ESI-mass spectra were collected both in positive (+) and negative (−) ion modes on an Agilent 6520 Q-ToF instrument by direct infusion of the Rh-glutathione reaction mixtures, as well as different parts of the orange band formed on the Sephadex G-15 column; water was used as the mobile phase. For technical details, see Ref. [22 ]. The assignments of the mass peaks were verified with the Isotope Distribution Calculator from Agilent and with a high resolution calculation method from Scientific Instrument Services. [23 ]
6520 qtof
Manufactured by Agilent Technologies
Sourced in United States, Germany
The Agilent 6520 QTOF is a quadrupole time-of-flight mass spectrometer designed for high-resolution accurate mass analysis. It provides precise mass measurements and structural information for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
1200 series nano lc system, by Agilent Technologies (4 mentions)
Q tof system, by Agilent Technologies (2 mentions)
Masshunter software, by Agilent Technologies (2 mentions)
Isotope distribution calculator, by Agilent Technologies (1 mentions)
Mass profiler professional, by Agilent Technologies (1 mentions)
Masshunter profinder b 08, by Agilent Technologies (1 mentions)
Agilent 6545 qtof, by Agilent Technologies (1 mentions)
Hplc chip q tof system, by Agilent Technologies (1 mentions)
Zorbax 300sb c18, by Agilent Technologies (1 mentions)
1200 series hplc system, by Agilent Technologies (1 mentions)
Chymotrypsin, by Promega (1 mentions)
1200 nano lc system, by Agilent Technologies (1 mentions)
1200 series, by Agilent Technologies (1 mentions)
1260 uplc, by Agilent Technologies (1 mentions)
Poroshell 120 ec 18 column, by Agilent Technologies (1 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions)
Agilent hplc system, by Agilent Technologies (1 mentions)
Quaternary pump, by Agilent Technologies (1 mentions)
Kinetex c18, by Phenomenex (1 mentions)
1100 series hplc system, by Agilent Technologies (1 mentions)
49 protocols using 6520 qtof
1
Rhesus Glutathione Reaction Analysis
2
Peptide Profiling by HPLC-Q-TOF
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Briefly, the peptide fraction was analyzed on a high-performance liquid chromatography-chip/quadrupole time-of-flight (Q-TOF) system (Agilent Technologies, Santa Clara, CA, USA), which included an Agilent 1200 series nano-LC system and an Agilent 6520 Q-TOF equipped with a chip cube interface. The sample was injected into a 360-nL enrichment column linked to a Polaris C18-A separation column (75 μm × 150 mm, 3 μm) at a flow rate of 0.3 μL/min for 120 min. MS/MS analysis was conducted in the positive ionization mode of ion source. The drying gas temperature and flow were 300°C and 3 L/min, respectively. The mass-to-charge ratio (m/z) MS scan range was m/z 300–2400, and the MS/MS scan range was m/z 100–3000, at a rate of 4 spectra/s.
3
Plasma Metabolome Profiling by LC-MS
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Samples from p100 fresh frozen plasma underwent LC-MS profiling in the laboratory of Nichole Reisdorph at the University of Colorado Anschutz Medical Campus as previously described.33 ,34 In brief, cold methanol was added to plasma sample aliquots containing internal standards to precipitate proteins. Supernatants were extracted using liquid-liquid extraction with methyl tert-butyl ether to obtain a lipid fraction and a small molecule aqueous fraction. Samples were analyzed in positive mode using C18 and HILIC on an Agilent 6545 quadrupole time-of-flight (QTOF) and 6520 QTOF, respectively. Spectral peaks were extracted using MassHunter Profinder B.08 (Agilent). Features were annotated using Mass Profiler Professional (Agilent) using either an in-house accurate mass and retention time (AMRT) database or exact mass and isotope ratios for the compounds that were not in the AMRT database. There were 10,561 features detected among the 81 samples.
4
LC-MS/MS Metabolite Quantification
5
Mass Spectrometric Analysis of Peptides
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For the preparation of samples for mass spectrometric analysis, the gel was serially sliced, minced and digested with trypsin and peptides were extracted [22] (link). Peptides extracted from trypsin treated samples were analyzed by LC-ESI-MS/MS using an Agilent 6520-QTOF. Peptides were taken in 3 μl of 0.1% formic acid (FA) (Solvent A). Typically, 2 μl of this sample was applied to an Agilent HPLC chip (G4240-62002). Nano-chip comprised of a 40 nl enrichment column and a 75 μm×150 mm separation column that was packed with Zorbax 300 SB-C18 (5 μm) material. After sample injection, the column was washed for 2 min with 0–3% Solvent B (90% acetonitrile in 0.1% formaldehyde), and peptides were eluted for 2–6 min with 1–30%, 6–15 min with 30–70%, 15–20 min with 70–95% Solvent B. Active exclusion was set-on for 0.5 min after each MS/MS spectrum.The m/z range used was 100–1700 for MS and 50–1700 for MS/MS. MS and MS/MS scan rate was 1.36 per second. For each MS, five most abundant precursor ions were sequenced.The mgf files were generated in MassHunter workstation software. The mgf files so obtained were submitted for protein identification searches against ‘H.sapien and E.coli databases from NCBInr’ using an in-house Mascot server.
6
HPLC-Chip/Q-TOF Mass Spectrometry Analysis
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A high-performance liquid chromatography (HPLC)-chip/Q-TOF system (Agilent Technologies) was used for sample analysis. The system consisted of an Agilent 1200 series nano-LC system and an Agilent 6520 Q-TOF coupled with a chip cube interface. The HPLC chip comprised a 160-nL enrichment column and a 75 µm (diameter) ×150 mm separation column packed with Zorbax 300SB-C18 (5 µm; Agilent Technologies, Santa Clara, CA, USA). The sample-loading capillary pump administered the solvent of 97% buffer A (water with 0.1% formic acid) and 3% buffer B (90% acetonitrile/0.1% formic acid) at a flow rate of 4 µL/min. A nano-pump was used to generate a separation-driven solvent composition gradient of 10% to 45% by pumping buffer B for 0 to 15 minutes. The buffer B percentage was then changed to 90% for 15 to 20 minutes and back to 10% for 20 to 30 minutes. The Q-TOF mass spectrometer was set to positive ionization mode. The drying gas (nitrogen gas) flow was set at 5 L/min and 300℃. Eluting peptides were selected for collision-induced dissociation during alternative procedures for MS scanning over the m/z range of 300 to 2,400 at a rate of 4 spectra/s and MS/MS scanning over the range of 100 to 3,000 m/z at 3 spectra/s. The isolation window was 4 m/z.
7
Quantitative Proteome Analysis by LC-Q-TOF
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The mass of each peptide fraction was analyzed using the high-performance liquid chromatography-chip/quadrupole time-of-flight (Q-TOF) system (Agilent Technologies, Santa Clara, CA, USA). The peptides from each patient's sample were analyzed without a pooling process to confirm individual protein expression patterns. The system consisted of an Agilent 1200 series nano-LC system and an Agilent 6520 Q-TOF coupled with a chip cube interface. The high-performance liquid chromatography-chip is comprised of a 160 nL enrichment column and a 75 µm×150 mm separation column packed with Zorbax 300SB-C18 (5 µm). Each sample was run for 52 min at a flow rate of 4 µL/min.
The Q-TOF mass spectrometer was set to positive ionization mode. The drying gas (nitrogen gas) flow was set at 5 L/min and 300℃. Eluting peptides were selected for collision-induced dissociation during alternative procedures of an MS scan over the m/z range of 300-2400 at the rate of 4 spectra/s and an MS/MS scan over the range of 100-3000 m/z at 3 spectra/s. The isolation window was 4 m/z.
The Q-TOF mass spectrometer was set to positive ionization mode. The drying gas (nitrogen gas) flow was set at 5 L/min and 300℃. Eluting peptides were selected for collision-induced dissociation during alternative procedures of an MS scan over the m/z range of 300-2400 at the rate of 4 spectra/s and an MS/MS scan over the range of 100-3000 m/z at 3 spectra/s. The isolation window was 4 m/z.
8
Mass Spectrometry Protocol with QTOF
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Mass spectrometry was performed with an Agilent 6520-QTOF, equipped with electrospray ionization (ESI) source operating at positive-ion (ESI+) and negative-ion (ESI-) electrospray ionization mode. The capillary voltage was 4.0 kV at ESI+ mode and 3.5 kV at ESI- mode. Nitrogen was used as the dry gas, and the desolvation gas flow was set at 10 l/min. The desolvation temperature was set at 330°C. Centroid data were collected in the full scan mode from 50 to 1000 m/z.
9
HILIC-MS Analysis of Extracted Samples
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Five microliters of each extracted sample
were injected onto a Luna Aminopropyl, 3 μm, 150 × 1.0
mm i.d. column (Phenomenex) with an Agilent 1200 series HPLC system.
