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

Christoph H. Borchers, Roopa Thapar, Evgeniy V. Petrotchenko, Matthew P. Torres, J. Paul Speir, Michael Easterling, Zbigniew Dominski, William F. Marzluff

Research output: Contribution to journalArticlepeer-review

40 Citations (Scopus)

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 Mr 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.

Original languageEnglish
Pages (from-to)3094-3099
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number9
DOIs
Publication statusPublished - 28 Feb 2006
Externally publishedYes

Keywords

  • Fourier transform ion cyclotron resonance mass spectrometry
  • Histone mRNA

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