Each sample was analyzed as previously described.(Wales et al., 2008 (link)) Briefly, the samples were digested online using a Poroszyme immobilized pepsin cartridge (2.1 mm x 30 mm, Applied Biosystems) at 15 °C for 30 s, then injected into a custom Waters nanoACQUITY UPLC HDX Manager™ and analyzed on a XEVO G2 mass spectrometer (Waters Corp., USA). The average amount of back-exchange using this experimental setup was 20–30%, based on analysis of highly deuterated peptide standards. Deuterium levels were not corrected for back-exchange and are therefore reported as relative.(Wales and Engen, 2006 (link)) All experiments were performed in duplicate. The error of measuring the mass of each peptide averaged ± 0.12 Da in this experimental setup (4). The HDX-MS data were processed using PLGS 3.0 and DynamX 3.0 (Waters Corp., USA).
Plgs 3
Manufactured by Waters Corporation
Sourced in United States
The PLGS 3.0 is a high-performance liquid chromatography (HPLC) system designed for separating and analyzing complex chemical mixtures. It is an advanced instrument that utilizes a combination of pressure and flow to efficiently transport and separate the components of a sample. The PLGS 3.0 is a reliable and precise tool for a wide range of analytical applications.
Automatically generated - may contain errors
2 protocols using plgs 3
1
Automated Online Pepsin Digestion and HDX-MS Analysis
2
LRRK2 Protein Deuterium Uptake Analysis
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RCKW proteins were expressed and purified from Sf9 cells [39 (link)]. Measurements were performed using a Waters Synapt G2Si system with nanoACQUITY UPLC and H/DX technology. The RCKW concentration was 5 µM in LRRK2 buffer (pH 7.4) containing 20 mM HEPES/NaOH, 800 mM NaCl, 0.5 mM TCEP, 5% Glycerol, 2.5 mM MgCl2, and 20 µM GDP. Deuterium uptake was measured in the presence and absence of kinase inhibitors MLi-2 (50 µM) or Rebastinib (50 µM). Peptide identification and analysis were performed using PLGS 3.0 and DynamX 3.0 (Waters Corporation) as described in previous publication. The HDX-MS data included at least three technical replicates. Data were corrected for back-exchange using a global back exchange correction factor. Deuterium uptake plots were generated using DECA (github.com/komiveslab/DECA), with data fitted using an exponential curve for visualization.
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