The HPLC system (1100/1200 series, Agilent, Waldbronn, Germany) consisted of a binary pump (G1312A), an autosampler (G1329B), and a DAD-detector (G1316A). It was coupled to a HCT Ultra Ion Trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization source (ESI). The anthocyanins and copigments were separated on a Luna C18(2) 3 μm column (150 × 2.0 mm, Phenomenex, Aschaffenburg, Germany) using water/acetonitrile/formic acid 95:3:2 v/v/v (eluent A) and water/acetonitrile/formic acid 48:50:2 v/v/v (eluent B) at a flow rate of 200 μL/min. Gradient elution was performed, starting with 6% eluent B and rising to 35% over 30 min. The level of eluent B was then set to 40% until minute 35 and then 90% until 45 min. This level was maintained for 5 min before being reduced to 30% until 55 min. Finally, the initial conditions (6% eluent B) were restored until 70 min. The ESI source was operated in positive mode (anthocyanins), negative mode (copigments) and alternating mode (+/− 3000 V), using nitrogen as the nebulizer gas (60 psi) and the drying gas (11 L/min, 330 °C). The sample extracts were dissolved in 2 mL eluent A. An aliquot of 5 μL was analyzed by the HPLC/DAD/ESI-MSn method described above using the Bruker Hystar V.3.2, Bruker ESI-Compass 1.3 for HCT/Esquire, and Data Analysis Version 3.0 software packages (Bruker Daltonics, Bremen, Germany).
Hct ultra ion trap mass spectrometer
Manufactured by Bruker
Sourced in Germany, United States
The HCT Ultra Ion Trap mass spectrometer is a laboratory instrument designed for high-performance mass analysis. It utilizes ion trap technology to trap and analyze ions, providing accurate mass measurements and detailed structural information about chemical compounds. The core function of the HCT Ultra is to serve as a versatile and sensitive analytical tool for a wide range of applications in various scientific fields.
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Lab products found in correlation
Agilent 1100 series lc, by Agilent Technologies (3 mentions)
Mascot search engine, by Matrix Science (2 mentions)
Hystar v 3, by Bruker (1 mentions)
Esi compass 1.3 for hct esquire, by Bruker (1 mentions)
Dataanalysis version 3, by Bruker (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Hystar software, by Bruker (1 mentions)
Zorbax sb c18 column, by Agilent Technologies (1 mentions)
Proxeon easy nanolc system, by Thermo Fisher Scientific (1 mentions)
Nupage gradient gel, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
Compass data analysis software, by Bruker (1 mentions)
Uplc system, by Bruker (1 mentions)
Acquity beh c18 column, by Waters Corporation (1 mentions)
Acquity uplc chromatograph, by Waters Corporation (1 mentions)
0.45 μm pvdf filter, by Merck Group (1 mentions)
14 protocols using hct ultra ion trap mass spectrometer
1
HPLC-DAD-ESI-MS analysis of anthocyanins and copigments
2
Comprehensive HPLC-MS Analysis of Phytochemicals
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The phenolic compounds and triterpenoids were identified by HPLC coupled with electrospray ionization-mass spectrometry (HPLC/ESI-MS) using a Waters ACQUITY UPLC system (Waters, Milford, MA, USA) coupled to an UV/Vis diode-array detector and an HCT ultra ion trap mass spectrometer equipped with an electrospray ionization source (Bruker Daltonics, Bremen, Germany). The HPLC conditions were the same as those used for the quantitative analyses (see below). Mass detection was conducted in negative electrospray ionization mode using m/z values from 100 to 1000. The MS conditions were as follows: capillary voltage 2 kV, nitrogen flow rate 10 litres min−1, desolvation temperature 365 °C, and nebulization pressure 50 psi.
The compounds were characterized according to their UV and mass spectra and their retention times. Co-chromatography was performed with known standards when available (ursolic and oleanolic acids, Extrasynthèse, Genay, France; corosolic and maslinic acids, Sigma-Aldrich, Saint-Quentin Fallavier, France).
The compounds were characterized according to their UV and mass spectra and their retention times. Co-chromatography was performed with known standards when available (ursolic and oleanolic acids, Extrasynthèse, Genay, France; corosolic and maslinic acids, Sigma-Aldrich, Saint-Quentin Fallavier, France).
