Project PXD011177

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Native mass spectrometry of calmodulin binding to the plasma-membrane Ca2+-ATPase ACA8

Description

Binding of two calmodoulin proteins to the integral membrane protein ACA8, a plasma-membrane Ca2+-ATPase von A. thaliana, was shown by native mass spectrometry.

Sample Processing Protocol

ACA8 was overexpressed with an N-terminal 8xHis tag in S. cerevisiae strain BJ5460 from a pYES2 plasmid. Cells were lysed and proteins were purified via Ni-NTA affinity chromatography and changed to 200 mM ammonium acetate solution pH 8.3 with 2x CMC DDM using centrifugal filter units (Vivaspin 500, 100.000 MWCO). Calmodulin (CaM7) from A. thaliana was produced from pET42a vector in E. coli BL21 Gold (DE3)and purified using a HiTrap Phenyl HP column. Purified ACA8 and CaM7 were mixed and analyzed on a high-mass QToF2 (Waters, MS Vision).

Data Processing Protocol

Acquired data were calibtrated with a CsI spectrum and analyzed using MassLynx (Waters) and Massign (Morgner & Robinson, 2012) software. Average masses and standard deviations were determined from at least three measurements and are listed together with the average full-width half-maximum (FWHM) value of the main peak as a measure of the spectral resolution.

Contact

Johannes Heidemann, Heinrich Pette Institute, Leibniz Institute for Experimental Virology
Charlotte Uetrecht, Heinrich Pette Institute, Leibniz Institute for Experimental Virology; European XFEL GmbH ( lab head )

Submission Date

24/09/2018

Publication Date

05/12/2018

Tissue

Not available

Instrument

Q-Tof ultima

Software

Not available

Quantification

Not available

Experiment Type

Top-down proteomics

Publication

    Nitsche J, Josts I, Heidemann J, Mertens HD, Maric S, Moulin M, Haertlein M, Busch S, Forsyth VT, Svergun DI, Uetrecht C, Tidow H. Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin. Commun Biol. 2018 1:206 PubMed: 30511020