Top-down proteomics, the direct analysis of intact proteoforms by tandem mass spectrometry is becoming more widely available. The ready availability of high resolution mass spectrometers such as the Orbitrap has widely allowed more laboratories to perform top-down proteomics. The vast majority of proteomic analyses, being performed by bottom-up methods, are unable to directly measure proteoforms due to enzymatic digestion. Although top-down proteomics is currently constrained by technical limitations, these can be partially overcome by integrating top-down and bottom-up data generated in parallel. Here we describe the data processing of top-down and bottom-up datasets from E. coli K12 using the software programs Proteoform Suite and MetaMorpheus (Lloyd Smith group, University of Wisconsin). To expand the repertoire of top-down proteomics data processing programs, this top-down data is also processed by the programs TopPIC Suite (Xiaowen Liu group, University Indiana-Purdue UI) and MASH Explorer (Ying Ge group, University of Wisconsin).
E. coli K12 lysis was performed under denaturing conditions (1% SDS/PBS) then extracted proteins were separated in two dimensions. The first dimension separation was performed by Gel-Eluted Liquid Fractionation Entrapment Electrophoresis (GELFrEE) fractionation, the second dimension was separated by reverse-phase chromatography (RPC). Additionally, extracted proteins were digested with trypsin, desalted with S-trap (Protifi), then peptides fractionated by High pH (RPC). Top-down and Bottom-up workflows were processed in parallel using GELFrEE and S-trap methods, respectively. Top-down and bottom-up datasets were searched by MetaMorpheus then combined using Proteoform Suite. Top-down data was deconvoluted and database searched using TopPIC suite and validated with MASH Explorer.