ABRF2007 Poster - Association of Biomolecular Resource

Report
The ABRF Edman Sequencing Research Group 2007 Study:
Results of Deacetylation Procedures used to Determine the N-Terminal Sequence of a Blocked Protein
B.S.
1
Hampton ,
2
Thoma ,
R.S.
D.
3
Brune ,
N.D.
4
Denslow ,
P.
5
Hunziker ,
K.D.
6
Linse ,
J.
7
Pohl ,
J.W.
8
Leone
1University
of Maryland School of Medicine, Baltimore, MD USA, 2Monsanto Co., St. Louis, MO, USA, 3Arizona State University, Tempe, AZ, USA
4University of Florida, Gainsville, FL, USA, 5University of Zurich, Zurich, Switzerland, 6University of Texas, Austin, TX, USA, 7Emory University, Atlanta, GA, USA, 8Pfizer Inc., St. Louis, MO, USA
Nature of the Test Sample
The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman
degradation for protein and peptide analysis. These studies provide a means for
participating laboratories to compare their analyses against a benchmark of those from
other laboratories that provide this valuable service. The main purpose of the ESRG 2007
study was to analyze techniques for chemically deblocking the N-terminus of an Nterminally acetylated protein thus making it susceptible to Edman degradation. The test
sample that was distributed to participating laboratories was bovine Histone H4, a protein
that is known to have an N-acetylserine at it’s N-terminus. The sample tube contained 450
picomoles; considered to be enough protein for three sequence determinations. The
participating laboratories were asked to deblock the protein, determine the sequence of
the first 20 amino acids including any modified amino acids encountered, and conduct a
database search to identify the protein. To aid participants in this study, references from
the literature, as well as two procedures used by the ESRG with some success were
provided. Participants were not limited to using these procedures and were encouraged to
use any procedures they have developed for the purpose of deblocking an N-acetylated
protein. An important aspect of this study was to compile the treatments and techniques
that were used for deblocking the ESRG 2007 sample to ascertain their effectiveness.
Therefore, participants were asked to provide detailed reporting of their experimental
protocols and techniques to ensure that successful strategies for deblocking can be
reproduced by the Edman sequencing community. Details about instruments and
parameters used in the analysis were also collected.
Amino acid sequence and feature table of bovine histone H4
Taken from http://www.expasy.org/uniprot/h4_bovin/
Position*
1
3
SGRGKGGKGLGKGGAKRHRK
VLRDNIQGITKPAIRRLARR
FLENVIRDAVTYTEHAKRKT
VTAMDVVYALKRQGRTLYGF
GG
*Numbering excludes the initiator methionine.
Tricine-SDS-PAGE
1
2
ESI QTOF MS Analysis
3
Deconvoluted Delta
Mass*
Mass**
75 kD 50 kD 37 kD 25 kD 20 kD -
Materials and Methods
Bovine histone H4 was purified by a combination of cation exchange and reversed phase
chromatography using modifications of Couppez(1) and Sarg(2) in a manner that allowed
the use of those columns and reagents that were on hand in the laboratory. Essentially, a
crude preparation of histones from bovine thymus was purchased from Worthington and
was dissolved in 25mM triethylamine phosphate pH 3, 6M urea, 0.3M guanidine-HCl and
subjected to cation exchange chromatography on a polysulphoethyl-aspartamede column.
Proteins were eluted in this buffer with a gradient of guanidine-HCl from 0.3M to 2.0M.
SDS-PAGE was used to assay these fractions for the presence histone H4 based on the
characteristic migration pattern of histones on a coomassie blue stained gel (3,4) where
histones H3, H2B, H2A, and H4 are the predominant stained bands with histone H4
migrating the fastest. Those fractions containing histone H4 were pooled and purified
further by reversed phase chromatography on a Phenomenex Jupiter C18 column. Final
purity was achieved after rechromatography on a Higgins Analytical TARGA C8 column.
