The epididymides were extracted in 50 μL of lysis buffer (8 M urea and 0.2 M ammonium bicarbonate) and sonicated. Each lysate was digested with trypsin and labeled with iTRAQ 4-plex reagent, according to the manufacturer’s instructions. An Orbitrap Q Exactive Plus spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and an EASY-nLC 1200 chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) were used to generate shotgun proteomics data. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis and database searches were conducted using APRO Science (Tokyo, Japan).
Orbitrap q exactive plus spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Orbitrap Q Exactive Plus spectrometer is a high-resolution mass spectrometer that combines a quadrupole mass filter with an Orbitrap mass analyzer. It provides accurate mass measurements and high-resolution capabilities for a wide range of analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Easy nlc 1 200 chromatograph, by Thermo Fisher Scientific (1 mentions)
Xcalibur 4, by Thermo Fisher Scientific (1 mentions)
1290 infinity uhplc, by Agilent Technologies (1 mentions)
Xcalibur 4.1, by Biocrates (1 mentions)
Absolute idq p400 hr kit, by Biocrates (1 mentions)
Ultrashield 400 mhz, by Bruker (1 mentions)
Silica gel 60 f254, by Merck Group (1 mentions)
4 protocols using orbitrap q exactive plus spectrometer
1
Shotgun Proteomics of Epididymis
2
Targeted Metabolic Profiling Using IDQ p400 HR
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The Absolute IDQ p400 HR kit (test plates in the 96-well format; Biocrates Life Sciences AG, Innsbruck, Austria) was used to perform targeted quantitative analysis of the metabolites, according to the manufacturer’s protocol. The kit allows the quantitative measurement of up to 408 compounds (or their isomer groups) covering 11 classes of metabolites (including amino acids, biogenic amines, hexoses, acylcarnitines, di- and triglycerides, (lyso)phosphatidylcholines, sphingolipids, and cholesteryl esters) in 10 µl of sample thanks to the combination of direct flow injection and liquid chromatography (LC) with mass spectrometry (MS). The MS analyses were performed using Orbitrap Q Exactive Plus spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), which was equipped with a 1290 Infinity UHPLC (Agilent, Santa Clara, CA, USA) system; Xcalibur 4.1 software (Thermo Fisher Scientific, Waltham, MA, USA) controlled the whole system. The concentrations of metabolites in µM were established by processing the registered spectra and chromatograms using Xcalibur 4.1. and MetIDQ DB110-2976 software (Biocrates Life Sciences AG) (19 (link)).
3
Analytical Characterization of Organic Compounds
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All analytical-grade reagents and solvents were purchased from Merck KGaA (Darmstadt, Germany) and were used without further purification. 1H and 13C NMR spectra were collected at 25 °C using a Bruker UltrashieldTM 400 MHz (Switzerland). The following abbreviations are used: Singlet (s), doublet (d), triplet (t) doublet of doublets (dd), and multiplet (m). Melting points (m.p.) were measured with a Leica Galen III microscope (Leica/Cambridge Instruments) and were uncorrected. Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates containing a fluorescent indicator (Merck Silica Gel 60 F254); spots were detected under UV light (254 nm). Evaporation was carried out under reduced pressure using a rotating evaporator, and Na2SO4 was always used as the drying agent. Mass spectra (ESI-MS) were recorded with a high-resolution Q Exactive plus Orbitrap spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), with a resolution of 140,000 at m/z 200.
4
High-resolution Metabolite Profiling by Orbitrap MS
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The solvents containing extracted metabolites were transferred into a 96-well plate, then 10 μL were directly infused into a high-resolution Q-Exactive plus Orbitrap spectrometer (Thermo Fisher Scientific, Hemel Hempstead, UK) via chip-based nanoelectrospray ionisation (Advion Biosciences, Ithaca, NY) at 1.5 kV and 0.6 psi gas pressure. Data was acquired for 1 min for each polarity using a scan range of m/z 70–1050. In full MS mode, the resolution was set to 140 000 at m/z 200, and the AGC target was set to 3 × 106 with a maximum ion injection time of 200 ms. The top 10 most intense ions were isolated within a 0.5 m/z window for data-dependent acquisition (DDA) at a resolution of 17 500, AGC target of 1 × 106 and a maximum ion injection time of 50 ms. For data-independent acquisition (DIA), the pre-selected ions were isolated within a 0.4 m/z window at a resolution of 35 000 and AGC target of 2 × 105. Stepped normalized collision energy (NCE) of 20, 30 and 40 was applied in both DDA and DIA. The pooled QC samples were analysed intermittently for the duration of the MS analysis.
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