Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 103, Issue 9, Pages 3094-3099Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0511289103
Keywords
Fourier transform ion cyclotron resonance mass spectrometry; histone mRNA
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Funding
- NCI NIH HHS [5P30CA016086, P30 CA016086] Funding Source: Medline
- NCRR NIH HHS [S10 RR019889, 1S10RR19889] Funding Source: Medline
- NIEHS NIH HHS [2P30ES010126, P30 ES010126] Funding Source: Medline
- NIGMS NIH HHS [GM58921, GM29832, R01 GM076660, R01 GM029832, R01 GM058921] Funding Source: Medline
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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 baculovirusexpressed 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.
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