After the NMR analysis, the sample in the NMR tube was returned to the tube containing the fecal pellet and then dried using a Speedvac concentrator at a vacuum pressure rate of 50 torr/min without heating (Savant SPD1010; Thermo Scientific, Waltham, MA, USA). BAs were then extracted from the fecal pellet by ethanol containing an internal standard of 20 nM nor-deoxycholic acid (NDCA; Santa Cruz Biotechnology, Dallas, TX, USA) at 60 °C for 30 min and subsequently at 100 °C for 3 min. Thereafter, the ethanol extract was purified using an Oasis HLB cartridge column and then subjected to LC-MSMS analysis (LCMS-8050, Shimadzu, Kyoto, Japan). The methods in detail are described by Tanaka et al. [47 (link)].
Savant spd1010
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Savant SPD1010 is a centrifugal evaporator designed for efficient sample concentration and drying. It features a compact benchtop design and digital controls for precise temperature and time settings.
Automatically generated - may contain errors
3 protocols using savant spd1010
1
Fecal Bile Acid Extraction and Analysis
2
Proteomics Analysis of Patient-Derived Neurons
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The same line of patient-derived neurons used for high content screening was used for proteomics analysis vis-à-vis a healthy control. At 21 DIV iPSC-derived neurons were suspended, lysed and the proteins reduced in 4% SDS, 100 mM DTT, 100 mM Tris pH 7.8 through heating for 5 min. Next, the proteins were alkylated by 100 mM iodoacetamide treatment for 30 min in the dark. Samples were further processed according to the Single-Pot Solid-Phase enhanced Sample Preparation (SP3) method of Hughes et al.79 (link). Digestion was carried out overnight at 37 °C using Trypsin/LysC mix (Promega) at a protein/enzyme ratio of 50:1 in a ThermoMixer under continuous mixing at 1000 rpm. After digestion, the tubes were placed on a magnetic rack, and the supernatant containing the peptides was collected and dried down in a centrifugal evaporator (Savant SPD 1010, Thermo scientific). The peptide mixtures were reconstituted in a solution of 2% (v/v) ACN/ 0.1% (v/v) formic acid and incubated for 3 min in a sonication water bath. Peptide concentration was determined by nanodrop absorbance measurement at 280 nm.
3
Nano-HPLC-MS/MS Analysis of Atlantic Cod Peptides
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The ethanol-soluble Atlantic cod (Gadus morhua) peptides mixture used for nano-HPLC-MS/MS analysis, which is reported by our previous study,15 and the nano-HPLC-MS/MS analysis methods were described as follows. The peptides in the sample were identified by using the Q Exactive™ coupled to an EASY-nano LC 1200 system (Thermo Fisher Scientific, MA, USA). Samples were first desalted with MonoSpin C18 column by following the manufacturer's protocol. Then the desalted sample was dried by using a centrifugal vacuum concentrator (Thermofisher, SAVANT SPD1010). Subsequently, samples were re-dissolved in 0.1% formic acid in water (buffer A) and loaded onto an analytical column (Acclaim PepMap C18, 25 cm, 75 μm i.d.) for nano-HPLC-MS/MS analysis. The analyzing parameters were set as: a total of 60 min washing gradient, starting at 2% buffer B (80% ACN with 0.1% FA) followed by a stepwise increase to 35% in 47 min, from 35% to 100% within 1 min and stayed for 12 min; 300 nL min−1 of flow rate; 40 °C of column temperature, and 2 kV of the electrospray voltage. Other mass spectrometer parameters were set as: data dependent acquisition (DDA) mode, automatically switched between MS and MS/MS mode, 17 500 of MS/MS resolution, 3 × 106 of automatic gain control target. Finally, the data were analyzed by PEAKS Studio version 10.6 (Bioinformatics Solutions Inc., Waterloo, Canada).
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