Auxin metabolome was assessed in young 4-day-old seedlings of Col-0 and the DR5 line treated with either 100 mM NaCl or 200 mM mannitol in 2.5 mM MES buffer (pH 5.7) for 3 h. Untreated seedlings grown on 2.5 mM MES buffer (pH 5.7) were used as controls. Furthermore, the roots of Arabidopsis plants were also analyzed after prolonged stress conditions (13-day treatments). After the long-term treatment, four to five biological replicates of plant samples were collected and weighed up to 10 mg fresh weight per replicate. Complete auxin profiling was performed using the ultra-rapid auxin metabolite profiling method, previously described [65 (link)]. All purified samples were analyzed by a 1260 Infinity LC system combined with a 6495 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA, USA) using stable isotope-labeled internal standards as a reference.
6495 triple quadrupole lc ms system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6495 Triple Quadrupole LC/MS system is a high-performance liquid chromatography-mass spectrometry (LC/MS) instrument. It is designed for quantitative analysis and identification of compounds in complex matrices. The system combines a triple quadrupole mass analyzer with an electrospray ionization (ESI) source to provide sensitive and selective detection of target analytes.
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Lab products found in correlation
1260 infinity lc system, by Agilent Technologies (1 mentions)
Db 5ms, by Agilent Technologies (1 mentions)
Hypersil gold reversed phase c18 column, by Thermo Fisher Scientific (1 mentions)
Hypersil gold reversed phase column, by Thermo Fisher Scientific (1 mentions)
1290 uhplc system, by Agilent Technologies (1 mentions)
5 protocols using 6495 triple quadrupole lc ms system
1
Auxin Profiling Under Abiotic Stress
2
GC-APCI-MS Quantification Protocol
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An Agilent 7890B gas chromatograph equipped with a split/splitess injector, autosampler (G453A), syringe 10 µL (9301-0713) and splitless liner (5190-3165) was coupled to a 6495 triple quadrupole LC/MS system from Agilent Technologies (Santa Clara, USA) using the new developed GC-APCI ion source. GC separation was performed on a nonpolar fused silica column DB-5MS (30 m, 0.25 µm film thickness and 0.25 mm i.d.) purchased from Agilent Technologies Inc. (Santa Clara, USA). The analyses were operated in constant flow mode. The make-up gas flow was controlled by a mass flow controller from ALLBORG model 325656.
3
Quantitative Analysis of CSF Proteins
4
Simultaneous Quantification of Synaptic Biomarkers
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The panel of synaptic biomarkers for simultaneous quantification included 14–3–3 epsilon, 14–3–3 eta, 14–3–3 zeta/delta, activating protein-2 (AP-2) complex subunit beta, complexin-2, beta-synuclein, gamma-synuclein, neurogranin, neuronal pentraxin-1 (NPTX1), neuronal pentraxin-2 (NPTX2), neuronal pentraxin receptor (NPTXR), rab GDP dissociation inhibitor (GDI) alpha, PEBP-1, syntaxin-1B, and syntaxin-7. The applied methodology has been described in detail elsewhere [10 ]. See Supplementary Table 1 for all analyzed peptides. In brief, 100μL CSF was mixed with stable isotope labeled peptide standards (internal standard), followed by sample preparation in a consecutive four-step process consisting of reduction, alkylation, tryptic digestion, and purification by solid-phase extraction. For multiple reaction monitoring MS quantitation, a micro-high-performance LC-MS system (6495 Triple Quadrupole LC/MS system, Agilent Technologies, Santa Clara, CA, USA), equipped with a Hypersil Gold reversed phase column (dim. 100×2.1 mm, particle size 1.9μm, Thermo Fisher Scientific, Waltham, MA, USA) was used. Pooled CSF samples were used as quality control and injected at regular intervals to monitor assay performance over time and to assess inter- and intra-assay variation.
5
Plasma Ceramide Measurement by LC-MS
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Blood samples for plasma ceramide measurements were obtained in ethylenediamine tetra-acetic acid (EDTA)-containing tubes both immediately before and after stress MPS.
Plasma was collected within two hours from withdrawal and stored at -80°C until analysis. The apparatus consisted of an Agilent 1290 UHPLC system coupled with an Agilent 6495 Triple Quadrupole LC/MS system. Mobile phases consisted in LC-MS grade water (A), acetonitrile with 0.1% formic acid (B) and 10 mM ammonium acetate in 2-propanol (C).
Plasma was collected within two hours from withdrawal and stored at -80°C until analysis. The apparatus consisted of an Agilent 1290 UHPLC system coupled with an Agilent 6495 Triple Quadrupole LC/MS system. Mobile phases consisted in LC-MS grade water (A), acetonitrile with 0.1% formic acid (B) and 10 mM ammonium acetate in 2-propanol (C).
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