Transcript No Slide Title

ABRF ESRG 2006 Study: Edman Sequencing as a Method for Polypeptide Quantitation
R.S. Thoma1, J.W. Leone2, J. Pohl3, R. Kobayashi4, K.D. Linse5, B. Hampton6, D. Brune7, N.D. Denslow8
1Monsanto
Co., St. Louis, MO, United States, 2Pfizer Inc., St. Louis, MO, United States, 3Emory University, Atlanta, GA, United States, 4UT MD Anderson Cancer Center, Houston, TX, United States, 5University of Texas, Austin, TX, United States,
6University
of Maryland Baltimore, School of Medicine, Baltimore, MD, United States, 7Arizona State University, Tempe, AZ, United States, 8 University of Florida, Gainesville, FL, United States.
300
15 liquid, 3 gas phase
440
Q
Chemistry Cycle
17 users seemed to use the cycles that matched the support (ie. GFF or PVDF)
except 1 PVDF with GFF cycle
DTT in S2
8
yes and 10 no
500
600
120%
Other Additives
1
TCEP to R5
100%
% Loaded
11 used 0.1% TFA/30% acetonitrile
3 used 0.1% TFA/20% acetonitirile
3 used 0.1% TFA/50% acetonitrile
1 used 1.0% FA/74% methanol
Ranged from 10 to 100%
Mean 81.9%
700
Sample Loaded
Sample Solvent
80%
800
60%
40%
880
20%
0%
100 110 200 220 300 330 400 440 500 550 600 660 700 770 800 880 900 990
Participent #
900
12
12
Correct
Incorrect
Unidentified
10
8
6
6
4
4
2
2
0
√
1
2
3
4
5
6
7
8
9
10
0
11
Cycle Number
160
140
25
Initial Yield (pmol)
30
80 pmol
120
100
80
60
40
20
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
20 pmol
20
15
10
5
5
4
3
2
1
990
900
880
800
770
700
660
600
550
500
440
400
100
5
4
3
2
1
990
900
330
0
0
880
√
180
800
√ √ √ √ √ √ √ √ √ √ √ √ √ √
6
B: Peptide B
770
nc √ √ √ √ √ √ √ √ √ √ √ √ √ nc
5
Figure 2. Graphical Representation of Initial Yields
√
(R) √ √ √ √ √ √ √ √ √ √ √ √ √ nc
4
KAQYARSVLLEKDAEPDILELATGYR
A: Peptides C + C*
√ √ √ √ √ √ √ √ √ √ √ √ √ √
K
3
X = AcLys or K
√
√ √ √ √ √ √ √ √ √ √ √ √ √ √ (r)
2
Cycle Number
R Q A X V L L Y S G R
√ √ √ √ √ √ √ √ √ √ √ √ √ √
1
300
√ √ √ √ √ √ √ √ √ √ √ √ √ √
220
√
8
200
√ √ √ √ √ √ √ √ √ √ √ √ √ √
Correct
Incorrect
Unidentified
10
110
√ √ √ √ √ √ √ √ √ √ √ √ fi √ nc
700
√
√
√
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E
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R
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√
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nc
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nc
(r)
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14
660
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nc
G
14
600
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
S V L
√ √ √
L Y S
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ nc
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
nc nc nc
√ √ √
G √ √
√ √ √
√ √ √
√ √ √
Y nc R
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ √ √
√ hyL √
nc √ L
L hyL √
16
550
√
√
√
√
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R
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R
L
16
500
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A
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V
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440
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Y
tmK
K
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K
√
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K
(k)
hyP
(y)
√
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√
H
hyL
(L)
18
Lab ID
Lab ID
√ √ √ √ √ √ (D) √ √ (E) √ (t) nc nc nc
A: Average = 53.6 pmol, 67%
√ √ √ √ √ √ √ √ √ √ √ √ √ √
B: Average = 13.1 pmol, 65.6%
√
Figure 3. Graphical Representation of Yield Ratios
√ √ √ √ √ nc nc nc nc nc nc nc nc nc nc
√ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √
√ √ √ √ √ √ √ √ √ √ √ √ √ √
√
√
√
√
√
√ √ √ √ √ √ √ √ √ √ √ √ √ nc nc nc
√ K D A E P D I L E L A G E hyL R
E
R nc √ √ √ √ √ √ √ √ √ √ G E hyL √
√ - Correct assignment; Lower case letter in parentheses - Tentative Correct assignment; Upper case letter Positive Wrong assignment; Upper case letter in parentheses - Tentative Wrong assignment; X - Unidentified
