Acetylation reactions for mass spectrometry analysis were performed like those for the aforementioned acetyltransferase assays. However, to obtain sufficient signal for detection after subsequent processing, 75 nM p300 or 200 nM CBP enzyme was used as well as 50 μM nonradioactive acetyl-CoA and 1 μM nucleosome. Reactions were quenched with 4 volumes of trichloroacetic acid, mixtures washed with acetone and dried, and free lysines blocked by treatment with propionic anhydride followed by pH adjustment with ammonium hydroxide and incubation for 1 h at 51 °C. The modified proteins were digested in trypsin overnight at 37 °C before separation of peptides on a Waters Acquity UPLC system with a BEH C18 column and identification on a Thermo TSQ Quantum Access triple-quadrupole mass spectrometer. Peptide elution was monitored by selected reaction monitoring for H3 and H4 acetylated and propionylated peptides.17 (link) Six time points were taken for each reaction, and the data were plotted with respect to time to determine rates and standard deviations. Statistical significance was determined using unpaired two-tailed t tests and the Holm–Sidak method to correct for multiple comparisons (α = 5.000%).
Tsq quantum access triple quadrupole mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TSQ Quantum Access triple quadrupole mass spectrometer is an analytical instrument designed for high-performance quantitative analysis. The core function of this device is to separate and detect ionized molecules based on their mass-to-charge ratio, enabling precise identification and quantification of chemical compounds within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Vacuum pump v 700, by Büchi (1 mentions)
Vacuum controller v 850, by Büchi (1 mentions)
Accela ultra high performance liquid chromatography system, by Thermo Fisher Scientific (1 mentions)
Rotavapor r 210, by Büchi (1 mentions)
Acquity h class uplc, by Thermo Fisher Scientific (1 mentions)
7 protocols using tsq quantum access triple quadrupole mass spectrometer
1
Acetylation Profiling of Histones
2
Quantification of Histone Acetylation
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Histones were extracted as described in the “Acid extraction of histones” section. Dry histone pellets were stored at −80°C until processing for LC/MS analysis. The unmodified lysines in dry histone were propionylated by propionic anhydride at pH 8 and 51°C for 1 hour followed by trypsin digestion at pH 8 and 37°C for overnight as in our previously published procedures (57 (link)). A Waters Acquity H-class Ultra-performance liquid chromatography (UPLC) coupled with a Thermo TSQ Quantum Access triple quadrupole mass spectrometer was used to quantify the acetylated lysines on H3 tryptic peptides. The UPLC and tandem mass spectrometry (MS/MS) settings, solvent gradient, and detailed mass transitions were reported previously (58 (link)). Retention time and specific mass transitions were both used to identify individual acetylated and propionylated peaks. The 13C-labeled acetyl-lysine peptide peaks were identified by considering the shift in mass by +2. The resolved peaks were integrated using Xcalibur software (version 2.1, Thermo Fisher Scientific). Relative quantitative analysis was used to determine the amount of modification on individual lysines.
3
Quantifying Advanced Glycation Endproducts in ApoA-IV
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LC-MS/MS was used to determine carboxymethyllysine (CML) and pyrraline (PYR) levels in apoA-IV as previously described [44 (link)]. Briefly, unmodified and AGE-apoA-IV were concentrated and digested using four different enzymes (pepsin, pronase E, aminopeptidase, and prolidase). Samples were separated and analyzed by high-performance liquid chromatography (HPLC) coupled directly to a TSQ Quantum Access triple quadrupole mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via an electrospray interface operating in the positive mode. Calibration curves generated with a range of concentrations (0-10 ng of CML/μL and 0-100 pg of PYR/μL) of unlabeled standards and a constant concentration of isotopically labelled standards (ISTD) were used to calculate the amount of CML and PYR in samples.
4
Quantitative Protein Hydrolysis Protocol
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For protein digestion, C and AGE-albumin were incubated with pepsin, pronase E, aminopeptidase and prolidase, under argon gas, at 37°C, for 4 days, according to Rabbani et al. (2014 (link)). A gradient solution [0.1% formic acid and 5 mM ammonium formate in water (mobile phase A) and 0.01% formic acid in acetonitrile (mobile phase B)] at the flow rate of 500 μl/min (0.7 min of 95% B, 4 min of 99% A, 5.3 min of 95% B) was used to separate the enzymatic hydrolysates at 40°C (SeQuant ZIC®HILIC HPLC column (IDLX: 2.1 × 100 mm; Particle size: 3.5 μm and Pore Size: 200 A -Merck Millipore).
