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7 protocols using lcq deca xp plus mass spectrometer

1

Comparative Metabolomics of Insect Herbivores

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For the comparison between insects from willows and those from the tree of heaven, 0.1 g dry weight of L. delicatula specimens was crushed with a spatula. 1 mL MeOH was added and the mixture was sonicated for 30 minutes. Specimens were classified according to developmental stage (nymph instars 3, 4 and adults) and the host plant species (the tree of heaven and the Korean willow).
Prepared sample was analysed by LCQ mass spectrometry by NICEM at Seoul National University. An Acquity HSS T3 column with 1.8 μm particles with Solvent A (Water, 0.2% formic acid) and solvent B (98% acetonitrile, 2% water with 0.2% formic acid) were used. Separation was carried out under the following condition: 5 min 90% A, 2.5 min 90–0% A, 10 min 0% A. then a fast return in 0.1 min to 100% A and 2 min for column equilibration. The flow rate was increased from 350 μL/min. Mass spectrometry was carried out with a Thermo Finnigan LCQ Deca XP Plus mass spectrometer operated in positive and negative mode. A full scan (m/z 50–2000) at 30,000 and 60,000 resolving power was conducted. Ions were generated through probe electrospray ionization (PESI). For collision induced dissociation (CID), normalized collision energy of 25% was used.
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2

Enzymatic Sulfation of 5α-Cyprinol

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Enzymatic sulfation of 5α-cyprinol was performed with zebrafish Sult2st3 and nonradioactive PAPS as the sulfate donor based on the above-mentioned procedure. The final reaction mixture was applied to a Waters Sep-Pak C18 cartridge. The bound 5α-cyprinol and its sulfated metabolite were eluted using 80% methanol, and the eluate was dried using a SpeedVac concentrator. The eluate reconstituted into 50% methanol/1% formic acid was analyzed by direct-infusion electrospray ion-trap mass spectrometry in the negative ion mode using a Thermo-Finnigan LCQ DECA XP Plus mass spectrometer. Ionization conditions employed were: spray voltage, 5 kV; sheath gas flow rate, 40; capillary temperature, 315 °C, and the infusion flow rate, 5 μL/min. For NMR analysis, the eluate from Waters Sep-Pak C18 cartridge was further loaded into Oasis WAX column. The bound 5α-cyprino sulfate was eluted into 80% methanol/1% NH4OH and dried using a SpeedVac concentrator. The isolated sample was dissolved in CD3OD, respectively, (Aldrich) and analyzed using a Bruker Advance 600 instrument (600 MHz) (Bruker) with 1H-NMR, 1H-1H COSY, and HSQC modes. The data obtained are summarized in Table S1.
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3

Quantification of Thiol Precursors in Grape Juice

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Freshly thawed juice obtained from fresh whole bunches (n = 21) was cold settled at 4 °C for 2 hours and aliquot was analysed for thiol precursors (Cys-3-SH, Glut-3-SH) in duplicate according to a previously reported method with modified reconstitution procedure (Capone & Jeffery, 2011) .
Analysis was performed on a Thermo Finnigan Surveyor HPLC fitted with an Alltima C18 HPLC column (250 × 2.1 mm i.d., 5 µm, 100 Å, Grace Davison Discovery Sciences, Rowville, VIC, Australia) connected to a Thermo Finnigan LCQ Deca XP Plus mass spectrometer using electrospray ionisation in positive ion mode. Chromatographic conditions and ion pairs were as described previously (Capone, Sefton, Hayasaka, & Jeffery, 2010) and helium was used as collision gas with the following source and mass spectrometer conditions: spray voltage of 4.5 kV, respective sheath and aux/sweep gas flow rates of 30 and 19, capillary voltage of 36 V, capillary temperature of 250 °C, single reaction monitoring mode with activation Q of 0.250, activation time of 30 ms, normalised collision energy of 35%, and isolation width m/z = 1.50. Xcalibur software (Thermo Finnigan, version 1.3) was used for instrument control and data acquisition. Cys-3-SH and Glut-3-SH concentrations were reported as the sum of the two respective diastereomers.
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4

