For the time-dependent adduct formation study and the concentration-dependent adduct formation study, the acquired UHPLC–Q-Exactive-Orbitrap-MS raw data (in full-scan mode) was analyzed by TraceFinder 5.0 (Thermo Fisher Scientific, Mississauga, ON, Canada). The potential atrazine-modified peptide and non-modified peptide ions were selected as target ions, and the peak areas in extracted chromatograms were compared.
Tracefinder 5
TraceFinder 5.1 is a software application developed by Thermo Fisher Scientific for data processing and analysis in analytical laboratories. It provides a comprehensive suite of tools for the identification and quantification of target analytes in complex sample matrices.
Lab products found in correlation
17 protocols using tracefinder 5
Identification of Histone Atrazine Adducts
For the time-dependent adduct formation study and the concentration-dependent adduct formation study, the acquired UHPLC–Q-Exactive-Orbitrap-MS raw data (in full-scan mode) was analyzed by TraceFinder 5.0 (Thermo Fisher Scientific, Mississauga, ON, Canada). The potential atrazine-modified peptide and non-modified peptide ions were selected as target ions, and the peak areas in extracted chromatograms were compared.
Targeted Metabolomics Data Processing
Quantification of 6-AmHap-acetamide by UPLC-PRM
was performed in a Thermo Scientific Vanquish UPLC coupled with a
Q-Exactive Quadrupole-Orbitrap detector. The water’s HSS T3
column (2.1 mm × 100 mm, 1.8 μm particle size; Waters,
Milford, MA) and the following mobile phases were used: A (water with
10 mM NH4COOH and 0.1% HCOOH) and B (MeOH with 0.1% HCOOH).
The UPLC gradient used is described in
The injection volume was 10 μL. All data were acquired using
positive electrospray ionization (ESI) in a parallel reaction monitoring
(PRM) mode. The electrospray and source settings were as follows:
3.5 kV (capillary voltage), 320 °C (capillary temperature), 25
AU (sheath gas flow rate), 10 AU (Aux gas flow rate), and 300 °C
(Aux gas temperature). The analyte (6-AmHap-acetamide) was detected
as [M + H]+ with the PRM transition of 473.2215 > 129.0004
at 5.73 min (chromatographic retention time). Quantification was performed
using the external calibration method with a 1/X2 weighting scheme in TraceFinder 5.1 (Thermo Scientific, Waltham,
MA).
Palmitic Acid Quantification in Fungal Hyphae
Quantification of hexose sugars in fungi
Quantitative Metabolomic Data Processing
Characterization and Purification of Immunomodulatory Lipids
and reagents used in mobile phases (methanol, water, and formic acid)
were Optima LC-MS grade and were purchased from Fisher Chemicals.
DMPC, DMPG, 3D-PHAD (synthetic monophosphoryl lipid A, MPLA), and
cholesterol for liposomal preparation were purchased from Avanti Polar
Lipids Inc. and were used without further purification. Triethylamine
(Et3N) used for the hydrolysis reaction was purchased from
Sigma-Aldrich (Saint Louis, Missouri). The QS-21 working standard
was prepared from in-house HPLC-purified QS-21 purchased from the
vendor Indena (through Desert King). The QS-21 HP working standard
was generated from the purified product of the base-mediated hydrolysis
of QS-21.
ALFQ was prepared following the established procedure.31 (link) cGMP-grade ALFQ was provided by the Pilot Bioproduction
Facility (PBF) at the WRAIR.
Purification of QS-21 and QS-21
HP was done using a Shimadzu UltraFast
Liquid Chromatograph (UFLC; LC-6AD) equipped with a Shimadzu Fraction
Collector (FRC-10A). Quantitative analyses were done using a Thermo
Scientific Vanquish Flex UHPLC system coupled with a Q-Exactive Quadrupole-Orbitrap
Mass Spectrometer, controlled by Xcalibur software version 4.4. The
data were processed using Thermo Scientific TraceFinder 5.1.
UHPLC-Q-Exactive Analysis of QS-21 Saponins
of QS-21 and QS-21 HP was done using a Thermo Scientific Vanquish
UHPLC coupled with a Q-Exactive Quadrupole-Orbitrap detector. The
separation was carried out in an Agilent Zorbax Eclipse Plus C18 column
(4.6 mm ID × 50 mm, 1.8 μm particle size), using water
with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) as
mobile phases with a constant flow of 0.5 mL/min at a controlled column
temperature of 35 °C. The UPLC gradient used is described in
All data were acquired using negative electrospray ionization (ESI)
in parallel reaction monitoring (PRM) mode. The electrospray and source
settings were as follows: 2.5 kV (capillary voltage), 320 °C
(capillary temperature), 25 AU (sheath gas flow rate), 10 AU (Aux
gas flow rate), and 300 °C (Aux gas temperature).
Intact
QS-21 1 and QS-21 2 were detected as [M – H]− with a PRM transition of m/z 1987.9169
> 485.3272 at 10.91 and 10.41 min (chromatographic RT), respectively.
QS-21 R1 and R2 derivatives eluted at 10.83 and 11.40 min, respectively,
were detected using the m/z 1855.8746
> 485.3268 PRM transition. The degradation product QS-21 HP was
detected
using a PRM transition of m/z 1511.6548
> 955.4549 at 7.19 min. Quantification was done using an external
calibration method with an equal weighting scheme in TraceFinder 5.1
(Thermo Scientific, Waltham, MA).
LC-MS Metabolomics Data Processing
Quantification of Solanidine and Metabolites
14 (link) but, in this case, applied for identification and quantification of solanidine and metabolites through retrospective reprocessing of the non‐selective full scan HRMS data, as described in detail elsewhere.
8 (link),
9 (link) Solanidine and metabolites (i.e., M414, M416, and M444) were identified in the HRMS data files by accurate mass (m/z, ±5 ppm) detection at the fourth decimal and isotope ratio. The identity of solanidine was confirmed using retention time and matched tandem mass spectrometry spectrum by analyzing a reference standard purchased from Phytolab (Vestenbergsgreuth, Germany). TraceFinder 5.1 (Thermo Fisher Scientific) was used for data processing. All peaks were integrated automatically. Undetectable levels of solanidine and solanidine metabolites were truncated to the lower limit of detection (LLOD; i.e., M414, 1564; M416, 2041; M444, 1257; and solanidine, 2556) found for the corresponding analyte to enable proper calculations of metabolic ratios.
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