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

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

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

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

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/

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

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)

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

ESI MS of Bovine Histone H4 Mass Range: 575-1300 m/z ; MaxEnt: 10-13 Kd; Zoom: 11.3-11.52 Kd 11348.00 100 m/z 600 700 800 900 1000 1100 1200 % 100 11306.50 11364.50 11404.50 11322.00 % 11446.50 11420.00 11334.00 11389.50 11462.00 11377.50 11432.50 11475.50 11502.00 11490.00 11518.00 mass 11300 11320 11340 11360 11380 11400 11420 11440 11460 11480 11500 11520

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 2000.17 2472.55 1922.10 2430.65 1938.18 S1 – R23 (2 acetyl + 1 dimethyl D85 – G102 D68 – M84 + M(O)84 Expected Masses 1921.98 2000.07 2360.43 5004.92 S1 – R23 (1 acetyl + 1 dimethyl)

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

Deblocking Procedures Number of Sites TFA/MeOH Bergman et al. (1996) FEBS Lett. 390, 199-202 9 GAS TFA On-Sequencer and off-line 10 Liquid TFA Wellner et al. (1990) Proc. Natl Acad. Sci. USA 87, 1947-1949 2 BrCN/formic acid 1

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

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

N-Terminal Sequence Call

Internal Sequence Call

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

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

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

Identification of Modifications

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/

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

Yield (log pmol vs. cycle)

Initial Yield (%)

Repetitive Yield (%)

Initial and Repetitive Yield Instrument type? Deblocking procedure?

Initial Yield (%) for Different Types of Instrument

Repetititive Yield (%) for Different Types of Instrument

Deblocking Procedures Number of Sites TFA/MeOH Bergman et al. (1996) FEBS Lett. 390, 199-202 10 GAS TFA On-Sequencer and off-line 11 Liquid TFA Wellner et al. (1990) Proc. Natl Acad. Sci. USA 87, 1947-1949 2

Initial Yield (%) for Different Deblocking Procedures

Repetitive Yield (%) for Different Deblocking Procedures

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

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)

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