Nanoflow system

An Eskigent nanoflow system (Sciex; Framingham, MA) was used for liquid chromatography (LC) and ThermoScientific TSQ Vantage triple quadrupole was used for mass spectrometry (Thermo Scientific; San Jose, CA). A 75 μM x 11 cm column packed with Phenometrix Jupiter C18 was used. 6 μL aliquots of sample were injected for each analysis. Peptides were eluted with a linear gradient of acetonitrile in water with 0.1% formic acid. For data analysis, the open-source program Skyline (developed by Dr. Michael MacCoss, University of Washington) was used. Total protein responses were calculated as the geometric mean of the abundance of peptides measured for each protein, and normalized to bovine serum albumin (BSA) total protein response. The final abundance of each protein is calculated as pmol/100 μg total protein based on the ratio to BSA and the amount of BSA added, and data expressed as the relative amount of protein in experimental groups relative to control.

An Eskigent nanoflow system (Sciex, Framingham, MA, USA) was used for liquid chromatography (LC) and ThermoScientific TSQ Vantage triple quadrupole was used for mass spectrometry (Thermo Scientific, San Jose, CA, USA). A 75 μM × 11 cm column packed with Phenometrix Jupiter C18 was used. Sample aliquots (6 μL) were injected for each analysis. Peptides were eluted with a linear gradient of acetonitrile in water with 0.1% formic acid. For data analysis, the open-source program Skyline (developed by Dr. Michael MacCoss, University of Washington) was used. Total protein responses were calculated as the geometric mean of the abundance of peptides measured for each protein, and normalized to bovine serum albumin (BSA) total protein response. The final abundance of each protein was calculated as pmol/100 μg total protein based on the ratio in relation to BSA and the amount of BSA added, and data were expressed as the amount of protein in experimental groups relative to control.