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

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

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
Absorbance 214 nm 60
0.80
% acetonitrile
0.60 40
0.40
0.20
0.00 20
0.00
10.00
20.00
30.00
40.00
50.00
Time (min)

8. SDS PAGE Lane 1: Marker Lane 2: Worthington histones preparation3
Lane 1: Marker
Lane 2: Worthington histones preparation
Lane 3: purified histone H4
Estimated purity: > 95%

9. ESI MS of Bovine Histone H4 Mass Range: 575-1300 m/z ; MaxEnt: 10-13 Kd; Zoom: 11.3-11.52 Kd
100
m/z
600
700
800
900
1000
1100
1200 %
100 %
mass
11300
11320
11340
11360
11380
11400
11420
11440
11460
11480
11500
11520

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

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,
Bergman et al. (1996) FEBS Lett. 390,
ESRG web site (on-sequencer deblocking cycle)
27 laboratories requested a sample
22 laboratories returned data

12. Deblocking Procedures
Number of Sites
TFA/MeOH
Bergman et al. (1996) FEBS Lett. 390, 9
GAS TFA
On-Sequencer and off-line 10
Liquid TFA
Wellner et al. (1990) Proc. Natl Acad. Sci. USA 87, 2
BrCN/formic acid 1

13. Equipment
Instrument
Number of Sites
AB 49x HT 17
AB 49x cLC 5

14. N-Terminal Sequence Call
Sites %
Correct N-terminus 17 77
Correct internal sequence 2 9
Wrong sequence 1 5
No sequence

15. N-Terminal Sequence Call

16. Internal Sequence Call

17. Protein Identification
Correct Protein
No Identification
No Search Performed
Number of Sites 17 3 2 % 77 14 9

18. Search Engines and Databases
Sites %
BLAST
(for short, nearly exact matches) nr 14 64
BLAST and FASTx
nr/uniprot 3
ProteinProspector
MS-Pattern
NCBInr 2 9
PROTEININFO PROWL
(Rockefeller University) 1 4 NA

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

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

28. Repetititive Yield (%) for Different Types of Instrument

29. Deblocking Procedures
Number of Sites
TFA/MeOH
Bergman et al. (1996) FEBS Lett. 390, 10
GAS TFA
On-Sequencer and off-line 11
Liquid TFA
Wellner et al. (1990) Proc. Natl Acad. Sci. USA 87, 2

30. Initial Yield (%) for Different Deblocking Procedures

31. Repetitive Yield (%) for Different Deblocking Procedures

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

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