Purity was estimated to be at least 95 percent based on the coomassie blue staining of
approximately 5 ug of the preparation loaded onto an SDS-PAGE gel. The protein
concentration was determined by absorbance at 280 nm after applying the extinction
coefficient that was calculated from the unmodified amino acid sequence
(http://www.expasy.org/tools/protparam.html). Additionally, the protein concentration
was also determined by amino acid compositional analysis following vapor phase acid
hydrolysis for 1.5 hr at 150°C and analysis on a Hitachi L-8800 with post column
ninhydrin derivitization. Identity of this protein as histone H4 was confirmed following
digestion with trypsin and analysis of the resulting peptides by LC MS/MS and searching
the Bos taurus (ENSEMBL) database using the search algorithm !XTandem (5). Aliquots
of 8 µl were dried by rotary evaporation and stored at -80°C until distributed to the
requesting laboratories.
Methods for deblocking histone H4 were based upon published studies of Wellner (6) and
Bergman(7). Two methods were used by the ESRG to validate that this protein could
indeed be deblocked. These methods involved exposure of the protein to vapor, or liquid
phase trifluoroacetic acid either in a test tube, or on the sequencing instrument itself. A
cycle printout of the on instrument gas phase deblocking method was posted on the
ABRF web site (8) Prior to acid treatment the protein was immobilized on either a PVDF
membrane or polybrene treated glass fiber filter according to established procedures.
The histone H4 preparation was characterized further to provide evidence that the Nterminus is blocked. One hundred fifty picomoles of histone H4 was immobilized on a
PVDF membrane and subjected directly to Edman sequencing to show the percent of the
protein preparation that was refractory to Edman chemistry. Additionally, histone H4 was
digested with endoproteinase Asp-N and the peptide corresponding to the N-terminus of
the protein was analyzed by MALDI MS, and finally the intact protein was analyzed by an
ESI QTOF-MS and the mass of the intact protein determined after deconvoluting the
multiply charged envelope of peaks with MaxEnt software.
References
1.
Couppez, M., Martin-Ponthieu, A., Sautière, P. (1987) Histone H4 from Cuttlefish Testis is
Sequentially Acetylated JBC 262, 2854-2860.
2.
Sarg, B., Koutzamani, E., Helliger, W., Rundquist, I. (2002) Postsynthetic Trimethylation of
Histone H4 at Lysine 20 in Mammalian Tissues is Associated with Aging JBC 277, 39195-39201
3.
Ball, D., Slaughter, C., Hensley, P., Garrard, W., (1983) Amino Acid Sequence of the N-terminal
Domain of Calf Thymus histone H2A.Z FEBS Lett. 154, 166-170
Web site for !Xtandem database search: http://www.thegpm.org/
6.
Wellner, D., Panneerselvam, C. and Horecker, B.L. (1990) Sequencing of peptides and proteins
with blocked N-terminal amino acids: N-Acetylserine or N-acetylthreonine Proc. Natl Acad.
Sci.USA 87, 1947-1949.
8.
Bergman, T., Gheorghe, M.T., Hjelmqvist, L. and Jörnvall, H. (1996) Alcoholytic deblocking of
N-terminally acetylated peptides and proteins for sequence analysis FEBS Lett. 390, 199-202.
Web site for on-sequencer deblocking cycle:
http://www.abrf.org/ResearchGroups/EdmanSequencing/Studies/DeblockCycle.pdf/
Possible Modifying Groups
11306.50
70.35
1 Ac + 1 diMe (42+28=70)
11348.00
111.85
2 Ac + 1 diMe (84+28=112)
11364.50
128.35 2 Ac + 1 diMe + 1 OxMet (84+28+16)=128)
11404.50
168.35
4 Ac =168
11446.50
210.35
5 Ac =210
* The mass 11236.15 corresponding to the mass of the unmodified
amino acid sequence was not observed for this sample.
10 kD -
** Delta mass is calculated by subtracting the mass calculated from
the unmodified amino acid sequence of bovine histone H4 from the
observed mass.
Lane 1 - BioRad Precision Plus Standards
Lane 2 - Worthington histones
Lane 3 - purified histone H4.
