DNA was purified and extracted from 235 saliva samples. Briefly, 4 ml of saliva was incubated in a water bath at 50°C overnight. Following incubation, the appropriate amount of purifier was added followed by 10 min of ice incubation. The samples were centrifuged for 20 min at maximum speed (4000 rpm) at room temperature. In order to precipitate the DNA, the yielded supernatant after centrifugation was transferred to new centrifuge tubes followed by adding an equivalent amount of ethanol and then kept in a refrigerator overnight. Samples were then centrifuged for 20 min at maximum speed (4000 rpm) at room temperature. The yielded supernatant was then discarded and the DNA pellet was dried for 30 min followed by adding 1 ml of Tris-EDTA buffer placed on a rotator (34 rpm) overnight. Finally, samples were prepared, labeled and analyzed for quality and quantity using an eight-multichannel Nano-drop (ND8000 V.2., Thermo Fisher Scientific, DE, USA). Eight candidate SNPs in five key genes were genotyped at the Biomedical Genomic Center at the University of Minnesota (MN, USA). Six SNPs were genotyped by Sequenom iPLEX Gold method using Bruker Autoflex II MALDI/TOF mass spectrometer (Bruker, MA, USA; rs505802, rs2231142, rs12129861, rs11942223, rs1014290, rs1183201). The other two SNPs (rs3733591, rs734553) were genotyped using TaqMan® (Applied Biosystems™ 7500 Real-Time PCR, CA, USA).
Autoflex 2 maldi tof mass spectrometer
Manufactured by Bruker
Sourced in United States
The Autoflex II MALDI-TOF mass spectrometer is a laboratory instrument developed by Bruker. It is designed to perform matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, a technique used for the analysis of biomolecules such as proteins, peptides, and other macromolecules.
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Lab products found in correlation
Surveyor hplc system, by Thermo Fisher Scientific (3 mentions)
Ls6500, by Beckman Coulter (2 mentions)
Micromass q tof ultima, by Thermo Fisher Scientific (1 mentions)
Q exactive hybrid quadrupole orbitrap, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 hplc system, by Macherey-Nagel (1 mentions)
Ltq orbitrap discovery, by Thermo Fisher Scientific (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000 liquid chromatography, by Thermo Fisher Scientific (1 mentions)
Cary 300 bio, by Agilent Technologies (1 mentions)
Flexanalysis 3, by Bruker (1 mentions)
Mtp 384 target plate polished steel bc, by Bruker (1 mentions)
Avance 400 spectrometer, by Bruker (1 mentions)
6 protocols using autoflex 2 maldi tof mass spectrometer
1
DNA Extraction and Genotyping from Saliva Samples
2
Analytical Techniques for Chemical Characterization
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All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) and were of the highest purity commercially available. HPLC was performed on a Dionex Ultimate 3000 HPLC system equipped with a diode array detector using Macherey-Nagel C18 reverse-phase column. Radiolabeled samples were counted in a Beckman LS6500 scintillation counter. NMR spectra were acquired on a Bruker AVANCE III 500 MHz high-field NMR spectrometer, and the data were processed using Topspin software (https://www.bruker.com/en/products-and-solutions/mr/nmr-software/topspin.html?gclid=EAIaIQobChMIi-T93OvA-QIVzmSLCh2ScAJOEAAYASAAEgLEgvD_BwE , accessed on 2 July 2022). Mass spectra were obtained with a Bruker Autoflex II MALDI-TOF mass spectrometer operated in positive ion reflectron mode. HRMS spectra were acquired with either a Waters Micromass Q-tof Ultima or a Thermo Scientific Q-Exactive hybrid Quadrupole Orbitrap.
3
Analytical Characterization of Chemical Compounds
4
MALDI-TOF Mass Spectrometry Protocol
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Mass spectra were obtained using an Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Measures were performed in linear positive mode, using a nitrogen laser (337 μm) at 50 Hz frequency. The acceleration voltage was 19.50 kV, with delay time acquisition. The analytical samples were obtained by the dry-droplet method. Briefly, 1 μL of an analyte solution in methanol (1 mg/mL) was loaded on the MALDI plate (MTP 384 target plate polished steel BC, Bruker Daltonics, Bremen, Germany) and allowed to dry at 23 °C. Each sample was covered with 2 μL of matrix (α-cyano-4-hydroxycinnamic acid) solution (10 mg/mL, 50% acetonitrile, water 47.5% and 2.5% trifluoroacetic acid) and allowed to dry at 23 °C before the plate was inserted into the vacuum chamber of the MALDI instrument. Data analysis was carried in FlexAnalysis 3.0 software (Bruker Daltonics, Bremen, Germany).
5
Ion-induced PML-R Modifications and Analysis
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Before analysis, the mixtures (0.5 ml) containing 20 μM purified PML-R and 200 μM ion (Zn2+, As3+, or Se4+) were incubated at 37 °C for 2 h. Then, Zn2+-, As3+-, or Se4+-containing solutions of PML-R were mixed with 50 mM IA in the dark at room temperature for 1 h. The residual IA and ions were washed out by NH4HCO3 (50 mM) using a centrifugal filter (3 kDa). The concentration of deionized PML-R was determined by the method of Bradford. The deionized PML-R was incubated with 5% trypsin overnight at 37 °C. Digested peptide fragments were dissolved in 60% acetonitrile and 0.1% (v/v) trifluoroacetic acid, lyophilized, and stored at −80 °C for analysis.27 (link), 41 (link)Before MS analysis, the lyophilized sample was dissolved in 10 μl H2O and mixed with 10 μl α-cyano-4-hydroxycinnamic acid matrix solution. The mixture was dried by spotting on a polished steel target plate. The masses of peptide fragments ware recorded on an AUTOFLEX II MALDI-TOF mass spectrometer (Bruker, Madison, WI,USA) with a 20-kV acceleration voltage.40 (link), 41 (link)
6
Synthesis and Characterization of Boron Compounds
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All reagents were purchased from commercial sources and used without further purification, unless otherwise stated. Reagent grade solvents were distilled prior to use. Column chromatography was performed on silica (silica gel, 230–400 mesh). 1H, 13C and 11B NMR, and 2D NMR spectra were recorded on a Bruker Avance 400 spectrometer. 1H and 13C NMR spectra were calibrated to the residual solvent signals. 11B NMR spectra were calibrated to the signal of boron trifluoride diethyl etherate (BF3·Et2O) as external standard. J values are given in Hz. The following abbreviations were used to designate multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet. High resolution mass spectra were obtained by electrospray ionization (ESI) and were recorded on an ESI microOTOF Focus spectrometer from Bruker Daltonics. Low resolution mass spectra were obtained by matrix-assisted laser desorption/ionization (MALDI) and were recorded on an Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics GmbH). 1,3-indandione was commercially available. Compounds 4 [29 (link),36 (link)] and 5 [29 (link)] were prepared according to reported procedures.
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