1. Christoph H. Borchers, Roopa Thapar, Evgeniy V. Petrotchenko, Matthew Michael Easterling, Zbigniew Dominski, and William F. Marzluff Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem–loop-binding proteins that contributes to high-affinity RNA binding Presented by Aliya Sadeque

2. stem–loop-binding protein  Binds metazoan replication-dependent histone mRNA  Not acetylated, stem-loop instead  Involved in cell-cycle regulation of histone mRNA Erkmann, J. A., Wagner, E. J., Dong, J., Zhang, Y., Kutay, U. & Marzluff, W. F. (2005) Mol. Biol. Cell 16, 2960–2971.

3. Previously known  dSLBP is stoichiometrically phosphorylated on multiple sites  Phosphatase treatment resulted in change in mobility on gel electrophoresis

4. Previously known  dSLBP is stoichiometrically phosphorylated on multiple sites  Multiple phosphorylations sites at the C- terminus  Based on M r determinations of intact and truncated proteins

5. Previously known  dSLBP is stoichiometrically phosphorylated on multiple sites  Multiple phosphorylations sites at the C- terminus  4 phosphorylated Ser residues identified in C terminus  Using Phosphatase-directed Phosphorylation-Site Determination

6. Outline of experiments  Where phosphorylation is occurring  To what extent  What are the biological implications?  How does this affect the overall function of SLBP  How does this affect RNA binding specifically?

7. Top or Bottom?  Top-down = intact protein molecular mass measurement  Bottom-up = proteolytic fragment identification Strader, M. B., VerBerkmoes, N. C., Tabb, D. L., Connelly, H. M., Barton, J. W., Bruce, B. D., Pelletier, D. A., Davison, B. H., Hettich, R. L., Larimer, F. W., et al. (2004) J. Proteome Res. 3, 965–978.

8. Torres, M. P., Marzluff, W. F.&Borchers, C. H. (2005) J. Proteome Res. 4, 1628– 1635.

9. Top-down  FTICR analysis of M r of RBD and RPD  Single major molecular species for RBD  9 Da lower than predicted |----------| |---------------|

10. Top-down  FTICR analysis of M r of RBD and RPD  Two ion series for RPD

11. Top-down  Collision-induced fragmentation of 4_P ions followed by M r analysis of fragments

12. Sequence tag carfuffle  N-term sequence tags did not correspond to predicted b-ions..??

13. Top-down De novo sequencing by CID revealed  Modified N-terminus

14. Top-down De novo sequencing by CID revealed  Modified N-terminus  Removal of N-term Met

15. Top-down De novo sequencing by CID revealed  Modified N-terminus  Removal of N-term Met  Additional P on T230

16. Top-down De novo sequencing by CID revealed  Modified N-terminus  Removal of N-term Met  Additional P on T230

17. 9 Da discrepancy…  loss of N-term Met (-131 Da)  Acetylation of new N-term Ser (+42 Da)  Additional Phosphorylation (+80 Da) -------------------------------------------------- = 9Da  Confirmed by analysis of tryptic digests of dSLBP

18. Top-down  Stoichiometric phosphorylation of T230  Baculovirus expressed dSLBP

19. Bottom-up  Stoichiometric phosphorylation of T230  Baculovirus expressed dSLBP  dSLBP-RPD from baculovirus and full- length dSLBP from Drosophila

20. Top-down  Stoichiometric phosphorylation of T230  M r analysis of corresponding hSLBP T171  Same 9 Da discrepancy  Same tag carfuffle  So now we know where they are… http://incolor.inebraska.com/stuart/riders/shocker.htm

21. Big ups

22. Effects on RNA Binding

23. Effect on RNA Binding  Treated with calf intestinal phosphatase  Full-length hSLBP  hSLBP-RBD  Used T230A mutant  Full-length dSLBP  dSLBP-RPD

24. Effect on RNA Binding  Gel mobility shift assay  Shows that they are binding RNA

25. Effect on RNA Binding  Filter-binding assay to measure affinity  Dephosphorylation results in up to 10-fold lower affinity

26. T230  Non-viable null mutant dSLBP is not rescued by T230A  Mutations that affect mRNA-SLBP affinity result in low expression of mRNA  Sea urchin TPNK studies: unable to phosphorylate Thr

27. Binding Mechanism  “One or more dSLBP phosphoryl groups contributes favorably to overall stability of the SLBP-RNA complex.”

28. Sketchiness (a.k.a. critique)  Why use top down at all  Insect cells - are they sure PTMs were done properly?  Filter binding assays assume 1:1 stoichiometry….why? Why even use this method?  Fraction bound curves: fit to standard is a stretch although mutant still worse  Acetylation…isn’t that a big deal?  How do these results reflect structural stability?  Extent of phosphorylation - cell cycle reg’d?