The proteome of Nicotiana benthamiana is shaped by extensive protein processing

Summary Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana , which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in‐gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor‐like kinases. Transient expression of double‐tagged candidate proteins confirmed processing events in vivo . This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana .