Figure 3

CCT5 is N-terminally processed by methionine excision and acetylation. (a) Schematic of the top-down MS experiment. The chaperonin is color-coded as though subunit identities are unknown. (b) Monomer region of the native mass spectrum of a hTRiC variant lacking subunits CCT2 and CCT4, containing predominantly CCT5 and trace amounts of other subunits (colored triangles). Each highlighted peak was fragmented. The inset is a magnified view of the most abundant charge state, 15⁺, showing that the resolution is high enough to distinguish sodium adducts. The CCT8 15⁺ peak is just 3 m/z to the left of the peak of interest. (c) Deisotoped peptide spectrum resulting from fragmentation of the 15⁺ peak in b. (d) Numbers of fragment ion matches from all highlighted peaks in b to sequences of all CCT subunits, divided into b- and y-ions. CCT2 and CCT4 are faded because they were not present in the sample, and can therefore serve as negative controls for the expected number of false positives. (e) Primary sequence of human CCT5 without the initiator methionine. Blue marks indicate at least one reported peptide mass (in data from all three highlighted peaks in b) supporting an N- or C-terminal ion cleaved at the given site. The red N at the N-terminus designates acetylation. (f) Measured and inferred N-terminal states of post-translationally processed recombinant hTRiC subunits expressed in insect cells. Red ovals denote acetylation.