For the detection of metabolites by LC-MS, a Dionex UltiMate 3000 LC System (Thermo Scientific) with a thermal autosampler set at 4 °C, coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) was used. Samples were resuspended in 50 μl of water and a volume of 10 μl of sample was injected on a C18 column (Acquity UPLC HSS T3 1.8 μm 2.1 × 100 mm). The separation of metabolites was achieved at 40 °C with a flow rate of 0.25 ml/min. A gradient was applied for 40 min (solvent A: 10 mM Tributyl-Amine, 15 mM acetic acid—solvent B: Methanol) to separate the targeted metabolites (0 min: 0% B, 2 min: 0% B, 7 min: 37% B, 14 min: 41% B, 26 min: 100% B, 30 min: 100% B, 31 min: 0% B; 40 min: 0% B). The MS operated in negative full scan mode (m/z range: 70–1050 and 300–700 from 5 to 25 min) using a spray voltage of 4.9 kV, capillary temperature of 320 °C, sheath gas at 50.0, auxiliary gas at 10.0. Data were collected using the Xcalibur software v4.0 (Thermo Scientific) and analyzed with Matlab for the correction of protein content and natural abundance, but also to determine the isotopomer distribution using the method developed by Fernandez et al.54 (link).
Xcalibur software v4
Manufactured by Thermo Fisher Scientific
Sourced in United States
Xcalibur software v4.0 is a data acquisition and analysis platform for Thermo Fisher Scientific's mass spectrometry instrumentation. The software enables users to control the instrument, acquire and process data, and interpret results.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000 lc system, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 uhplc, by Thermo Fisher Scientific (1 mentions)
Pierce ltq velos esi positive ion calibration solution, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ultimate ncs 3500rs nanouhplc, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap rslc, by Thermo Fisher Scientific (1 mentions)
Nanospray flex ion source, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy nanolc 1200 system, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap 100 pre column, by Thermo Fisher Scientific (1 mentions)
4 protocols using xcalibur software v4
1
Metabolite Detection by LC-MS
2
UHPLC-HRMS Analysis of Compounds
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The UHPLC-HRMS system consisted of a Dionex UltiMate 3000 UHPLC coupled to a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) operating with a heated electrospray ionization (HESI II) source in positive ionization mode. For mass calibration of the instrument, Pierce™ LTQ Velos ESI Positive Ion Calibration Solution (Thermo Fisher Scientific, Waltham, MA, USA) was used.
During the HRMS optimization and in both FIA-MSX-MS/MS and UHPLC-MS/MS methods, the ion-source parameters were spray voltage 4 kV; sheath gas (N2) 50; auxiliary gas (N2) 20; sweep gas (N2) 1; capillary temperature 300 °C; S-lens RF level 40; and auxiliary gas heater temperature 400 °C. Value for automatic gain control (AGC) target was set at 1 × 106, with a resolution of 35,000 FWHM (full width at half maximum, m/z = 200). The accumulation time limit of the ions per scan event was 10 ms.
The instrument control and the data processing were performed using QExactive Plus Tune 2.8 and TraceFinder General Quan 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA), while Xcalibur software v. 4.0 (Thermo Fisher Scientific, Waltham, MA, USA) was applied for the spectral examinations.
During the HRMS optimization and in both FIA-MSX-MS/MS and UHPLC-MS/MS methods, the ion-source parameters were spray voltage 4 kV; sheath gas (N2) 50; auxiliary gas (N2) 20; sweep gas (N2) 1; capillary temperature 300 °C; S-lens RF level 40; and auxiliary gas heater temperature 400 °C. Value for automatic gain control (AGC) target was set at 1 × 106, with a resolution of 35,000 FWHM (full width at half maximum, m/z = 200). The accumulation time limit of the ions per scan event was 10 ms.
The instrument control and the data processing were performed using QExactive Plus Tune 2.8 and TraceFinder General Quan 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA), while Xcalibur software v. 4.0 (Thermo Fisher Scientific, Waltham, MA, USA) was applied for the spectral examinations.
3
Peptide Analysis by Nano-UHPLC-MS/MS
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Peptides were analysed on a Dionex UltiMate NCS-3500RS nanoUHPLC coupled to a Q-Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer equipped with nanospray ion source in positive mode as described. [39, 40] Peptides were loaded (Acclaim PepMap100 C18 5 m beads with 100 Å pore-size, Thermo Fisher Scientific) and separated (1.9-µm particle size C18, 0.075 × 250 mm, Nikkyo Technos Co. Ltd) with a gradient of 2-28% acetonitrile containing 0.1% formic acid over 110 mins at 300 nL min -1 at 55°C (butterfly portfolio heater, Phoenix S&T). An MS1 scan was acquired from 350-1,650 m/z (60,000 resolution, 3 × 10 6 automatic gain control (AGC), 128 msec injection time) followed by MS/MS data-dependent acquisition (top 30) with collision-induced dissociation and detection in the ion trap (15,000 resolution, 1 ×10 5 AGC, 60 msec injection time, 28% normalized collision energy, 1.3 m/z quadrupole isolation width). Unassigned precursor ions charge states and slightly charged species were rejected and peptide match disabled. Selected sequenced ions were dynamically excluded for 30 sec. Technical replicates were performed for all analyses (n=2). Data was acquired using Xcalibur software v4.0 (Thermo Fisher Scientific). A list of samples and RAW data is available in ProteomeXchange. #PXD023570.
4
Peptide Separation and Mass Spectrometry Analysis
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Mass spectrometry analyses were conducted using a Q-Exactive HF-X mass spectrometer (Thermo Scientific, Bremen, Germany) connected to an EASY-nanoLC 1200 System (Thermo) using a nanospray flex ion source (Thermo). Desalted peptides were loaded onto an Acclaim PepMap100 precolumn (75 μm × 2 cm, Thermo Scientific) connected to an Acclaim PepMap RSLC (75 μm × 25 cm, Thermo Scientific) analytical column34 (link). To elute peptides from the column, we used the following gradient: 120 min from 2.4 to 24%, 2 min from 24 to 32% and 12 min at 80% acetonitrile in 0.1% formic acid at a flow rate of 300 nL min−1. Full MS scans were obtained from m/z 375 to 1800 with a resolution of 60,000 at m/z 200. The 10 most intense ions were fragmented by higher energy C-trap dissociation with a normalised collision energy of 28, and MS/MS spectra were documented with a resolution of 15,000 at m/z 200. The maximum ion injection times were 50 ms and 100 ms, whereas AGC target values were 3 × 106 and 1 × 105 for survey and MS/MS scans, respectively. To avoid repeat sequencing of peptides, dynamic exclusion was applied for 20 s. Singly charged ions, ions with unassigned charge states and ions with charge states above 5 were also ignored from MS/MS. Data were obtained using Xcalibur software (v.4.0) (Thermo Scientific)34 (link).
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