Photo-tunable Protein Nitration by Sensitizer Tris(bipyridine)-Ruthenium(II) Chloride complex_Glu-C digestion Dataset

Published: 5 January 2022| Version 1 | DOI: 10.17632/w57h2hrftt.1
Contributors:
Ezequiel Giménez,
Henning Urlaub,
Lisandro J. Falomir Lockhart

Description

Post-translational modifications of proteins are a diverse source of variability that impacts on their functions, localization, regulation, and lifetime; and oxidative modifications are usually associated to pathological and degenerative conditions. Photosensitizers have been employed to promote oxidative modifications and model molecular aspects of diseases. Therefore, we provide the MS/MS raw data from the application of the novel light-controlled Ru(bpy)3+2/APS/NaNO2 system to nitrate four different model proteins. Each protein dataset contains raw data from treated and untreated samples.

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Protein samples (200 µl, 300 µg/ml) were irradiated in quartz cuvettes with a 450nm beam in the presence of 10 µM Ru(bpy)3Cl2, 300 µM APS and 10 mM NaNO2 for 200 s. Proteins were precipitated with cold ethanol, dried and dissolved in 10 µl of 1 % (m/v) Rapigest (Waters). Samples were reduced with 10 µl of 50 mM DTT, and thiols were alkylated (10 µl IAA, 100 mM). Protein digestion was carried out with Glu-C (1:20 enzyme to substrate mass ratio). For LC-MS/MS analysis, samples were dissolved in 10 µL loading buffer (5 % V/V acetonitrile, 1 % V/V TFA). Chromatographic separation was performed with a Dionex UltiMate 3000 UHPLC system (Thermo Scientific), equipped with an in house-packed C18 column (ReproSil-Pur 120 C18-AQ, 1.9 µm pore size, 75 µm inner diameter, 30 cm length, Dr. Maisch GmbH), coupled online to the mass spectrometer. Peptides were loaded at a flow rate of 10 μL/min in buffer A (0.1 % TFA in H2O, V/V) and subsequently eluted and separated with a gradient of 5 90 % buffer B (95 % acetonitrile, 0.1 % TFA in H2O, V/V) over an elution time of 58 m and a flow rate of 300 nL/min. Electrospray ionization (ESI) followed by tandem MS was performed in a Q−Exactive HF-X Hybrid Quadrupole-Orbitrap system (Thermo Scientific) operating in positive data-dependent mode. MS scans were recorded in the m/z range of 350-1600 and the 30 most intense ions were selected for subsequent fragmentation (TOP30 method). Fragmented ions were generated by higher-energy collisional dissociation, and recorded from m/z=110. Precursors and fragment ions were scanned in the Orbitrap detector.

Institutions

Instituto de Investigaciones Bioquimicas de La Plata, Universidad Nacional de la Plata Facultad de Ciencias Medicas, Max-Planck-Institut fur biophysikalische Chemie

Categories

Mass Spectrometry

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