Ac = Acetyl; diMe = diMethyl; OxMet = oxidized Methionine
Amino Acid Sequence Assignment Accuracy
1
2
3
4
5 6 7 8
9 10 11 12 13 14 15 16
17
18 19
20
Indicate d
Facility No. Se que nce r% Loade d PC TC PW TW NC CIS S G R G K G G K G L G K G G A K
R
H R
K
Ide ntity
100
492
50
13 1
0
1
5
S G R G K G G K - L G K - - A (K)* R
- ( R ) Histone H4
200
494
16
19 0
0
0
1
S G R G K G G K G L G K G G A
K
R
H R
- Histone H4
300
494HT
33
0
0
0
2 13 - Vºº V Y A L K R Q G R T L Y
Histone H4
400
494-HT
33
0
0
0
5 10 T¤ R G V L K - F L E N - - - Histone H4
500
494HT
33
1
3
0
0 11 - (s) (g) R (g) - - - - - - - - - Unknow n
600
492
33
10 1
3
0
1
- (S) G T G K G V K G A G K - G A
Histone H4
700
492
33
0
0
0
0
8
- - - - - - None
800
492
33
5
0
2
0
3
S G R G K K - A None
900
492
33
1
0
9
0
0
T K Y A F K R K L A
None
1000
494cLC
33
19 1
0
0
0
S G R G K G G K G L G K G G A K*
R Hà R (K)à Histone H4, type VIII
1100
492HT
33
18 2
0
0
0
S G R G K G G K G L G K G G A X¡
R
H R (K) Histone H4
1200
492HT
33
18 2
0
0
0
S G R G K G G K G L G K G G A X¡
R
H R (K) Histone H4
1300
492cLC
30
6
2
0
0
2
S G R G - G G - (G) (L)
Histone H4 (bovine)
1400
494
33
14 0
0
0
0
S G R G K G G K G L G K G G
Histone H4-3
1500
494HT
33
11 0
0
0
1
- G R G K G G K G L G K
Histone H4
1600
494HT
33
11 1
0
0
0
- (S) G R G K G G K G L G K
Histone H4
1700
494cLC
33
19 0
1
0
0
S G R G K* G G K* G L G K* G G A K*
R
H R G Histone H4
1800
494cLC
20
15 3
0
1
1
S G R G K G G K G L G K G G A (A)# (R)# (H) (R) - Histone H4-3
1900
494HT
33
19 0
1
0
0
S G R G K G G K G L G K G G A K*
R
H R
R Histone H4
2000
494HT
33
18 1
0
0
1
- (S) G R G K G G K G L G K G G A
K
R
H (R) - Histone H4
2100
492cLC
<60
18 1
0
1
0
- (S) G R G K G G K G L G K G G A
K
R
H R (T) Histone H4 (dog)
Sum
235 18 16 3 41 23
Note: A "-" indicates a cycle that w as run for w hich no call w as made, w hile empty cells indicate that sequencing stopped bef ore these positions.
PC = Positive correct
PW = Positive Wrong
NC = Sequencing cycle f or w hich No Call w as made
TC = Tentative Correct TW = Tentative Wrong
CIS = Correct Internal Sequence
* Acetylated amino acid also indicated to be present
º º Sequence is f rom histone H4 starting at residue 87; sample w as treated w ith CNBr.
¤ 5 additional short sequences (none N-terminal) also reported -sequence is f rom histone H4 starting at residue 55.
à Methylated f orm of identif ied amino acid also suggested to be present
¡ X "probably acetyl-K"
# Unidentif ied peak w ith retention time of PTH-acetly-Lys (bef ore PTH-Ala) also observed
Color code
Positive Correct
Tentative Correct
Positive Wrong
Tentative Wrong
Correct Internal Sequence -not N-terminal sequence
Initial Yield Calculation for Histone H4 Sequence
Cycle
1
Log(10)
S
100
0.556
200
0.875
300
N.R.
400
1.901
500
0.881
600
N.R.
700
N.R.
800
0.645
900
N.R.
1000
2.046
1100
0.752
1200
0.893
1300
-0.222
1400
0.262
1500
N.R.
1600
0.622
1700
1.890
1800
0.602
1900
-0.277
2000
-0.572
2100
0.859
2200
N.R.
