Absolute quantification of peptides from TIM23 or PINK1-3HA using AQUA peptides

To quantify TIM23 and PINK1-3HA in the immunoprecipitates, control or PINK1-3HA expressing HeLa cells were treated with CCCP and subjected to immunoprecipitation with anti-HA antibody bound to magnetic beads. After spiked with 200 fmol stable isotope-labeled AQUA peptides (Cosmo Bio Co Ltd) as standards, proteins on the beads were digested with 400 ng
trypsin/LysC mixture at 37ºC overnight. The digests were reduced, alkylated, acidified, desalted, evaporated, and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile. The absolute amounts of tryptic peptides derived from TIM23 and PINK1-3HA were measured by PRM. Targeted MS/MS scans were acquired by a time-scheduled inclusion list at a resolution of 70,000, an AGC target of 2e5, an isolation window of 2.0 m/z, a maximum injection time of 300 ms, and a normalized
collision energy of 27. Time alignment and quantification of the transitions of three biological replicates were performed using Skyline software.