Samples were measured using a combination of UltiMate 3000 binary RS nano-liquid chromatography system (Thermo Scientific) with an EASY-Spray ion source connected to an on-line Q Exactive HF mass spectrometer. All plasma samples were stored lyophilized and resuspended by the autosampler. A total of 1 µg peptide weight was loaded onto an Acclaim PepMap 100 trap column (75 µm × 2 cm, C18, 3 µm, 100 Å, Thermo Scientific), washed 5 min at 5 µl/min with 100% of solvent A (3% ACN, 97% H2O, 0.1% FA) and then separated by a PepMap RSLC C18 column (75 µm x 25 cm, 2 µm, 100 Å, Thermo Scientific). The peptides were eluted with a linear 33 min gradient of 1-30% solvent B (95% ACN, 5% H20, 0.1% FA) at a flow rate of 0.400 µl/min followed by a 2 min increase to 43% and then 1 min increase to 99% of solvent B. Column was washed for 8 min with 99% of solvent B at a flow rate of 0.750 µl/min and then equilibrated for approximately 10 min with 3% of solvent B at a flow rate of 0.400 µl/min. The analytical column was kept at 35 °C by the in-source temperature controller.
Ultimate 3000 binary rs
Manufactured by Thermo Fisher Scientific
The UltiMate 3000 binary RS is a high-performance liquid chromatography (HPLC) system designed for advanced analytical applications. It features a binary-gradient pump capable of delivering precise and reproducible mobile phase compositions, supporting a wide range of flow rates and pressures. The system is designed to provide reliable and consistent performance, making it suitable for a variety of analytical tasks.
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2 protocols using ultimate 3000 binary rs
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Plasma Peptide Separation Workflow
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Nano-LC-MS proteomics workflow
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Samples were measured using the combination of UltiMate 3000 binary RS nano-liquid chromatography system (Thermo Scientific) with an EASY-Spray ion source connected to an on-line Q Exactive HF mass spectrometer. All plasma samples were stored lyophilized and resuspended by the autosampler. A total of 1 µg peptide weight was loaded onto an Acclaim PepMap 100 trap column (75 µm × 2 cm, C18, 3 µm, 100 Å, Thermo Scientific), washed 5 min at 5 µl/min with 100% of solvent A (3% ACN, 97% H2O, 0.1% FA) and then separated by a PepMap RSLC C18 column (75 µm x 25 cm, 2 µm, 100 Å, Thermo Scientific). The peptides were eluted with a linear 120 min gradient of 3-35% solvent B (95% ACN, 5% H20, 0.1% FA) at a flow rate of 0.300 µl/min followed by a 7 min increase to 99% of solvent B. The column was washed for 6 min with 99% of solvent B at a flow rate of 0.300 µl/min followed by a flow increase to 0.500 µl/min for 4 min and then equilibrated for approximately 10 min with 3% of solvent B at a flow rate of 0.300 µl/min. The analytical column was kept at 35 °C by the in-source temperature controller.
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