2300
N.R.
ESRG1
ESRG2 1*
0.288
ESRG2 4*
0.641
ESRG2 9*
0.520
ESRG2 H** 0.794
ESRG3
0.881
ESRG4
0.978
ESRG5
0.184
2
G
0.699
0.926
N.R.
2.195
0.888
N.R.
N.R.
0.695
N.R.
2.280
0.968
0.956
0.470
0.683
0.996
1.119
1.709
1.301
0.134
0.363
0.913
N.R.
N.R.
1.130
0.658
1.614
1.253
1.378
1.442
1.307
0.594
3
R
0.778
0.959
0.857
2.116
0.484
N.R.
N.R.
0.435
N.R.
1.920
1.090
0.897
0.415
-0.018
1.041
0.958
1.342
1.301
-0.513
0.154
1.126
N.R.
N.R.
1.276
0.458
1.546
1.180
1.277
1.190
1.267
0.301
4
G
0.653
0.834
0.903
1.974
0.876
N.R.
N.R.
0.710
N.R.
2.184
1.085
0.993
0.462
0.675
1.114
1.276
1.223
1.342
0.206
0.583
0.973
N.R.
N.R.
1.121
0.659
1.651
1.281
1.403
1.594
1.415
0.645
5
K
0.875
0.885
0.806
1.834
N.R.
N.R.
N.R.
0.364
N.R.
2.614
1.020
1.009
0.260
1.104
1.046
1.526
1.362
-0.187
0.333
1.043
N.R.
N.R.
0.875
0.286
1.601
1.093
1.258
1.423
1.332
0.454
6
G
0.708
0.744
0.845
1.498
N.R.
N.R.
N.R.
N.R.
N.R.
1.987
1.083
0.988
0.420
0.555
1.149
1.292
1.037
1.204
0.235
0.527
0.806
N.R.
N.R.
1.137
0.700
1.634
1.242
1.391
1.519
1.420
0.640
7
G
0.623
0.889
0.756
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
2.055
1.060
0.787
0.476
0.694
1.143
1.253
1.182
1.279
0.243
0.530
0.867
N.R.
N.R.
1.137
0.790
1.663
1.297
1.425
1.509
1.412
0.698
8
K
0.903
0.730
0.643
1.343
N.R.
N.R.
1.129
2.292
1.074
1.053
0.299
1.182
1.151
1.387
1.301
-0.034
0.384
1.015
N.R.
N.R.
0.886
0.367
1.613
1.034
1.258
1.407
1.350
0.509
N.R. indicates no data was reported for any amino acid
- (dash) indicates data was reported for an incorrectly assigned PTH-amino acid
9
G
N.R.
0.646
0.663
1.614
N.R.
N.R.
N.R.
N.R.
1.837
1.133
0.977
0.423
0.742
1.158
1.324
0.857
1.176
0.283
0.519
0.908
N.R.
N.R.
1.093
10
L
0.477
0.695
0.477
1.161
N.R.
N.R.
N.R.
N.R.
1.807
1.084
1.010
0.276
0.396
1.017
1.119
1.108
1.204
-0.157
0.205
0.890
N.R.
N.R.
0.826
11
G
0.000
0.606
1.164
0.458
N.R.
N.R.
N.R.
1.730
1.054
0.973
N.R.
0.605
1.185
1.331
0.973
1.114
0.302
0.511
0.894
N.R.
N.R.
1.093
12
K
0.398
0.692
0.820
N.R.
N.R.
N.R.
N.R.
2.175
1.131
1.041
0.301
1.233
1.198
1.203
1.204
-0.007
0.490
0.978
N.R.
N.R.
