The ABRF Edman Sequencing Research Group 2007 Study

1. The ABRF Edman Sequencing Research Group 2007 Study Procedures used to Determine the N-Terminal Sequence of a Blocked Protein

2. ESRG Members • Joseph W. Leone (Chair) Pfizer • Daniel Brune Arizona State University • Brian Hampton University of Maryland School of Medicine • Peter Hunziker University of Zurich • Klaus Linse University of Texas at Austin • Jan Pohl Emory University School of Medicine • Richard S. Thoma Monsanto • Nancy D. Denslow (EB liaison) University of Florida - Gainesville

3. Introduction • ~80% of cellular proteins are N-terminally blocked • N-acetylation is very common • 35 % Ac-Ser • 4 % Ac-Thr • 5 % Ac-Gly • 33 % Ac-Ala • 10% Ac-Met • N-terminally blocked proteins are resistant to Edman degradation

4. Histone H4 ESRG 2007 Study • Establish techniques to chemically deblock N-acetylated proteins • Detection of modified amino acids

5. Posttranslational Modifications of Histone H4 (Bovine) S G R G K G G K G L G K G G A K R H R K FT MOD_RES 1 N-acetylserine FT MOD_RES 3 Symmetric dimethylarginine (alternate) (By similarity) FT MOD_RES 5 N6-acetyllysine (By similarity) FT MOD_RES 8 N6-acetyllysine (By similarity) FT MOD_RES 12 N6-acetyllysine (By similarity) FT MOD_RES 16 N6-acetyllysine FT MOD_RES 20 N6,N6-dimethyllysine (alternate) FT MOD_RES 20 N6-methyllysine (alternate) Taken from http://www.expasy.org/uniprot/h4_bovin/

6. Posttranslational Modifications of Histones Peterson CL et al, Current Biology, 2004

7. Sample Preparation • Purification of H4 from a commercially available histone preparation by ion exchange and reversed-phase chromatography 100 1.60 1.40 80 1.20 1.00 60 Absorbance 214 nm 0.80 % acetonitrile 0.60 40 0.40 0.20 20 0.00 0 0.00 10.00 20.00 30.00 40.00 50.00 Time (min)

8. 1 2 3 SDS PAGE Lane 1: Marker Lane 2: Worthington histones preparation Lane 3: purified histone H4 Estimated purity: > 95%

9. 100 % 0 m/z 600 700 800 900 1000 1100 1200 ESI MS of Bovine Histone H4Mass Range: 575-1300 m/z ; MaxEnt: 10-13 Kd; Zoom: 11.3-11.52 Kd 11348.00 100 11306.50 11364.50 11404.50 11322.00 % 11446.50 11420.00 11334.00 11389.50 11462.00 11377.50 11432.50 11502.00 11475.50 11518.00 11490.00 0 mass 11300 11320 11340 11360 11380 11400 11420 11440 11460 11480 11500 11520

10. Expected Masses 1921.98 2000.07 2360.43 5004.92 D85 – G102 2000.17 100 80 60 S1 – R23 (2 acetyl + 1 dimethyl D68 – M84 % Intensity 2472.55 40 1922.10 S1 – R23 (1 acetyl + 1 dimethyl) + M(O)84 2430.65 20 1938.18 1915.0 2034.8 2154.6 2274.4 2394.2 2514.0 m/z Asp-N Digestion

11. Sample distribution • H4 quantification by amino acid analysis • Aliquots of 450 pmol were freeze-dried • Three methods for deblocking • Wellner et al. (1990) PNAS 87, 1947-1949. • Bergman et al. (1996) FEBS Lett. 390, 199-202. • ESRG web site (on-sequencer deblocking cycle) • 27 laboratories requested a sample • 22 laboratories returned data

12. Deblocking Procedures

13. Equipment

14. N-Terminal Sequence Call

15. N-Terminal Sequence Call

16. Internal Sequence Call

17. Protein Identification

18. Search Engines and Databases

19. Species Identification Three sites reported the identification of histone H4 for a particular species  ?

20. Identification of Modifications

21. Identification of Posttranslational Modifications S G R G K G G K G L G K G G A K R H R K FT MOD_RES 1 N-acetylserine FT MOD_RES 3 Symmetric dimethylarginine (alternate) (By similarity) FT MOD_RES 5 N6-acetyllysine (By similarity) FT MOD_RES 8 N6-acetyllysine (By similarity) FT MOD_RES 12 N6-acetyllysine (By similarity) FT MOD_RES 16 N6-acetyllysine FT MOD_RES 20 N6,N6-dimethyllysine (alternate) FT MOD_RES 20 N6-methyllysine (alternate) Taken from http://www.expasy.org/uniprot/h4_bovin/

22. Initial and Repetitive Yield • Average initial yield around 10% • Average repetitive yield around 95%

23. Yield (log pmol vs. cycle)

24. Initial Yield (%)

25. Repetitive Yield (%)

26. Initial and Repetitive Yield • Instrument type? • Deblocking procedure?

27. Initial Yield (%) for Different Types of Instrument (n=4) (n=11)

28. Repetititive Yield (%) for Different Types of Instrument (n=4) (n=11)

29. Deblocking Procedures

30. Initial Yield (%) for Different Deblocking Procedures n=11 n=2 n=10

31. Repetitive Yield (%) for Different Deblocking Procedures n=11 n=2 n=10

32. Mechanisms for deblocking Wellner et al. (1990) Proc. Natl Acad. Sci. USA 87, 1947-1949 Bergman et al. (1996) FEBS Lett. 390, 199-202

33. Conclusions • A majority of participating laboratories obtained sequence information • The majority of the labortatories that reached residue 16 could identify Ac-Lys at this position • One laboratory reported the presence of Ac-Lys at all suggested positions • One laboratory reported the presence of methylated Arg in position 20 • All deblocking procedures used were successfull • None of the partcipants suggested an additional method • Yield of deblocking is highly variable irrespective of method (and instrument)

34. Acknowledgement • All participating laboratories • Renee Crawford and Olga Stuchlik • The American Peptide Society • The Protein Society