Pierce quantitative colorimetric or fluorescent peptide assay

Cell pellets were thawed on ice and subjected to sample preparation with the PreOmics iST kit (PreOmics, Planegg, Germany) according to the manufacturer’s protocol. Samples were resuspended in LC-Load Buffer from the iST kit and peptide concentration determined (Pierce Quantitative Colorimetric or Fluorescent Peptide Assay, ThermoFisher Scientific, Waltham, Massachusetts). Sample concentration was normalized to 100 ng/μl and 2 μl was loaded onto the instrument.

Sorted cells were pelleted and washed twice with ice-cold PBS to remove any remaining FBS (1,300 rpm x 5 min, 4°C). PBS was aspirated away, and the pellets were snap frozen with liquid nitrogen prior to storage at −80°C. Prior to lysis, cells were thawed on ice and subjected to sample preparation with the PreOmics iST NHS kit (PreOmics, Planegg, Germany) according to literature protocol. (To normalize lysis across cell number, 10 μL of lysis buffer was added for every 100,000 cells). The only additional modification made to the protocol was scaling down in volume of digest buffer to align with amount of lysis buffer (for example, 20 μL digest buffer for 20 μL lysis buffer). Samples were resuspended in 12 μL of LC-Load Buffer from the iST NHS kit and peptide concentration determined (Pierce Quantitative Colorimetric or Fluorescent Peptide Assay, ThermoFisher Scientific, Waltham, Massachusetts). Sample concentration was normalized to 100 ng/μL and 2 μL was loaded onto the instrument.