1. ABOUT THE DATASET -------------------- Title: Nanopipettes enable native mass spectrometry studies of the intrinsically disordered protein alpha-synuclein in biochemical buffers Creator(s): Emily J. Byrd1•, Emma L. Norgate1•, Joel A. Crossley1, Chalmers C. C. Chau2,3, Bob Schiffrin1, Alexander Kulak4, Sheena E. Radford1, Paolo Actis2,3*, Antonio N. Calabrese1*, Frank Sobott1* Organisation(s): 1Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. 2School of Electronic and Electrical Engineering, University of Leeds, LS2 9JT, UK. 3Bragg Centre for Materials Research, University of Leeds, LS2 9JT, UK. 4School of Chemistry, University of Leeds, LS2 9JT, UK. Rights-holder(s):Unless otherwise stated, Copyright 2026 University of Leeds Publication Year: 2026 Description: This data set includes native mass spectrometry and ion mobility mass spectrometry data collected on a Synapt G1 HDMS instrument (Waters Corporation) at the University of Leeds, processed using MassLynx 4.1. Cite as: Byrd. E.J., Norgate. E.L., Crossley. J.A., Chau. C.C.C., Schiffrin.B., Kulak. A., Radford. S.E., Actis. P., Calabrese. A.N and Sobott. F (2026) Dataset for 'Nanopipettes enable native mass spectrometry studies of the intrinsically disordered protein α-synuclein in biochemical buffers'. University of Leeds [Dataset] https://doi.org/10.5518/1833 Related publication: Contact: 2. TERMS OF USE --------------- [A standard copyright notice and licence statement with URL can be used, e.g. Copyright [publication year] [University of Leeds, name of other rights-holder(s)]. Unless otherwise stated, this dataset is licensed under a Creative Commons Attribution 4.0 International Licence: https://creativecommons.org/licenses/by/4.0/.] 3. PROJECT AND FUNDING INFORMATION ---------------------------------- Title: [Elucidating the Structural Dynamics of Alpha-Synuclein by Structural Mass Spectrometry] Dates: [Oct 2019 - Dec 2023] Funding organisation: BBSRC, Wellcome and the Royal Society, MRC, Royal Society Grant no.: BB/M011151/1, 220628/Z/20/Z, BB/Y00034X/1, BB/X003086/1, MR/W031515/1, RSRP/R1/211057, BB/E012558/1 4. CONTENTS ----------- File listing IM-MS data: RAW files acquired on a Synapt G1 HDMS instrument (Waters Corporation), analysed using MassLynx V4.1. 5. METHODS ---------- Native IM-MS experiments were performed on a Waters Synapt HDMS mass spectrometer with travelling (T-wave) ion mobility and a nano-ESI source. N-terminally acetylated αS, expressed and purified, was analysed at a concentration of 20 µM in 20 mM AmAc, 20 mM Tris-HCl, 1 x PBS (Dulbeccos A; Thermo Fisher Scientific), 10 mM potassium phosphate, 20 mM sodium phosphate, 100 mM AmAc, and 100 mM Tris-HCl each at a pH of 7.2. For salt titrations, NaCl was added at 12.5 mM, 25 mM and 125 mM. Instrument parameters were set at: capillary voltage 1.0 kV, source temperature 30 °C, backing pressure 0.0 – 0.3 Bar, sampling cone 18 V, extraction cone 1.0 V, trap collision energy 5 V, transfer collision energy 2.0 V, trap DC bias 30 V, IM wave velocity 300 m/s, IM wave height 7.0 V. Gas pressures in the instrument were: trap cell 0.0258 mbar, IM cell 0.36 mbar. TWCCSN2 signifies CCS values calculated using traveling wave ion mobility in N2 buffer gas using calibrants acquired in N2 buffer gas. Lysozyme was analysed at 10 µM under identical conditions except for: sampling cone 10 V and trap DC bias 12 V. MassLynx V4.1 (Waters Corporation) was used for data processing. The IM spectra were calibrated according to the Bush database using denatured cytochrome C (charge states 13+ to 19+), myoglobin (charge states 15+ to 24+) and ubiquitin (charge states 7+ to 13+) at 10 µM in 50 % (v/v) acetonitrile, 0.1 % (v/v) formic acid. Calibrated intensity values were normalised using a scaling factor determined by the signal intensity in the mass spectrum as well as the area under the arrival time distribution peak. Hen egg white lysozyme IM spectra were also calibrated55 using native cytochrome C (charge states 6+ and 7+) and myoglobin (charge states 7+ to 9+) measured in 100 mM AmAc. Arrival-time distributions were extracted in MassLynx using fixed m/z windows around the labelled monomeric charge states. Unless otherwise noted, ATDs were not corrected for potential overlap from oligomer ions (e.g., 8+ monomer overlapping with 16+ dimer). Therefore, reported CCS values represent apparent CCS for the extracted m/z window. Measurements were taken with n = 3 replicates and the standard error was calculated for plotting as shaded areas in the graphs. The apparent radius of gyration (Rapp) was calculated using the following equation: R_app=√((Average CCS)/π)