Native MS dataset for: "Caldendrin and myosin V regulate synaptic spine apparatus localization via ER stabilization in dendritic spines."

Native mass spectrometry dataset used in: Caldendrin and myosin V regulate synaptic spine apparatus localization via ER stabilization in dendritic spines. Anja Konietzny, Jasper Grendel, Alan Kadek, Michael Bucher, Yuhao Han, Nathalie Hertrich, Dick H. W. Dekkers, Jeroen A. A. Demmers, Kay Grünewald, Charlotte Uetrecht and Marina Mikhaylova. The EMBO Journal (2021) e106523. doi:10.15252/embj.2020106523

Description:
Native mass spectrometry (MS) analysis of the stoichiometry and ion occupancy of recombinant human calmodulin (CaM) and recombinant rat caldendrin (CaD) complex with synthetic mouse myosinV IQ1 (myoIQ) motif in the presence / absence of excess Ca2+ and Mg2+ ions.
Sample processing:
Full-length CaD and CaM as well as the synthetic myoVa peptide were buffer exchanged into 150 mM aqueous ammonium acetate solution (pH 7.4). CaM was twice passed through a Bio-Spin P-6 gel filtration spin column (6 kDa cut-off, Bio-Rad), CaD and the myoVa peptide were buffer exchanged through five cycles of tenfold dilution and re-concentration using centrifugal concentrators Vivaspin 500 (10 kDa cut-off, Sartorius) or Amicon Ultra 0.5mL (3 kDa cut-off, Merck/Millipore), respectively. Desalted proteins were introduced into an Orbitrap Q Exactive UHMR mass spectrometer (Thermo Scientific) via static nanoelectrospray ionization from in-house prepared gold-coated borosilicate glass capillaries Kwik-Fil 1B120F-4 (World Precision Instruments). Proteins were sprayed and analysed at 8.5 µM concentration in ammonium acetate alone or supplemented with 200 µM calcium acetate and 100 µM magnesium acetate (both for trace metal analysis, Sigma-Aldrich). For interaction analysis, CaM and/or caldendrin were mixed with myoVa peptide which had final concentration of 8.5 µM (low concentration) or 34 µM (high concentration). The mass spectrometer was tuned for best signal quality and intensity, keeping ion activation and unfolding minimal. Namely, electrospray voltage was kept at 1.3 kV, source desolvation temperature 250°C, in-source desolvation -50 V, ion transfer profile "high m/z", analyzer profile "low m/z", analyzer target resolution 12500 acquiring in mass range 500 – 9000 m/z. Nitrogen was used as collision gas in HCD cell at relative gas pressure setting 7.0 with gentle collisional activation by 10 V HCD voltage gradient.
Data processing:
Raw spectra were averaged over at least 50 scans for mass deconvolution and peak assignment in UniDec 4.4.1 package (Marty et al., 2015). The averaged spectra were exported for ZENODO deposition using Thermo Scientific FreeStyle 1.5.93.34 as single-scan Thermo .raw files (including instrumental parameters metadata) as well as in plain m/z vs intensity .txt files.