Mass-Spectrometry analysis of ATP13A2 samples

Pool purified ATP13A2 sample after SEC (sample should be in MS buffer after SEC)

Concentrate the protein to ~1.2 mg/mL using an Amicon Ultrafilter (cut-off 100kDa) at 4°C

Prepare standards of spermidine (Sigma-Aldrich) and spermine (Sigma-Aldrich) at a concentration of 10 µM in MS buffer

Flash-freeze samples in liquid nitrogen and store at −80 °C until use

Dilute each sample 1:1 into acetonitrile with 1% formic acid (volume/volume)

Analyze by nanoelectrospray ionization (nanoESI) high-resolution mass spectrometry, using an LTQ-Orbitrap-XL mass spectrometer (Thermo Fisher Scientific)

Performed by QB3/Chemistry Mass Spectrometry Facility (University of California, Berkeley)

Mass spectrometer was equipped with a nanoESI source and operated in the positive ion mode

Mass spectra were acquired at a mass resolution setting of 100,000, as measured at mass-to-charge ratio (m/z) = 400, full width at half-maximum peak height

Mass spectrometry data acquisition and processing were performed using Xcalibur software (version 2.0.7, Thermo)