A total of 50 μL of serum and approximately 10mg of hippocampal tissues (wet weight) were used for sample preparation following previously reported methods (51 (link), 52 (link)). Samples were analyzed using an Agilent 6550 iFunnel Q-TOF LC/MS system (Agilent) with an Acquity UPLC HSS T3 column (53 (link)). Raw data were converted to mzXML format by ProteoWizard 3.0 package and uploaded to XCMS online (54 (link)). Metabolites were identified against METLIN (55 (link)) and Human Metabolome Database (56 (link)). Multivariate analysis was performed with SIMCA 13.0 (Umetrics). VIP scores were assessed. Discriminated metabolites were defined with a VIP > 1.0 by partial least squares-discriminant analysis (PLS-DA), P < 0.01, FDR-adjusted P < 0.05. Pathway enrichment analysis were analyzed using Metaboanalyst 4.0 (57 (link)).
Agilent 6550 ifunnel qtof lc ms system
Manufactured by Agilent Technologies
Sourced in United States
The Agilent 6550 iFunnel QTOF LC/MS system is a high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) instrument. It is designed to provide accurate mass measurements and high-resolution analysis of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Hp 0921, by Agilent Technologies (2 mentions)
Matlab r2015b, by MathWorks (2 mentions)
Simca 13, by Sartorius (1 mentions)
Uplc hss t3 column, by Agilent Technologies (1 mentions)
3 amino 1 propanesulfonic acid, by Merck Group (1 mentions)
Mps2 autosampler, by Gerstel (1 mentions)
Trypsin gold, by Promega (1 mentions)
Zorbax eclipse plus c18 column, by Agilent Technologies (1 mentions)
Agilent 1260 infinity system, by Agilent Technologies (1 mentions)
Kieselgel 60, by Merck Group (1 mentions)
Rp 18 f254s plates, by Merck Group (1 mentions)
Agilent 400 mr nmr, by Agilent Technologies (1 mentions)
7 protocols using agilent 6550 ifunnel qtof lc ms system
1
Metabolomic analysis of serum and hippocampal tissues
2
High-Resolution Metabolic Profiling by FIA-TOFMS
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Cell extract samples were analyzed by flow-injection analysis time-of-flight mass spectrometry (FIA-TOFMS) on an Agilent 6550 iFunnel Q-TOF LC-MS System (Agilent Technologies, Santa Clara, CA, USA), as described by Fuhrer et al.14 (link). This method allows generating high-resolution spectral profiles in less than one minute per sample, allowing for sensitive high-throughput profiling of large sample collections. In brief, a defined sample volume of 5 µL is injected using a Gerstel MPS2 autosampler into a constant flow of isopropanol/water (60:40, v/v) buffered with 5 mM ammonium carbonate (pH 9), containing two compounds for online mass axis correction: 3-Amino-1-propanesulfonic acid, (138.0230374 m/z, Sigma Aldrich, cat. no. A76109) and hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine (940.0003763 m/z, HP-0921, Agilent Technologies, Santa Clara, CA, USA). The sample plug is delivered directly to the ion source for ionization in negative mode (325 °C source temperature, 5 L/min drying gas, 30 psig nebulizer pressure, 175 V fragmentor voltage, 65 V skimmer voltage). Mass spectra were recorded in the mass range 50–1000 m/z in 4 GHz high-resolution mode with an acquisition rate of 1.4 spectra per second. Raw MS profiles were processed to align spectra and pick centroid ion masses using an in-house data processing environment in Matlab R2015b (The Mathworks, Natick).
3
Proteomic Analysis of Differentially Expressed Proteins
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Differentially expressed protein spots were manually excised from the 2DE gels. In-gel digestion with trypsin and analysis using Agilent 6550 iFunnel QTOF LC/MS system (Agilent, Santa Clara, CA, USA) were performed as earlier described by Lee et al. (2016) (link).
