RNase-dependent gradient fraction reveals RNA-binding character of proteins. (A) The Salmonella lysate was digested with RNase A/T1, and sedimented in a glycerol gradient that yielded RNA-dependent shifts for RNA-binding proteins (RBPs). The profile shows changes in protein abundance, color-coded from black (shifting from) to red (shifting toward). (B) Sedimentation profile of the global RNA-binding proteins CsrA, Hfq, and ProQ were detected by FLAG-tagged variants and western blotting. After RNase treatment, RBPs shifted to top fractions. The Hfq monomer accumulated after RNase digestion in top fractions. GroEL sedimentation was not affected by RNase treatment. n = 2. (C) One-third of the Salmonella proteome was recovered by MS. RNA-binding proteins (red) were enriched in the group of proteins with a high relative shift to the top (<−2 fractions). n = 2. (D) The global RBPs correlated well in MS quantification with B. Protein abundance was normalized to total protein levels per gradient. Reference and RNase treated sedimentation profiles were represented as black and red bars, respectively. n = 2.
