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

doi:10.1073/pnas.0511289103

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

*Department of Biochemistry and Biophysics and
^‡Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599; and
^§Bruker Daltonics, Billerica, MA 01821

Communicated by Richard V. Wolfenden, University of North Carolina, Chapel Hill, NC, December 29, 2005 (received for review July 22, 2005)

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.

Footnotes

^†To whom correspondence may be addressed. E-mail: christoph_borchers{at}med.unc.edu or marzluff{at}med.unc.edu
Author contributions: C.H.B. and W.F.M. designed research; R.T., M.P.T., J.P.S., Z.D., and M.E. performed research; J.P.S. and M.E. contributed new reagents/analytic tools; C.H.B., E.V.P., J.P.S., and M.E. analyzed data; and C.H.B., R.T., and W.F.M. wrote the paper.
Conflict of interest statement: No conflicts declared.
Abbreviations:

CID,

collision-induced dissociation;

ECD,

electron capture dissociation;

FTICR,

Fourier transform ion cyclotron resonance;

MS/MS,

tandem MS;

PTM,

posttranslational modification;

SLBP,

stem–loop-binding protein;

dSLBP,

Drosophila SLBP;

hSLBP,

human SLBP;

RBD,

RNA-binding domain;

RPD,

RNA processing domain.