All reagents were purchased from commercial sources and were used without further purification. Pre-coated ANALTECH uniplate was used for Thin layer chromatography (TLC) and monitored under UV light at 254 nm and with potassium permanganate stain. Silica gel (230–400 mesh, grade 60, Fisher scientific, USA) was used for Column chromatography. 1H NMR and 13C NMR spectra were recorded in chloroform-d or DMSO-d6 on a Bruker-400, Bruker-500 and Bruker-600 spectrometer. The purity of final compounds was determined by analytical HPLC and was found to be ≥95% pure. Analysis of sample purity was performed on a Waters Alliance 2695 HPLC system Phenomenex Luna-2 RP-C18 (5 μm, 4.6 mm × 250 mm, 120 Å, Torrance, CA) column. HPLC conditions: solvent A, H2O containing 0.1% formic acid (FA); solvent B, CH3CN containing 0.1% FA; gradient, 10% B to 100% B over 15 min followed by 100% B over 4 min with flow rate, 1 mL/min. Mass spectrometric data was acquired using Quattro Micro triple quadrupole mass spectrometer with electron-spray ionization (ESI) technique and as TOF mass analyzer. HRMS data was generated on an Agilent 6230 LC/TOF system with UV detector (254 nm).
6230 lc tof system
Manufactured by Agilent Technologies
The 6230 LC/TOF system is a high-performance liquid chromatography and time-of-flight mass spectrometry (LC/TOF-MS) instrument designed for accurate mass analysis. The system combines liquid chromatography separation with precise mass detection to provide detailed information about the molecular composition of samples.
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2 protocols using 6230 lc tof system
1
Analytical Characterization of Organic Compounds
2
Analyzing V272M DBD-PAT Interaction by MS
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Protein buffer of V272M DBD was replaced by 200 mM ammonium acetate buffer (pH 7.0) using dialysis device. V272M DBD was mixed with PAT at a molar ratio of 1 : 1 and the mixture was divided into two same samples. The mass of the two samples were immediately determined, without an incubation step, by native MS and denatured MS, respectively. The mass of V272M DBD without PAT treatment was also determined in the same way. Native MS was carried out on a 1290 Infinity II LC coupled with a 6230 LC/TOF system equipped with an Agilent Jet Stream source. LC separation was obtained with PolyHYDROXYETHYL ATM column (200 3 2.1 mm, 5 mm, 200 A ˚). In denatured MS, a 1290 Infinity II LC coupled with a 6530 LC/QTOF system and an Agilent 300SB-C8 column (50 3 2.1 mm, 3.5 mm) were used for denatured protein determination. All experimental MS data of the samples were processed using Agilent MassHunter Qualitative Analysis 7.0 software. Experimental molecular weights (MW) were determined from MS peak maxima with average MW and standard deviations calculated from identified charge states.
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