Jul 20, 2022
Mass-Spectrometry analysis of ATP13A2 samples
- 1University of California, Berkeley
Protocol Citation: Sue Sim, eunyong_park 2022. Mass-Spectrometry analysis of ATP13A2 samples. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldzdyxv5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 17, 2022
Last Modified: July 20, 2022
Protocol Integer ID: 66883
Abstract
Preparing mass-spectrometry samples to analyze polyamine content in purified ATP13A2 samples
Materials
MS Buffer
25 mM Tris pH 7.5
100 mM NaCl
1 mM EDTA
1 mM DTT
0.03% DDM/ 0.006% CHS
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)