0.748
1.619
1.222
1.393
1.486
1.461
0.700
1.585
1.134
1.264
1.391
1.396
0.380
1.605
1.052
1.365
1.484
1.428
1.560
0.923
1.226
1.511
1.396
0.497
Amt. of
Sample
0.500
0.160
0.330
0.330
0.330
0.330
0.330
0.330
0.330
0.330
0.330
0.330
0.300
0.330
0.330
0.330
0.330
0.200
0.330
0.330
0.600
0.330
0.330
1.000
0.330
0.330
0.330
1.000
1.000
Log(10)
Init. Yield
y-int (pmol)Cycle 0
0.873
14.92
1.004
63.09
0.831
20.52
2.387
738.22
0.767
17.70
0.802
19.20
2.313
1.015
0.921
0.503
0.443
1.018
1.071
1.572
1.376
-0.090
0.344
1.005
623.14
31.36
25.29
10.62
8.41
31.56
35.71
113.03
118.79
2.46
6.70
16.84
1.242
0.589
1.622
1.318
1.375
1.392
1.303
0.536
52.89
11.77
41.88
62.98
71.81
74.75
20.09
3.43
*Indicates one, four or nine cycles of gas phase deblocking
**Four cycles of gas phase deblockiing with TFA:HFIP (80:20)
2
1
4
9
4
1 .8
1 .6
M ethod
Category
Number of
Experiments
TFA/MeOH
+(microwave)
Gas TFA
(ABRF)
Liquid TFA
BrCN Formic
acid
11
2.1, 2.4, 7.3, 13.8, 16.4 , 20.09, 23.3, 24.1, 58.6, 150, 492
18
(0), (0), (0), 6.7, 10.6, 11.8, 15.2, 25.0, 25.5, 31.6, 41.9, 52.9, 62.98, 71.8,
74.8, 85.4, 116.0, 738.2
(0), 110.0, 118.8
20.5 (Did not produce N-terminal sequence)
3
1
Initial Yields (pmoles)
M ean Initial
Yield
(pmoles)*
28.9
41.5
114.5
-
* Mean values were calculated after omitting zero values and those values above the theoretical maximum.
Results
The primary goal of this study was to assess the ability of the Edman sequencing community to
deblock the N-terminus of histone H4 (a protein known to have N-acetylserine at its N-terminus),
sequence the first 20 residues and identify any modified amino acids that may be present. A source
of histone H4 was not readily available so the ESRG purified bovine histone H4 from a crude
preparation to high purity as judged by SDS-PAGE. It was demonstrated that the N-terminus was
blocked following Edman sequencing of the untreated sample where no amino acid sequence was
observed (data not shown). Analysis of the untreated protein by ESI QTOFMS and deconvolution
of the multiply charged envelope of peaks showed masses of (11306.50, 11348.00, 11364.50,
11404.50 and 11446.50) indicating that the histone H4 present in the sample contained a number of
posttranslational modifications consistent with a combination of acetylation, methylation, and
oxidation of amino acid side chains. The mass 11236.15 corresponding to the calculated mass of
the unmodified protein was absent from these spectra. Since the untreated protein was refractory to
Edman degradation it was assumed that the N-terminus was also modified.
All but one participant used TFA to deblock the N-terminus. Nine used a mixture of TFA and
methanol, nine used gas phase delivery on the sequencing instrument, and three used TFA. Fifteen
of the twenty three participants who returned data were able to deblock histone H4 and determine
enough of the N-terminal amino acid sequence to identify the protein by a database search. Serine
was positively identified by eleven, and tentatively identified by five participants in cycle one. Of the
ten participants who provided amino acid sequence beyond sixteen cycles, one participant observed
acetylated lysine in position 16 and methylated lysine in position 20, nine were able to identify an
acetylated lysine in position 16, and one participant observed acetylated lysine in positions 5, 8, 12,
and 16.
The addition of methanol to the TFA used to deblock the protein improved the ability to positively
identify serine in cycle one and to identify acetylated lysine in cycle sixteen. Of those participants
who used either gas or liquid phase TFA without methanol, three positively identified serine and two
identified acetylated lysine in cycles one and sixteen respectively, whereas seven participants
positively identified serine and five identified acetylated lysine in cycles one and sixteen respectively
when methanol was included in the TFA. The ESRG tested four cycles of deblocking with gas
phase delivery of TFA containing twenty percent hexafluoroisopropanol and demonstrated some
improvement in overall yield and ease of identifying N-terminal serine and the modified lysines in
the sequence.