The HPLC system was directly coupled to a TSQ Quantum Access triple quadrupole mass spectrometer (Thermo Fisher Scientific); sheath gas pressure and auxiliary gas pressure were 30 and 15 (Thermo Fisher Scientific arbitrary units), respectively, and capillary temperature 245°C; collision-induced dissociation was performed in Q2 operated with argon at 2 kPa. For each analyte two most intense selected reaction-monitoring transitions were chosen. XCalibur software was used for the quantification and the calibration curves generated by analysis of unlabeled standard with a constant concentration of isotopically labeled standards were used to calculate the compound concentration in the samples (Woods et al., 2013 (link)).
The HPLC system was directly coupled to a TSQ Quantum Access triple quadrupole mass spectrometer (Thermo Fisher Scientific); sheath gas pressure and auxiliary gas pressure were 30 and 15 (Thermo Fisher Scientific arbitrary units), respectively, and capillary temperature 245°C; collision-induced dissociation was performed in Q2 operated with argon at 2 kPa. For each analyte two most intense selected reaction-monitoring transitions were chosen. XCalibur software was used for the quantification and the calibration curves generated by analysis of unlabeled standard with a constant concentration of isotopically labeled standards were used to calculate the compound concentration in the samples (Woods et al., 2013 (link)).
5
Quantitative Phenolic and Carotenoid Analysis
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Extract condensation was performed under vacuum and heat-assisted evaporation, in temperatures below 35 °C, using a Büchi Rotavapor R-210 apparatus, equipped with a Büchi vacuum pump V-700, Vacuum controller V-850 (all obtained from Büchi, Flawil, St Gallen, Switzerland), and Julabo F12 (Seelbach, Germany) cooling unit.
The estimation of Total Phenolic Content (TPC) and Total Tannin Content (TTC) was implemented using an Infinite® 200 PRO microplate reader (Tecan Group Ltd., San Jose, CA, USA) and the estimation of Total Carotenoid Content (TCC) was performed using an x-ma 100 spectrophotometer (Human Corporation, Seoul, Republic of Korea).
An Accela Ultra High-Performance Liquid Chromatography system equipped with an autosampler and coupled with a TSQ Quantum Access triple-quadrupole mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for the determination of the phenolic and carotenoid compounds qualitative and quantitative content.
The estimation of Total Phenolic Content (TPC) and Total Tannin Content (TTC) was implemented using an Infinite® 200 PRO microplate reader (Tecan Group Ltd., San Jose, CA, USA) and the estimation of Total Carotenoid Content (TCC) was performed using an x-ma 100 spectrophotometer (Human Corporation, Seoul, Republic of Korea).
An Accela Ultra High-Performance Liquid Chromatography system equipped with an autosampler and coupled with a TSQ Quantum Access triple-quadrupole mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used for the determination of the phenolic and carotenoid compounds qualitative and quantitative content.
6
Quantification of Histone Acetylation by UPLC-MS/MS
7
Characterization of Organic Compounds
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Proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded on a Varian 500 spectrometer (at 500 MHz for 1 H and 125 MHz for 13 C) using CDCl 3 or DMSO-d 6 as solvents. Chemical shifts are given in parts per million ( ppm) (δ relative to the residual solvent peak for 1 H and 13 C), coupling constants (J) are given in Hertz, and the attributions are supported by heteronuclear single-quantum coherence (HSQC) and correlation spectroscopy (COSY) experiments. Chemical shifts are reported in ppm relative to the residual CHCl 3 peak (7.26 ppm) or residual DMSO peak (2.49 ppm). Combustion analyses were carried out on a FISONS EA1108 elemental analyzer, and mass analysis of final products (1a and 1b) was performed with a TSQ-Quantum access Triple Quadrupole Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a HESI (Heated ElectroSpray Ionization) source; analyses were run in positive mode. Mass spectrometer parameters were: sheath gas flow rate, 30 (arbitrary units); aux gas flow rate, 15 (arbitrary units); spray voltage, 5.00 kV; capillary temperature, 250°C; tube lens voltage, 55 V; heater temperature, 270 °C; and scan mode: full scan.
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