Targeted proteomics was carried out as previously described (Stergachis, MacLean, Lee, Stamatoyannopoulos, & MacCoss, 2011 ) using either a 1200 Series LC (Agilent Technologies, USA) combined with a QTRAP 3200 MS/MS system (Sciex, USA), or a Shimadzu HPLC combined with a QTRAP 6500 MS/MS system (Sciex). Bovine serum albumin (BSA) was used as an internal control (10 ng/µl; target peptide: YLYEIAR). This was added to the crude extract before 200 µg of total protein was digested per sample. The targeted proteomics method was developed with Skyline (MacLean et al., 2010 ), and the peptide VDITQIK was used to detect chloramphenicol acetyltransferase (CatP, backbone marker), whereby the peptide VLSVITEPILPFER was used to detect IspS. The peptides were separated in a linear gradient by changing the ratio between buffer A (2% acetonitrile, 98% water, 0.1% formic acid) and buffer B (98% acetonitrile, 2% water, and 0.1% formic acid) from 95:5 to 5:95 with a flow rate of 0.4 ml/min.
Qtrap 6500 ms ms system
Manufactured by AB Sciex
Sourced in United States
The QTRAP 6500+ MS-MS system is a high-performance tandem mass spectrometer designed for quantitative and qualitative analysis. It features a triple quadrupole configuration with an enhanced linear ion trap for improved sensitivity and selectivity.
Automatically generated - may contain errors
Lab products found in correlation
Lcad u hplc system, by AB Sciex (2 mentions)
1200 series lc, by Agilent Technologies (1 mentions)
Nexera x2 uhplc system, by Shimadzu (1 mentions)
C18 solid phase extraction cartridge, by Waters Corporation (1 mentions)
Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions)
C18 spe cartridge, by Waters Corporation (1 mentions)
5 protocols using qtrap 6500 ms ms system
1
Targeted Proteomics for Biomarker Detection
2
Quantitative Analysis of Methamphetamine and Metabolites
3
Bioactive Lipid Mediator Extraction and Analysis
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The extraction protocol and analysis of bioactive lipid mediators were performed as described by Le Faouder et al. (17 (link)), adapted by the Ambiotis SAS (Toulouse, France) standard operating procedure. Briefly, samples of plasma, cell supernatants and purified EVs were mixed with 0.4 mL of ice-cold methanol and held at -80°C for protein precipitation. Samples were then centrifuged and supernatants were collected. After removal of the organic solvent under a stream of nitrogen, samples were suspended in methanol and rapidly acidified to pH 3.5 with HCl. Acidified samples were then loaded into C-18 solid-phase extraction cartridges (Waters, Milford, MA), rapidly neutralized, and eluted with methyl formate. Eluate solvents were evaporated under a stream of nitrogen and residues were suspended in mobile phase for liquid chromatography analyses using an Exion LCAD U-HPLC system coupled to a Sciex QTRAP 6500+ MS-MS system (AB Sciex, Framingham, MA), equipped with an ESI source in negative ion mode.
4
ABA Quantification in Plant Samples
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Abscisic acid (ABA) extraction was performed according to the method by Errard et al. (2016) (link) with modifications. ABA was extracted from 10 mg homogenized sample using methanol/water (3:2, v/v). Deuterated ABA was added as an internal standard followed by sonication in cold. Combined supernatants were filtered through PTFE filters (0.2 µm) and diluted with 0.1% acetic acid in ultrapure water (1:1, v/v). The extracts were analyzed using an Agilent Technologies 1260 Infinity HPLC coupled with a triple quadrupole, Q-Trap® 6500-MS/MS system (AB Sciex LLC, Framingham, USA) equipped with a Zorbax Eclipse Plus C18 column (1.8 µm, 2.1 mm x 50 mm; Agilent Technologies, Waldbronn, Germany). Elution was performed in gradient mode using solvent A: 0.1% acetic acid and solvent B: acetonitrile and 0.1% water. Ionization was performed in negative mode using ESI (electrospray ionization) at 500°C with the following settings: ionization voltage, -4,500 V; curtain gas, 50 psi; drying gas, 50 psi; nebulizer gas, 50 psi; auxiliary gas, 65 psi; and multi reaction monitoring (MRM) at a dwell time of 0.3781 s. Identification and quantification were based on MRM transitions (263→153 quantifier, 263→203 and 263→122 qualifier). ABA was quantified using external calibration with internal standard.
5
Extraction and Analysis of Bioactive Lipid Mediators
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The extraction protocol and analysis of bioactive lipid mediators were performed as described by Le Faouder et al.,(15 ) adapted by the Ambiotis SAS (Toulouse, France) standard operating procedure. Briefly, plasma (1 mL) was mixed with 0.4 mL of ice‐cold methanol and held at −80°C for protein precipitation. Samples were then centrifuged and supernatants collected. After removal of the organic solvent under a stream of nitrogen, samples were suspended in methanol and rapidly acidified to pH 3.5 with HCl. Acidified samples were then loaded into C‐18 SPE cartridges (Waters, Milford, MA), rapidly neutralized, and eluted with methyl formate. Eluate solvents were evaporated under a stream of nitrogen and residues suspended in mobile phase for LC/MS‐MS analyses using an Exion LCAD U‐HPLC system coupled with a Sciex QTRAP 6500+ MS‐MS system (AB Sciex, Framingham, MA), equipped with an ESI source in negative ion mode. Quantification was performed using calibration curves with synthetic standards for each of the SPMs included in the analysis.
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