3
LC-MS Analysis of Iridoid Glucosides
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LC-MS analysis was performed using an Agilent 1100 Series LC (Agilent Technologies) coupled to a Bruker HCT-Ultra ion trap mass spectrometer (Bruker Daltonics). The mass spectrometer was run in positive electrospray mode and loganin was detected from integration of extracted ion chromatograms. All iridoid glucosides were detected as single-charged sodium adducts [M + Na+]: 7-deoxyloganic acid, m/z 383; loganic acid, m/z 399; secologanin, m/z 411; and loganin, m/z 413. For details on the LC set-up, see Supplementary Table S2 .
4
Proteomic Profiling of SRO Allergens
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Bands corresponding to highly IgE‐reactive proteins of raw and cooked SRO extracts with visceral mass were excised from SDS‐PAGE gels for mass spectrometric analysis (Monash Biomedical Proteomics Facility, Monash University, Australia). Briefly, the protein bands were destained, reduced, and alkylated. The gel pieces were washed and dehydrated, followed by trypsin digestion. The gel pieces were then sonicated and analyzed by LC‐MS/MS on a HCT ULTRA ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled online with an Ultimate 3000 nano HPLC (Dionex Corp., Sunnybrook, CA, USA). Data were exported in Mascot generic file format and searched against the Swiss‐Prot databases using the MASCOT search engine (version 2.1, Matrix Science Inc., London, UK) with all taxonomy selected.
5
Proteomic Analysis of Spinal Cord Injury
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Tryptic digests were analysed by LC-MS/MS using the HCT ULTRA ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled online with nanoflow HPLC (Ultimate 3000, Thermo Scientific, Breman, germany). Samples injected onto a pepmap100, 75 µm id, 100 Å pore size, reversed phase nano column with 95% buffer A (0.1% Formic acid) at a flow rate of 300 nl/minute. The peptides were eluted over a 30-minute gradient to 70% B (80% Acetonitrile 0.1% formic acid). The eluant is nebulised and ionised using the Bruker ESI electrospray source via the nanoflow ESI sprayer with a capillary voltage of 4000 V, dry gas at 200°C and flow rate of 5.0l/minute and nebuliser gas at 6psi. Peptides are selected for MSMS analysis in autoMSn mode with smart parameter settings selected with a target mass of 900 m/z and active exclusion released after 1 minute. Data obtained from LC-MS/MS were searched against a custom database downloaded from the National Center for Biotechnology Information (NCBI) ftp site and Swiss-Prot databases using the MASCOT search engine (version 2.1, Matrix Science Inc., London, UK) with all taxonomy selected.
Identified proteins were categorized by relevance to spinal cord injury, obtained from search of relevant literature published in PubMed (http://www.ncbi.nlm.nih.gov/pubmed ).
Identified proteins were categorized by relevance to spinal cord injury, obtained from search of relevant literature published in PubMed (
6
Direct Infusion Mass Spectrometry Protocol
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Acquisition parameters, unless stated otherwise, were as follows. The direct infusion flow rate, provided by means of a syringe pump, was 5 μL/min with additional solvent provided at a flow rate of 100 μL/min by a HPLC pump placed in-line. A Bruker Daltonics HCTultra Ion-Trap mass spectrometer (Bruker Daltonics, Breme, Germany) was used. The spectra were obtained in the positive and negative mode. An average of two microscans and a rolling average of four spectra were acquired. The nitrogen drying-gas temperature was 350 °C, and its flow rate was 1 L/min. The nitrogen nebulizing-gas flow rate was 12 L/min. The internal capillary voltage was between –100 and −1500 V. The end-plate potential was –500 V. The N2 curtain gas flow rate was varied between 0.5 and 6 L/min. The ionization sources parameters were fixed as follows: ESI ionization voltage (3000 V) was applied via a home-built external power supply to the spray needle, the APCI needle current was 3000 nA, and the SACI surface potential was 47 V used to obtain the in source ionization and the inter capillary voltage gradient was employed to produce the CIMS effect. HyStar software (Bruker Daltonics, Breme, Germany) was used for data acquisition. DataAnalysis (Bruker Daltonics) was used for data treatment.