4
FIA-TOFMS Analysis of Metabolites
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FIA-TOFMS analysis was performed as described in ref. 23 (link) on an Agilent 6550 iFunnel Q-TOF LC/MS System (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ion source operated in negative ionization mode. In this setup, the samples are injected into a constant flow of an isopropanol/water mixture (60:40, v/v) buffered with 5 mM ammonium carbonate at pH 9 using a Gerstel MPS2 autosampler (5 µL injection volume). Two compounds were added to the solvent for on-line mass axis correction: 3-amino-1-propanesulfonic acid, (HOT, 138.0230374m/z, Sigma Aldrich, cat. no. A76109) and hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine (940.0003763m/z, HP-0921, Agilent Technologies, Santa Clara, CA, USA). The ion source parameters were set as follows: 325 °C source temperature, 5 L/min drying gas, 30 psig nebulizer pressure, 175 V fragmentor voltage, 65 V skimmer voltage, 750 V octopole voltage. The TOF detector was operated in 4 GHz high-resolution mode with a spectral acquisition rate of 1.4 spectra per second. Mass spectra were recorded in the mass range 50–1000m/z. Alignment of MS profiles and picking of centroid ion masses were performed using an in-house data processing environment in Matlab R2015b (The Mathworks, Natick)23 (link).
5
Proteomic Identification of Proteins via Tryptic Digestion
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Lyophilised protein samples were reconstituted in 37 μl of 50 mM ammonium bicarbonate with 4 mM of DTT. The samples were reduced at 95°C for 5 min. Alkylation was performed by incubating 7.5 mM iodoacetamide at room temperature in dark for 20 min. The protein sample was digested using 0.3 μg of Trypsin Gold (Promega, Madison, WI, USA) for 16 hours at 37°C. The reaction was quenched by freezing the sample. Digested peptides were desalted using μ-C18 ZipTips (Billerica, MA, USA) and eluted in 50% acetonitrile containing 1% formic acid. The peptides were subjected to QTOF LC/MS analysis using Agilent 6550 iFunnel QTOF LC/MS system (Agilent, Santa Clara, CA, USA) for peptides identification. Tryptic peptides were separated using the C18 HPLC-Chip with a linear gradient of water/acetonitrile. Spectra were analysed to identify proteins of interest according to the Uniprot database.
6
Aflatoxin Quantification in Aspergillus Extracts
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To prepare A. flavus and A. oryzae extracts, conidia were inoculated on sterilized maize grain and cultured for 4–5 days at 25°C. After cultivation, each grain covered with fungal cells was dipped into 1 ml 70% wt/vol, methanol, and secondary metabolites, including aflatoxins, were extracted by mixing vigorously. The supernatants obtained from each strain were used in the following analyses. Extract aflatoxin content was estimated using a MycoJudge Total Aflatoxin ELISA kit (NH Foods Ltd., Osaka, Japan) according to the manufacturer’s instructions and detected by liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF) as described below. Samples were separated by LC using the Agilent 1260 infinity system (Agilent Technologies, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse Plus C18 column (Agilent Technologies) maintained at 40°C. Ammonium acetate (5 mM) (A) and methanol (B) were used as mobile phases. Each 3 μLμl sample was separated with LC, using a gradient of 10% A/90% B to 100% B over 30 min with a flow rate of 0.2 mLml/min. The Agilent 6550 iFunnel QTOF LC/MS system (Agilent Technologies) was used for MS analysis.
7
Spectroscopic Characterization of Compounds
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All NMR spectra were recorded on an Agilent 400-MR-NMR (Agilent technologies, Santa Clara, CA, USA) spectrometer operated at 400 and 100 MHz for hydrogen and carbon, respectively. Data processing was carried out with the MestReNova ver.6.0.2 program. HRESIMS spectra were obtained using an AGILENT 6550 iFunnel Q-TOF LC/MS system (Agilent technologies, Santa Clara, CA, USA). Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. Preparative HPLC was carried out using an AGILENT 1200 HPLC system. Column chromatography was performed on silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck) or YMC RP-18 resins (30–50 µm, Fuji Silysia Chemical Ltd., Aichi, Japan). For thin layer chromatography (TLC), a pre-coated silica-gel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254S plates (0.25 mm, Merck, Darmstadt, Germany) were used.
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