across 596 unique blood samples. The 1846 meta-genes correspond to the genes being measured across the five datasets and form a virtual microarray platform (TOL-HUMAN-G1846). The 596 examined samples can be divided into 62 HV, 96 TOL, 32 MIS, 311 STA, 81 CR, and 14 AR.

Dataset Information

GNPS - Transient expression of NHP-Glc biosynthetic genes in tomato

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets