Each prepared metabolite sample was injected onto a Thermo Fisher Scientific Vanquish UHPLC with a Waters XBridge BEH Amide column (100 mm x 2.1 mm, 3.5 μm) coupled to a Thermo Fisher Q-Exactive mass spectrometer. Mobile phase (A) consisted of 97% H2O, 3% ACN, 20 mM ammonium acetate, and 15 mM ammonium hydroxide pH 9.6. Organic phase (B) consisted of 100% ACN. Metabolites were resolved using the following linear gradient: 0 min, 85% B, 0.15 ml/min; 1.5 min, 85% B, 0.15 ml/min; 5.5 min, 40% B, 0.15 ml/min; 10 min, 40% B, 0.15 ml/min; 10.5 min, 40% B, 0.3 ml/min; 14.5 min, 40% B, 0.3 ml/min; 15 min, 85% B, 0.15 ml/min; 20 min, 85% B, 0.15 ml/min. The mass spectrometer was operated in positive ionization mode with a MS1 scan at resolution = 70,000, automatic gain control target = 3 × 106, and scan range = 60–186 m/z and 187–900 m/z. This protocol was adapted from Mentch et al., 2015 (link). Individual metabolite data were called using MAVEN (Clasquin et al., 2012 ; Melamud et al., 2010 (link)) with retention times empirically determined in-house. Peak Area Top values were analyzed to determine metabolite expression.
Vanquish uhplc
Manufactured by Waters Corporation
Sourced in United States
The Vanquish UHPLC is a high-performance liquid chromatography system designed for advanced analytical applications. It features a modular design, allowing for customization and configuration to meet specific requirements. The Vanquish UHPLC system provides reliable and efficient separation of complex samples, enabling precise and accurate analysis.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion system, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (1 mentions)
Beh130 c18 column, by Waters Corporation (1 mentions)
Q exactive obitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Waters acquity uplc beh c18 column, by Waters Corporation (1 mentions)
4 protocols using vanquish uhplc
1
Targeted Metabolite Profiling by UHPLC-MS
2
High-Resolution Mass Spectrometry for Compound Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HPLC-UV/HRMS was performed using a Thermo Orbitrap Fusion system coupled with a Thermo Vanquish UHPLC using a Waters Cortecs UPLC C18 1.6 M (2.1 × 150 mm) column. The mobile phase consisted of 0.4% aqueous (deionized water) formic acid (phase A) and acetonitrile: methanol (50:50 v/v) LC-MS grade with 0.4% formic acid (phase B). At a flow rate of 0.2 mL/min, the linear gradient was as follows: 0.00–40.00 min, 70–30% (A–B%) to 15–85% (A–B%) followed by a 5 min column wash with 15–85% (A–B%) and 5 min equilibration period with 70–30% (A–B%). UV detection wavelength of 254 nm, a column temperature of 40 °C, and an injection volume of 2 µL were applied.
MS parameters in positive ionization mode were: ionization voltage 3500 V, electrospray ionization (ESI), ion transfer tube temperature 350°, vaporizer temperature 350 °C, 3 scans, resolution of 30000, HCD collision energy (%) 50. System control and data evaluation were performed with Thermo® Xcalibur for LC-MS.
MS parameters in positive ionization mode were: ionization voltage 3500 V, electrospray ionization (ESI), ion transfer tube temperature 350°, vaporizer temperature 350 °C, 3 scans, resolution of 30000, HCD collision energy (%) 50. System control and data evaluation were performed with Thermo® Xcalibur for LC-MS.
3
Proteomic Analysis of NCAP-1 Protein Digestion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NCAP-1
aliquots were digested overnight at 37 °C with trypsin (1:20
w/w) or chymotrypsin (1:50 w/w). Resultant peptides were separated
on a 2.1 × 150 mm BEH130 C18 column (Waters, Milford, MA) with
a water:acetonitrile gradient in the presence of 0.1% formic acid
delivered by a Thermo Fisher Scientific Vanquish UHPLC (San Jose,
CA). Eluting peptides were detected with an Orbitrap Fusion Lumos
(ThermoFisher Scientific). The ESI needle voltage was held at +3600
V and the sheath, auxiliary, and sweep gas flows were 35, 10, and
2, respectively (arbitrary units). The ion transfer tube and vaporizer
temperatures were set to 275 and 250 °C, respectively. Survey
scans were acquired from m/z 350–1400
at 60 000 resolution and RF 30%. Fragment spectra were generated
via CID (35% normalized CE) and detected in the ion trap. Data analysis
proceeded in MaxQuant 1.618 (link) employing the Cricetulus griselus Uniprot reference proteome database
supplemented with the nucleocapsid protein sequence and those of common
contaminants. Proteins detected by MS/MS with ≥3 unique peptides
were considered positive identifications. Relative host-cell protein
abundances were estimated using iBAQ19 (link) and
are shown inTable S1 .
aliquots were digested overnight at 37 °C with trypsin (1:20
w/w) or chymotrypsin (1:50 w/w). Resultant peptides were separated
on a 2.1 × 150 mm BEH130 C18 column (Waters, Milford, MA) with
a water:acetonitrile gradient in the presence of 0.1% formic acid
delivered by a Thermo Fisher Scientific Vanquish UHPLC (San Jose,
CA). Eluting peptides were detected with an Orbitrap Fusion Lumos
(ThermoFisher Scientific). The ESI needle voltage was held at +3600
V and the sheath, auxiliary, and sweep gas flows were 35, 10, and
2, respectively (arbitrary units). The ion transfer tube and vaporizer
temperatures were set to 275 and 250 °C, respectively. Survey
scans were acquired from m/z 350–1400
at 60 000 resolution and RF 30%. Fragment spectra were generated
via CID (35% normalized CE) and detected in the ion trap. Data analysis
proceeded in MaxQuant 1.618 (link) employing the Cricetulus griselus Uniprot reference proteome database
supplemented with the nucleocapsid protein sequence and those of common
contaminants. Proteins detected by MS/MS with ≥3 unique peptides
were considered positive identifications. Relative host-cell protein
abundances were estimated using iBAQ19 (link) and
are shown in
4
Metabolite Profiling via UHPLC-HRMS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each prepared metabolite sample was injected onto a Thermo Fisher Scientific Vanquish UHPLC with a Waters Acquity UPLC BEH C18 column (1.7μm, 2.1×100mm; Waters Corp., Milford, MA, USA) and analyzed using a Thermo Fisher Q Exactive obitrap mass spectrometer in negative ionization mode. LC separation was performed over a 25 minute method with a 14.5 minute linear gradient of mobile phase (buffer A, 97% water with 3% methanol, 10mM tributylamine, and acetic acid-adjusted pH of 8.3) and organic phase (buffer B, 100% methanol) (0 minute, 5% B; 2.5 minute, 5% B; 17 minute, 95% B; 19.5 minute, 5% B; 20 minute, 5% B; 25 minute, 5% B, flow rate 0.2mL/min). A quantity of 10μL of each sample was injected into the system for analysis. The ESI settings were 30/10/1 for sheath/aux/sweep gas flow rates, 2.50kV for spray voltage, 50 for S-lens RF level, 350C for capillary temperature, and 300C for auxiliary gas heater temperature. MS1 scans were operated at resolution = 70,000, scan range = 85–1250m/z, automatic gain control target = 1 × 106, and 100ms maximum IT. Metabolites were identified and quantified using El-MAVEN (v0.12.1-beta) with metabolite retention times empirically determined in-house. Metabolite levels were compared using the peak AreaTop metric.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!