7
Hydroxynitrile Glucoside Profiling in Plants
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For metabolite profiling of hydroxynitrile glucosides, plant material was extracted by boiling in 85% methanol, essentially as described in Takos et al. (2011 ). Samples of extracts prepared at CIAT were dried down in 96‐wells microtiter plates and shipped to Copenhagen for analysis, where they were redissolved in 85% methanol (v/v) and filtered prior to analysis. Analytical LC–MS was performed using an Agilent 1100 Series LC (Agilent Technologies) coupled to a Bruker HCT‐Ultra ion trap mass spectrometer (Bruker Daltonics). A Zorbax SB‐C18 column (Agilent, 2.1 mm × 50 mm, 1.8 μM) was used with chromatography conditions as described previously (Takos et al., 2010 ). Compounds were localized in extracted ion chromatograms as sodium adduct ions: linamarin (m/z 270) and lotaustralin (m/z 284).
8
Proteomic Identification of Peptides
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Samples for mass spectrometry were separated on 4%–12% gradient NuPAGE gels (Invitrogen, Carlsbad, CA, United States), followed by colloidal Coomassie blue staining. Gel lanes were cut into 14 slices and then digested with trypsin. Peptides were separated using the Proxeon EasyNanoLC system (Thermo Fisher, Waltham, MA, United States) fitted with a trapping column (Hydro-RP C18 (Phenomenex, Torrance, CA, United States), 100 μm × 2.5 cm, 4 μm) and an analytical column (Reprosil C18, 75 μm × 15 cm, 3 μm, 100 Å). The outlet of the analytical column was coupled directly to an HCT Ultra Ion Trap mass spectrometer (Bruker Daltonics, Billerica, MA, United States) using the ESI nanoflow source in positive ion mode. Peptides were identified via Mascot (Matrix Science, London, United Kingdom) using the Swiss-Prot database.
9
Identifying G3BP1 Interactors via IP-MS
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HeLa cells were grown under control conditions or exposed to DEM, and proteins were reversibly cross-linked with DSP. Protein complexes were isolated by immunoprecipitation with antibodies against HspBP1. Proteins were separated in a 7.5% to 15% SDS gel and visualized with GelCode Blue Stain (Pierce), following the company’s protocol. Stained bands were excised from the gel, reduced (10 mM DTT, 10 min), alkylated (55 mM iodoacetamide, 30 min), and digested overnight with trypsin (12 ng/μL) [50 (link)]. The resulting peptide digests were subjected to reverse phase separation, followed by MSMS on a Bruker HCT Ultra ion trap mass spectrometer. Data files were formatted to mgf files with Bruker ‘Compass Data Analysis’ software (standard settings) and searched on the Homo sapiens NCBI database (version NCBInr 2008), using Mascot v. 2.2 (Matrix Sciences). Protein identifications are based on unambiguous peptides with a Mowse score better than 47 (random probability value p < 0.05). Several peptides derived from G3BP1 were identified with this approach. Each peptide passed the confidence test, with a cutoff set at p < 0.05.
10
UHPLC-MS Analysis of Apple Pomace Polyphenols
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UHPLC-DAD-ESI-MS n analysis of polyphenol oxidation products was performed on apple pomace procyanidins fractions using an Acquity Ultra performance LC (UHPLC) apparatus from Waters (Milford, MA, USA), equipped with a photodiode array detector (detection at 280, 320, 350 and 520 nm) coupled with a Bruker Daltonics (Bremen, Germany) HCT ultra ion trap mass spectrometer with an electrospray ionization source. Separations were achieved using a Kinetex ® column (100 x 4.6 mm, 2.6 µm, C18, 100 Å) operated at 30 °C, and using water/formic acid (99:1, v/v) (eluent A) and acetonitrile (eluent B) in the conditions previously described (Section 2.3). A capillary voltage of 2 kV was used in the negative ion mode for polyphenol analysis. Nitrogen was used as drying and nebulizing gas with a flow rate of 12 L/min. The desolvation temperature was set at 365 °C and the nebulization pressure at 0.4 MPa. The ion trap was operated in the Ultrascan mode from m/z 100 to 1000.
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