Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding

Abstract

The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.

Fig. 1.

dSLBP-RPD and -RBD are each a single major molecular species. The M_r determination of truncated his-tagged dSLBP proteins expressed in insect cells by FTICR-MS of (A) dSLBP-RBD and (B) dSLBP-RPD. The minor peak at m/z 922.2 has one fewer phosphoryl group than the major peak at m/z 927.5.

Fig. 2.

Top-down analysis of dSLBP-RPD by FTICR-MS. (A) De novo sequencing of the 15+ charge state (m/z 927.5) of phosphorylated dSLBP-RPD, by CID outside the FTICR cell. The fragment ions did not match the predicted sequence, but de novo sequencing showed that they corresponded to a modified N terminus. (B) FTICR-MS³ analysis of dSLBP-RPD by skimmer-induced fragmentation and CID. The first MS stage was performed by skimmer-induced fragmentation, and ions with the mass corresponding to the doubly charged b₁₃ ion (see Table 2) were selected for CID. biotools software assignments show that the N-terminal methionine has been removed (MSYYHHHHHHDYDI…), and the new N terminus is acetylated (Ac-SYYHHHHHHDYDI…). (C) ECD analysis of dSLBP-RPD. The phosphorylation site T230 was directly confirmed by the observation of the z ion z₄₇. N-terminal (b ions by CID, c ions by ECD), C-terminal (y ions by CID, z ions by ECD), and abundant internal fragment ions are assigned.

Fig. 3.

Bottom-up analysis by MALDI-MS/MS of Drosophila and human SLBP reveals conserved phosphorylation at threonine in the TPNK motif. (A) MALDI-MS/MS spectrum of m/z 1649.8 (amino acids 224–236) from a tryptic digest of dSLBP isolated from Drosophila cultured cells. (B) MALDI-MS/MS spectrum of m/z 1571.8 (amino acids 164–176) from an Arg-C digest of hSLBP expressed in baculovirus (this peptide displayed anomalous cleavage by Arg-C after K176). (C) MALDI-MS/MS spectrum of m/z 1296.6 (amino acids 164–174) from an in-gel tryptic digest of hSLBP purified from HeLa cells.

Fig. 4.

Effect of phosphorylation on the affinity of SLBP for stem–loop RNA. (A) Schematic of proteins used for RNA-binding measurements. (B) An electrophoretic mobility-shift assay of hSLBP and dSLBP proteins with a 26-nt hairpin from mouse histone H4–12 mRNA. Lane 1 is free probe. Lanes 2–5 are reactions with baculovirus-expressed hSLBP-RPD, dephosphorylated hSLBP-RPD, dSLBP, and T230A dSLBP, respectively. (C) Representative plots of fraction of protein bound to RNA as a function of increasing SLBP concentration are shown. The data were fit to a standard two-state binding model, as described in Materials and Methods.