Analytical Characterization of Compounds

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All general chemicals and reagents were of analytical grade and were purchased from Merck (Damstadt, Germany) and J.T.Baker (Deventer, The Netherlands).
Low-resolution HPLC-ESI-MS measurements were carried out by a Surveyor HPLC system (Thermo Fisher, San Jose, CA, USA) connected by a T splitter to a photodiode-array detector and an LCQ Deca XP Plus mass spectrometer (Thermo Fisher). The mass spectrometer was equipped with an ESI source. High-resolution HPLC-ESI-MS/MS experiments were carried out by an Ultimate 3000 Micro HPLC apparatus (Dionex, Sunnyvale, CA, USA) equipped with a FLM-3000-Flow manager module and coupled to an LTQ Orbitrap XL apparatus (Thermo Fisher) (12 (link)).
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5

Protein N- and C-terminal Sequencing

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N- and C-terminal sequencing of the proteins was carried out by ProtTech (People’s Republic of China). In brief, GH18A and GH18B-CBM14 were dissolved in 6 M guanidine–HCl. Cysteine residues were reduced by adding 20 mM DTT and were then alkylated by reaction with iodoacetamide. After desalting, the protein samples were separated by SDS–PAGE. The protein gel bands were excised and subjected to trypsin and chymotrypsin digestion. The resulting peptides from each digestion reaction were analysed by nano-ESI-LC-MS/MS. In the analysis, the peptide samples were separated by reverse-phase HPLC coupled online to an LCQ Deca XP Plus mass spectrometer (Thermo, Waltham, USA). The mass-spectrometric data were analysed using proprietary peptide-mapping software from ProtTech to map the N- and C-termini of GH18A and GH18B-CBM14.
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6

Analytical Instrumentation for Multidisciplinary Research

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Tecan infinite M1000 system (Tecan, Zurich, Switzerland). AB TripleTOF 5600plus System (AB SCIEX, Framingham, USA), coupled to a Waters ACQUITY UPLC™ system (Waters, MA, USA). Finnigan LCQ DecaXPplus mass spectrometer equipped with an ESI source (Thermo, MA, USA) coupled to Agilent 1100 liquid chromatography (Agilent, Waldbronn, Germany). Agilent 1200 preparative performance liquid chromatography (Agilent, Waldbronn, Germany). Leica DMI 3000B Fluorescence Inversion Microscope System (Leica Microsystems Inc., USA), Andor Zyla 5.5 sCMOS Cameras (Oxford Instruments plc, Tubney Woods, Abingdon, UK). IX-100F Zebrafish system (iWorx Systems, Inc., USA).
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7

Radiolabeling of NOTA-Conjugated Oligonucleotides

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The 20-mer PO oligonucleotides bearing a 5-aminohexyl tether were purchased from Beijing Tsing Biotech Co., Ltd. (Beijing, China). The antisense and sense sequences used here were 5'-GGGAGTTACTTGCCAACTTG-3' and 5'-CAAGTTGGCAAGTAACTCCC-3', respectively. (HEPES), HCl (30%), acetonitrile (ACN), sodium chloride, sodium acetate, methanol, ammonium acetate, and ethanol (96%) were obtained from Merck (Darmstadt, Germany). p-SCN-Bn-NOTA) was purchased from Macrocyclics (Dallas, TX, USA). Foetal bovine serum (FBS) was obtained from Biosharp. Glen-Pak DNA puri cation cartridges were purchased from Glen Research. Pure water (18.2MΩ cm) was used in all reactions. Unless stated otherwise, all other chemicals used for tracer synthesis were obtained either from Acros (Geel, Belgium) or Sigma-Aldrich.
Aqueous triethylammonium acetate (TEAA) (1 M, pH 7.0) was prepared with 1 M acetic acid and 1 M TE (Sigma-Aldrich). A Thermo LCQ Deca XP plus Mass Spectrometer was used to record ESI-MS spectra.
Products were isolated and puri ed using a HPLC C18 column (1200 series, Zorbax ODS 4.6 × 250 mm). HPLC conditions were as follows: A/B gradient; A: 2% acetonitrile in 0.1 M TEAA (pH 7.0); B: 50% acetonitrile in 0.1 M TEAA (pH 7.0); elution: 1 mL/min. A 68 Ge-68 Ga generator (ITG Isotope technologies) was used to produce [ 68 Ga] Cl 3 .
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