H‐D exchange experiments were conducted with a Waters Synapt G2S system. Total volume of 5 μl samples containing 10 μM protein in gel filtration buffer were mixed with 55 μl of the same buffer made with D2O for several deuteration times (0 s, 1 min, 2 min, 5 min, 10 min) at 15°C. The exchange was quenched for 2 min at 1°C with an equal volume of quench buffer (3 M guanidine HCl, 0.1% formic acid). Proteins were cleaved with pepsin and separated by reverse‐phase chromatography, then directed into a Waters SYNAPT G2s quadrupole time‐of‐flight mass spectrometer. Peptides were identified using PLGS version 2.5 (Waters, Inc.), deuterium uptake was calculated using DynamX version 2.0 (Waters Corp.), and uptake was corrected for back‐exchange using DECA.53 Uptake plots were generated in Prism version 8.
Plgs version 2
Manufactured by Waters Corporation
PLGS version 2.5 is a liquid chromatography-mass spectrometry (LC-MS) data processing and analysis software. It is designed to provide automated and robust processing of proteomic data obtained from LC-MS/MS experiments.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using plgs version 2
1
Protein Deuterium Exchange Mass Spectrometry
2
Hydrogen-Deuterium Exchange Mass Spectrometry
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H-D exchange experiments were conducted with a Waters Synapt G2S system. 5 μL samples containing 10 μM protein in gel filtration buffer were mixed with 55 μL of the same buffer made with D2O for several deuteration times (0 sec, 1 min, 2 min, 5 min, 10 min) at 15°C. The exchange was quenched for 2 min at 1°C with an equal volume of quench buffer (3M guanidine HCl, 0.1% formic acid). Proteins were cleaved with pepsin and separated by reverse-phase chromatography, then directed into a Waters SYNAPT G2s quadrupole time-of-flight (qTOF) mass spectrometer. Peptides were identified using PLGS version 2.5 (Waters, Inc.), deuterium uptake was calculated using DynamX version 2.0 (Waters Corp.), and uptake was corrected for back-exchange using DECA51 (link). Uptake plots were generated in Prism version 8.
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