The samples were kept at 4 °C in the autosampler and injected
onto a column maintained at room temperature. The column was used
in hydrophilic interaction liquid chromatography (HILIC) mode with
the following buffers and linear gradients: A = 95%
H2O, 5% ACN, 10 mM ammonium hydroxide, 10 mM ammonium acetate,
pH 9.45; B = 100% ACN; 85% B from 0 to 7 min (min),
85% to 60% B from 7 to 27 min, 60% to 40% B from 27 to 32 min, and
40% to 20% B from 32 to 40 min. Mass spectrometry (MS) detection was
carried out on an Agilent 6520 Q-TOF in negative ESI (electrospray
ionization) mode with the following settings: gas temperature 325
°C, drying gas 5 L/min, nebulizer 15 psi, fragmentor 120 V, skimmer
65 V, capillary voltage −3500 V, and scan rate 1.06 spectra/s.
Tandem MS (MS/MS) analyses were carried out with identical ESI parameters,
and the following fragmentation and precursor ion selection settings:
collision energy 10 V, precursor isolation window 1.3 amu, and scan
rate 1.00 spectra/s.
were injected onto a Luna Aminopropyl, 3 μm, 150 × 1.0
mm i.d. column (Phenomenex) with an Agilent 1200 series HPLC system.
The samples were kept at 4 °C in the autosampler and injected
onto a column maintained at room temperature. The column was used
in hydrophilic interaction liquid chromatography (HILIC) mode with
the following buffers and linear gradients: A = 95%
H2O, 5% ACN, 10 mM ammonium hydroxide, 10 mM ammonium acetate,
pH 9.45; B = 100% ACN; 85% B from 0 to 7 min (min),
85% to 60% B from 7 to 27 min, 60% to 40% B from 27 to 32 min, and
40% to 20% B from 32 to 40 min. Mass spectrometry (MS) detection was
carried out on an Agilent 6520 Q-TOF in negative ESI (electrospray
ionization) mode with the following settings: gas temperature 325
°C, drying gas 5 L/min, nebulizer 15 psi, fragmentor 120 V, skimmer
65 V, capillary voltage −3500 V, and scan rate 1.06 spectra/s.
Tandem MS (MS/MS) analyses were carried out with identical ESI parameters,
and the following fragmentation and precursor ion selection settings:
collision energy 10 V, precursor isolation window 1.3 amu, and scan
rate 1.00 spectra/s.
10
Comprehensive Protein Identification Protocol
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Protein digestion: Proteins were denatured in 1∶1 v/v trifluoroethanol (TFE), reduced, alkylated and digested with ChymoTrypsin (Promega) according to published protocols [17] .
LCMS/MS analysis: An Agilent 6520 QTOF equipped with an Agilent chip-cube nano-electrospray interface and an Agilent 1200 nano-LC system was used: Samples were trapped on an Agilent High capacity Chip (part #: G4240-62010) at 4 uL/min 99% buffer A (0.1% formic acid in water) and 1% buffer B (0.1% formic acid in acetonitrile), then eluted at 0.3 uL/min from 1% to 85% buffer B. The QTOF was operated in Auto MS/MS mode with the standard parameters for chip cube interface. MS spectra were acquired from 300 to 2500 m/z and MS/MS was on the six most intense ions in each MS scan, in the range 300–3000 m/z.
Data analysis: Data was searched using X-Tandem! (version: TORNADO 2009.04.01.1). The following parameters were used: Fragment Tolerance: 50 PPM (Monoisotopic), Parent Tolerance: 25 PPM (Monoisotopic), Fixed Modifications: +57 on C (Carbamidomethyl), Variable Modifications: +1 on NQ (Deamidation), +16 on MW (Oxidation), +32 on M (Sulphone), +42 on n (Acetyl), Database: the Human Ensembl database and crap.fasta.pro database (unknown version, 76704 entries), Digestion Enzyme: ChymoTrypsin, Max Missed Cleavages: 3
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Protein digestion: Proteins were denatured in 1∶1 v/v trifluoroethanol (TFE), reduced, alkylated and digested with ChymoTrypsin (Promega) according to published protocols [17] .
LCMS/MS analysis: An Agilent 6520 QTOF equipped with an Agilent chip-cube nano-electrospray interface and an Agilent 1200 nano-LC system was used: Samples were trapped on an Agilent High capacity Chip (part #: G4240-62010) at 4 uL/min 99% buffer A (0.1% formic acid in water) and 1% buffer B (0.1% formic acid in acetonitrile), then eluted at 0.3 uL/min from 1% to 85% buffer B. The QTOF was operated in Auto MS/MS mode with the standard parameters for chip cube interface. MS spectra were acquired from 300 to 2500 m/z and MS/MS was on the six most intense ions in each MS scan, in the range 300–3000 m/z.
Data analysis: Data was searched using X-Tandem! (version: TORNADO 2009.04.01.1). The following parameters were used: Fragment Tolerance: 50 PPM (Monoisotopic), Parent Tolerance: 25 PPM (Monoisotopic), Fixed Modifications: +57 on C (Carbamidomethyl), Variable Modifications: +1 on NQ (Deamidation), +16 on MW (Oxidation), +32 on M (Sulphone), +42 on n (Acetyl), Database: the Human Ensembl database and crap.fasta.pro database (unknown version, 76704 entries), Digestion Enzyme: ChymoTrypsin, Max Missed Cleavages: 3
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