Updated publication reference for PubMed record(s): 30336045. Persisters, a dormant and multi-drug tolerant subpopulation that are able to resuscitate after antibiotic treatment, have recently received considerable attentions as the major risk of the relapse of various infectious diseases in clinics. However, due to their low abundance and inherent mutability, it is extremely difficult to study them by proteomics. Here, we developed a magnetic beads-based separation approach to enrich Escherichia coli persisters and then subject them to Filter-Aided Sample Perparation (FASP) followed by LC-MS/MS analysis. We applied spectral counting-based quantitative proteomics to study the proteomic changes of E. coli persisters under high concentration of ampicillin treatment.