amino acid. Abbreviations: acK - N--Acetyl lysine; tmK - N--Trimethyl lysine; pC - Cysteine-S--propionamide; hyP - Hydroxyproline; hyL - 5-Hydroxylysine; dmR - Dimethylarginine; nc - no call; fi - failed injection
A: (Ac-Lys/Lys) Ratio at cycle 4
B: Initial Yield Ratio (C + C*/B)
14
4
3.5
3
2.5
2
1.5
1
0.5
0
12
10
8
6
4
2
Figure 4. Graphical Representation Yield Ratios from Mass Spectrometry
A: Mass Spec Ratio (C/C*)
B: Mass Spec Ratio (C + C*/B)
3.5
120
3.0
100
2.5
80
2.0
Table 2: Initial and RepetitiveYields, Calculated Peptide Ratios, and Mass Spectrometry Peak Areas from Participating Facility
0
60
1.5
40
1.0
20
0.5
0
4
3
2
1
880
800
770
700
600
500
440
400
330
200
110
100
0.0
100
110
200
330
400
440
500
600
700
770
800
880
1
2
3
4
15 used all instrument manufacturer reagents
2 used some mfg. R1, R2c, R4, R5, & Premix
1 said S4 home made
√
C
√
√
√
√
√
√
√
√
√
A
√
√
√
√
√
A
√
√
√
√
√
(A)
√
√
√
√
√
nc
√
√
√
nc
√
(q)
√
√
√
nc
√
nc
√
√
A
√
√
A
√
√
nc
√
(H)
A
20
100
110
200
220
300
330
400
440
500
550
600
660
700
770
800
880
900
990
1
2
3
4
5
990
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C K A
C* √ √
B R Q
C √ √
C* √ √
B √ √
C K R
C* √ √
B dmR √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C nc nc
C* √ √
B √ √
C √ √
C* √ √
B √ √
C T E
C* ßpC √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* √ √
B √ √
C √ √
C* D A
B √ E
20
400
8 ABI 49X-HT (5-14 years old: avg age of 8.6 yrs)
10 ABI 49X-cLC (0.25-11 years old: avg age of 7.0 yrs)
15 GFF, 3 PVDF
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
R
R
E K D A E P D I L E L A T G Y R
330
770
Sample Support
10
G
G
L
300
Sequencer Information
TFA Cleavage
9
S
S
L
220
400
660
Reagents
8
Y
Y
V
200
330
both unmodified peptides were supplied by the ABRF Peptide Standards Committee
Manufacturer and Model
7
L
L
S
110
[ 2 ]
[ 2 ]
[ 1 ]
550
Peptide C* (0.2 mmol) was synthesized on a Milligen 9050+ peptide synthesizer using Fmoc chemistry .
After synthesis, the peptide was cleaved from the resin using 92.5% TFA containing 2.5% each of
triisopropylsilane, water, and ethanedithiol for 3 h, followed by precipitation and washing three times with
diethyl ether. The crude peptide was dissolved in water and purified by HPLC using a 2.12x25cm Jupiter
Proteo C12 column (Phenomenex) using a gradient of 15% to 28% acetonitrile in water containing 0.1% TFA
over 15 minutes and monitoring absorbance at 220 nm. The purified peptide eluted at 12 min (ca. 25%
acetonitrile). This peptide dried in a SpeedVac and redissolved in water to give a 525 µM stock solution. The
concentration was determined by amino acid analysis performed at the Keck Biotechnology Resource
Laboratory at Yale University.
Peptides B and C were obtained from the ABRF Peptide Standards Research Group. One mg of each was
dissolved in 5 mL of 30% acetonitrile in water containing 10 mM TFA, yielding stock solutions containing 67.8
µM Peptide B and 155 µM Peptide C. The final peptide mixture was then prepared by adding 15.25 µL C*, 59
µL B, and 51.6 µL C to 1874 µL 30% acetonitrile in water with 10 mM TFA producing a solution containing
4.0 µM C*, 4.0 µM C, and 2.0 µM B. Ten µL samples of this mixture were placed in 0.6 mL Eppendorf tubes
and dried in a SpeedVac. These sample tubes, each of which contained 40 pmol each of peptides C and C* and
20 pmol of peptide B, were stored at -20 C until mailing to study participants.
Initial and repetitive yields were determined using the Excel trend line function to plot logs of the picomolar
amounts of Ala, Val, Leu, and Tyr (residues 3, 5, 6, 7 & 8 in peptides C+C*, and residues 2, 4, 5, 8, 9 & 10 in
peptide B) as a function of the sequencing cycle. Picomolar quantities for these calculations were obtained by
dividing reported peak areas from the appropriate cycles by the area/pmol of the corresponding amino acid
standard. The antilog of the y-intercept of the log plot is the initial yield, and thus the picomolar amount
theoretically present in the sample loaded, while the antilog of the slope of the log plot is the repetitive yield.
Initial yields were corrected for the percentage of the sample loaded to determine the total amount in the
sample.
