Cells were harvested as single-cell suspension and prepared for MS analysis using Nano-ProteOmic sample Preparation (nPOP) as described by Leduc et al.50 ,51 . The automated collection of prepared samples had not been developed yet26 (link) and so samples were manually collected using a pipette (using 5μl of mass spectrometry grade Acetonitrile then water respectively) and transferred into a 384-well plate (Thermo AB1384). The samples were then dried down in a SpeedVac vacuum evaporator and resuspended in 1.07μl of 0.1% Formic Acid (buffer A) and tightly sealed using an aluminium foil cover (Thermo Fisher AB0626).
For the bulk experiments, cells were harvested (in MS grade water, at roughly 2000 cell/μl) and frozen at −80C. The samples were prepared using mPoP52 , following guidelines for the digest of carriers as outlined in Petelski et al.30 (link). Post digest, the samples were dried down in a SpeedVac vacuum evaporator and resuspended at a concentration of 1 μg/μl in 0.1% Formic Acid (buffer A) in a glass insert with polyspring within an HPLC vial.
For the bulk experiments, cells were harvested (in MS grade water, at roughly 2000 cell/μl) and frozen at −80C. The samples were prepared using mPoP52 , following guidelines for the digest of carriers as outlined in Petelski et al.30 (link). Post digest, the samples were dried down in a SpeedVac vacuum evaporator and resuspended at a concentration of 1 μg/μl in 0.1% Formic Acid (buffer A) in a glass insert with polyspring within an HPLC vial.