Analysis of internal cleavages of histone H4 that were generated during the acid deblocking
procedure was done by collating the amino acids reported to be observed in each cycle and matching
them with the sequences that would be generated by Ser-N, Thr-N or Asp-C cleavage of the protein.
This analysis revealed four “hot spots” of internal cleavage at threonine in positions 30, 54, 80, and
96. Threonine in these positions is flanked on either side by a charged amino acid. Data from only
one participant indicated cleavage at the single internal serine, and evidence for cleavage at threonine
in position 71 was not apparent in any of the participants data. Both of these sites are flanked on
both sides by non-polar amino acids.
Conclusions
Comparison of Gas Phase Deblocking
Sarg, B., Helliger, W., Talasz, H., Koutzamani, E., Lindner, H. (2004) Histine H4
Hyperacetylation Precludes Histone H4 Lysine 20 Trimethylation JBC 279, 53458-53464
5.
7.
15 kD -
Deblocking
Deblocking
Deblocking
Deblocking
Cycle
Cycles
Cycles
Cycles w /HFIP
1 .4
Log10 pmole
4.
Modification
N-acetylserine
Symmetric dimethylarginine
(alternate by similarity)
N6-acetyllysine (By similarity)
N6-acetyllysine (By similarity)
N6-acetyllysine (By similarity
N6-acetyllysine
N6,N6-dimethyllysine(alternate)
N6-methyllysine (alternate)
Phosphoserine (By similarity
5
8
12
16
20
20
47
GGVKRISGLIYEETRGVLKV
Summary of Deprotection Methods and Initial Yields
Results of Deblocking Methods
Introduction
1 .2
1
A majority of participating laboratories were able to deblock the protein and obtain enough amino
acid sequence information to identify it as histone H4. Identification of the modified amino acids in
the sequence was less successful as less than half of the participants were able to identify acetyllysine-16, the major site of lysine acetylation observed in this preparation.
Both liquid and gas phases of TFA were used successfully to deblock histone H4. In accordance
with a previous study by Bergman et. al. (7), adding methanol or in one case hexaflurorisopropanol,
to the TFA improved the deblocking results based on the increased number of participants that
identified serine in cycle one and acetyllysine in cycle sixteen.
ESI MS analysis of the intact protein by the ESRG revealed five distinct mass-resolved forms.
Based on these data and the following observations that a mass corresponding to unmodified histone
H4 was not observed, the untreated protein was refractory to Edman degradation and susceptibility
to Edman degradation occurred only following an acid treatment procedure, it was deduced that the
blocking group could be an acetyl moiety. Therefore, removal of the blocking group during the acid
treatment procedures could have occurred by the acid-catalyzed N  O shift reaction mechanism.
However, the precise nature of the blocking group itself was not determined in this study.
The deblocking efficiency of histone H4 was less than 10 percent for most participants and the
ESRG combined, but the initial yields varied significantly between participant's laboratories. Multiple
experiments performed by the ESRG showed similar initial yields compared to those obtained by
many of the participants, however, the initial yields in the ESRG experiments were fairly consistent
with each other even though slight variations in the procedure were employed.
In addition to cleavage of the N-terminal blocking group at serine-1, internal peptide bond cleavage
was observed primarily at the N-terminal side of threonine, and this cleavage appeared to occur
more frequently when threonine was flanked by a charged amino acid. These specific internal
peptide bond cleavages may be occurring by the same N  O shift reaction mechanism that also
deblocks the protein but seems to occur at a reduced rate allowing one to discern the N-terminal
sequence.
0 .8
Acknowledgments
0 .6
Thanks to all the participating laboratories for taking the time to analyze the test sample and sending
in their results. Without their participation, this effort would not have been successful.
Thanks to the American Peptide Society (www.americanpeptidesociety.org) and the Protein Society
(www.proteinsociety.org) for announcing the ESRG2007 Study to their members. Thanks also to
Renee Crawford and Olga Stuchlik for removing identifiers from the responding laboratories.
0 .4
0 .2
0
G
R
G
K
G
G
K
G
L
G
K

similar documents