The peptide C*/C ratio was calculated from the ratio of the picomolar amounts of Acetyl-Lys to Lys
observed on sequencing cycle 4. Picomoles of Ac-Lys were calculated by assuming that its peak area/pmol was
1.11 times the average area/pmol for Ala and Tyr. This assumption is based on the fact that the observed Ac-Lys
peak area was found to average 1.11 times the area whose log lay on the trend line of a plot of log area vs.
sequencing cycle using logs of regularly spaced Ala and Tyr residues in the ESRG 2004 study. Although
different sequencers in that study gave different ratios between the observed Ac-Lys peak area and the trend line
peak area on the cycle where Ac-Lys occurred, the factor of 1.11 was the average for both HT and cLC
sequencers from ABI. Picomolar amounts of Lys on cycles 1, 4, and 12 were calculated from the Lys area/pmol
obtained from the standards data supplied by each facility. In most cases, the picomolar amounts of Lys
reported on cycles 1 and 12 (from peptide B) were below the trend line values. Therefore, a Lys correction
factor for each facility was calculated by averaging the numbers by which the Lys 1 and Lys 12 amounts needed
to be multiplied to give areas whose logs lay on the trend line. The picomolar amount of Lys 4 was multiplied
by this correction factor, and the picomolar amount of Ac-Lys then divided by the corrected Lys 4 value to
obtain the peptide C*/C value.
6
L
L
R
100
110
200
220
300
330
400
440
500
550
600
700
770
800
880
900
1
2
3
4
5
220
Applied Biosystems Procise cLc
200
The test sample contained a mixture of 3 Peptides (100 pmol/vial) lyophilized [Ratio]
Materials and Methods
3 4 5
A K V
A acK1 V
Q Y A
100
100
Nature of the Test Sample
40 pmol RQAKVLLYSGR
40 pmol RQA(Ac-Lys)VLLYSGR
20 pmol KAQYARSVLLEKDAEPDILELATGYR
2
Q
Q
A
Facility ID
110
C
C*
B
1
R
R
K
Figure 1: Accuracy of Identification. A. Peptides C+C*; B. Peptide B.
Initial Yield (pmol)
 Determine how accurate Edman sequencing is for the quantitative analysis of
polypeptides.
 Compare quantitative results obtained by Edman sequencing to those obtained using
mass spectrometry techniques.
 Test the ability of participating laboratories to identify a modified amino acid residue
Cycle Number
Expected Peptide C
Expected Peptide C*
Expected Peptide B
Applied Biosystems Procise HT
The Edman Sequencing Research Group (ABRF) of the Association of Biomolecular Resource
Facilities (ABRF) has directed 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 2006 study was to determine how accurate Edman sequencing is for the quantitative
analysis of polypeptides. Secondarily, participants were asked to identify the modified amino acid
residue, -N-acetyl lysine present within one of the peptides. The ESRG 2006 peptide mixture
consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two.
The sequences of the two standard peptides were: (1) KAQYARSVLLEKDAEPDILELAT GYR
(Peptide B), and (2) RQAKVLLYSGR (Peptide C). The third peptide (Peptide C*) was identical to
peptide 2 but with acetyl lysine in position 4. The mixture consisted of 20% peptide 1 and 40% each
of peptide 2 and its acetylated form. Participating laboratories were provided with two tubes, each
containing 100 picomoles of the peptide mixture and were asked to provide amino acid assignments,
peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each
peptide in the mixture. Details about instruments and parameters used in the analysis were also
collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were
asked to provide information about the relative peak areas of the peptides in the mixture as a
comparison with the peptide quantitation results from Edman sequencing. The results from this study
may be viewed as a poster presentation during the ABRF 2006 conference and in the ESRG section
of the ABRF website.
Table 1: Submitted Amino Acid Calls by Participating Facilities
Objectives of the Study
Abstract
Results and Conclusions
Acknowledgements
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 ABRF Peptide Standards committee for supplying two of the synthetic peptides.
Thanks also to Renee Schrauben for removing identifiers from the responding laboratories.
 Participating facilities did well in calling the sequences of the peptides in the sample and in
identifying the acetylated lysine residue present in one of the peptides (Table 1 and Figure 1).
 Calculated initial yields were approximately 2/3 of the amounts of peptide expected (Table 2 and
Figure 2). The observed losses were between 25 to 30%.
 Data from 20 of the 23 participating facilities indicated a ratio within 25% of the expected 4/1
ratio for the 2 forms of peptide C (C + C*) relative to peptide B (Table 2 and Figure 3B).
 The average calculated C*/C ratio was 1.49±0.56, and 16 of the 23 facilities provided data
yielding values within 50% of the expected 1/1 ratio (Table 2 and Figure 3A).
 Relative peak areas from MALDI-TOF and ESI mass spectrometry of the peptides in the mixture
varied widely, and were not a good indication of the relative amounts of the peptides in the
mixture (Table 2 and Figure 4 A and B).