Fig. 1: Pupylomes are biased by protein size and abundance.

a In vitro pupylation of four different substrates followed by polyacrylamide gel electrophoresis (PAGE) and Coomassie-staining. All four substrates were identified as pupylation substrates in pupylome studies but demonstrate different reactivity in vitro. Representative gels of three individual experiments are shown. Source Data (uncropped gels) are provided as a Source Data file. b Protein abundance as determined in a previous mass spectrometry study³⁵ is plotted against protein molecular weight for members of the Mtb proteome. Green circles represent proteins that have been identified at least once as pupylated in one of the three mycobacterial pupylomes. Purple circles represent non-pupylated proteins in mycobacteria. c Proteins that were reconstituted and pupylated in vitro are depicted as circles and plotted versus abundance (left) and molecular weight (right). The histogram in the background (beige) depicts the whole proteome with the red line marking the median of non-pupylated substrates. Substrates were categorized into good (+++), intermediate (++) and bad (+) substrates. Good substrates are efficiently pupylated in vitro and/or have been shown to accumulate in PPS knockout studies. For intermediate substrates, slower pupylation is observed in vitro and to a lower extent compared to good substrates. Bad substrates, although identified in pupylome studies, are not significantly pupylated in vitro. A full list of substrates can be found in Supplementary Table 1. The size of the circle represents the number of mycobacterial pupylome studies that identified the protein among the substrates. Lime green circles depict proteins with known physiological effect caused by pupylation, the individual proteins are labeled. Where no effect is known, open circles are shown.