Transcript Document

ABRF 2006 ESRG Study
Edman Sequencing as a Method
For Polypeptide Quantitation
ESRG Committee Members
Daniel C. Brune (Chair) - Arizona State Univ.
Brian Hampton - Univ. of Maryland, Baltimore,
School of Medicine
Ryuji Kobayashi - Univ. of Texas, M.D.
Anderson Cancer Center
Joseph W. Leone (Chair elect) - Pfizer
Klaus D. Linse - Univ. of Texas, Austin
Jan Pohl - Emory Univ.
Richard S. Thoma - Monsanto Co.
Nancy D. Denslow (ABRF liaison) - Univ. of Florida
Objectives of the Study
- 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
Description of the Test Sample
Mixture of 3 Peptides (100 pmol/vial) lyophilized
Peptide C
40 pmol
RQAKVLLYSGR
Peptide C* 40 pmol
RQA(Ac-Lys)VLLYSGR
Peptide B
KAQYARSVLLEKDAEPD
ILELATGYR
20 pmol
- both unmodified peptides were supplied by the
ABRF Peptide Standards Committee
Sample Preparation
1. Peptide C* synthesis and purification
a. Synthesis on a Milligen 9050+
peptide synthesizer - FMOC chemistry
b. Purified by HPLC - Jupiter Proteo C12
column (Phenomenex)
c. Mass was verified using MALDI-TOF and
Edman Sequencing
d. Concentration determined by AAA
2. Peptides B & C each solubilized to 1mg/5mL in
10mM TFA/30% Acetonitrile
Sample Preparation (cont.)
3. Three peptides were mixed
a. 15.25L C*, 59L B, 51.6L C
b. 1874L 10mM TFA/30% Acetonitrile
4. 10L aliquots placed in 0.6mL Eppendorf tubes
(100 pmol/tube)
5. Dried in a Speed-Vac
6. Distributed 2 tubes per lab
Requested Information
- The amino acid sequence of each peptide
- Areas for peaks in each cycle
- Areas, picomolar yields and retention times
for each amino acid peak in the standard
- Instrument information
a. sequencer
e. Gradient
b. sample loading
f. Solvents
c. HPLC equipment
g. Flow rate
d. Column
- 34 facilities requested the sample
- 18 facilities returned sequencing data
Sequencer Information
Manufacturer
and Model
8
Reagents
15
2
10
1
ABI 49X-Ht
(5-14 yrs, Ave = 8.6)
ABI 49X-cLC
(0-11 yrs, Ave. = 7.0)
all Manufacturers reagents
used some (R1, R2C, R4, R5
and Premix)
S4 was homemade
TFA Cleavage
15 liquid, 3 gas-phase
Sample Support
15 GFF, 3 PVDF
DTT in S2
Other additives
8 yes, 10 no
1 TCEP to R5
1988
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2002
2003
2004
2005
2006
Number of
Participating Labs
Labs Participating in ESRG Studies
100
90
80
70
60
50
40
30
20
10
0
Year
Accuracy of Identification
20
Short
Peptide
(C* + C)
18
16
14
Q
R
12
A
K
Ac-K
V
L
L
Y
S
G
R
10
8
Correct
Calls
6
Accuracy
4
Long
Peptide
(B)
2
0
20
18
16
14
12
10
8
6
4
2
0
1
2
3
4
5
6
7
8
9
10
11
Incorrect
Calls
No Calls
K A Q Y A R S V L L E K D A E P D I L E L A T G Y R
1
3
5
7
9
11
13
15
Cycle
17
19
21
23
25
Initial Yield Calculation
1. Log10 (AA pmol yield)
Short Peptide
3(A),5(V),6(L),7(L),8(Y)
Long Peptide
2(A),4(Y),5(A),8(V),9(L),10(L)
2. Plot data and determine best fit line
Short Peptide
2
Long Peptide
1
0.8
1.5
0.6
1
0.4
0.5
y = - 0.0338x + 1.57
R2 = 0.9284
0
0
0
2
4
y = - 0.0334x + 0.946
R2 = 0.834
0.2
6
8
0
2
4
6
8
10
3. I.Y. = (antilog of y-intercept)/(% of sample tested)
R.Y. = (antilog of slope)
Initial Yield Performance
180
160
140
120
80 pmol
Short Peptide (C* + C)
Ave.
= 53.6pmol
67.0%
100
60
40
20
100
110
200
220
300
330
400
440
500
550
600
660
700
770
800
880
900
990
1
2
3
4
5
0
30
25
20 pmol
Long Peptide (B)
Ave.
= 13.1pmol
65.6%
20
15
10
5
0
100
110
200
220
300
330
400
440
500
550
600
660
700
770
800
880
900
990
1
2
3
4
5
Initial Yield (pmol)
80
Lab ID
Initial Yield Ratio
Short (C* + C) vs. Long (B) Peptide
14
12
Ratio
10
8
Ave.
= 4.27/1
4/1
ratio
6
4
2
100
110
200
220
300
330
400
440
500
550
600
660
700
770
800
880
900
990
1
2
3
4
5
0
Lab ID
20 of 23 labs were between 3/1 and 5/1 ratios
Reasons for Low Initial Yield
- Sample Washout
- N-termini blocked
a. interaction with the membrane
b. chemical modification
- Quality of reagents
a. Biobrene (age or cycles performed)
b. EtOAc (> 6 months)
- Poor solubility during reconstitution
- Specific Low Yielding Amino Acids
(e.g. Cys, Trp, Ser, Arg)
- Non-linear Repetitive Yield
- Small amount of sample analyzed
Log 10 (pmol)
Initial Yield - Short Peptide (C* + C) Slope Calculations
Sequence - RQAK*VLLYSGR
2
100
1.6
2.5
1.2
1.5
0.8
1
y = -0.0386x + 1.6901
0.4
110
2
200
1.5
1.45
1.4
1.3
R = 0.8306
4
6
0
8
1.4
1.2
2
4
6
400
1.7
1.65
1
0.8
0
1.6
330
y = -0.0388x + 1.3156
R2 = 0.9488
0.2
0
0
1.75
2
4
6
y = -0.0233x + 1.656
1.45
0
440
1.52
1.4
0
2
4
6
0
8
660
2
4
6
8
1.72
0
0
2
4
6
1.74
880
1.72
1.7
1.56
0
8
700
R2 = 0.0001
2
4
6
1.44
8
900
1.42
0
2
4
6
0.8
990
0.6
1.4
y = -0.01x + 1.7324
1.66
y = -0.0053x + 1.431
R2 = 0.5505
2
4
6
8
0
2
4
6
8
3
2
ESRG2006
4
6
1.5
0
2
4
6
4
1.8
1.75
1.7
1
y = -0.0211x + 1.7568
1.6
2
R = 0.9284
0
2
4
6
8
0
2
4
6
8
Cycle #
4
6
8
800
1.72
1.7
y = -0.0122x + 1.7282
1.64
R2 = 0.7389
1.62
0
2
4
6
0
8
1.65
1
1.6
1.55
2
4
6
8
1.4
2
1.36
1.32
y = -0.0164x + 1.6185
y = -0.0094x + 1.3771
1.28
R2 = 0.1359
R2 = 0.1706
1.24
0
2
4
6
0
8
1.64
1.62
5
1.6
1.58
y = -0.0151x + 1.6287
1.52
1.5
R2 = 0.9985
1.55
0
2
1.74
1.66
0
1.56
1.54
1.65
y = -0.0338x + 1.57
0.5
R2 = 0.8351
1.68
R2 = 0.8348
8
y = -0.0493x + 0.3229
0
8
1.35
0
1.38
0
2
y = -0.0365x + 1.346
0.4
1.4
R2 = 0.932
R2 = 0.4285
1.64
0
770
1.45
y = -0.0409x + 0.7282
0.2
550
-0.1
1.5
0.4
1.68
8
-0.2
0
8
6
0.1
y = -0.0075x + 1.5469
1.68
1.6
R2 = 0.8749
R2 = 0.4904
4
0.2
1.2
y = -0.0003x + 1.6881
2
0.3
0.8
y = -0.0835x + 1.7353
0.5
R2 = 0.8364
0.4
1.6
1.76
y = -0.0139x + 1.6837
0
8
1.4
1.64
1.5
6
R2 = 0.0616
1
1.55
4
500
1.55
R2 = 0.8296
1.36
1.6
y = 0.0225x + 1.5177
2
1.6
1.45
y = -0.0181x + 1.5448
1.4
R2 = 0.5445
1.5
1.65
8
1.5
2
600
1.7
6
1.44
1.5
8
4
1.48
1.55
0.6
0.4
2
1.56
300
1.66
1.64
1.58
1.56
R2 = 0.3287
1.85
8
1.7
1.68
1.62
1.6
y = -0.0187x + 2.0896
1.9
R2 = 0.9615
1.25
0
2
2.05
1.95
y = -0.0215x + 1.4787
2
0
2.1
2
1.35
y = -0.122x + 2.1403
R2 = 0.8023
0.5
0
220
2.15
R2 = 0.4432
0
2
4
6
8
2
4
6
ABRF
8
Initial Yield - Long Peptide (B) Slope Calculations
Sequence - KAQYARSVLLEKDAEPDILELATGYR
1.2
100
1
0.8
110
1.2
1
0.6
0.4
y = -0.0457x + 1.0882
0.2
0
2
4
6
8
0.8
330
0.6
4
6
8
400
1
0.8
y = -0.0462x + 0.7373
0.4
R2 = 0.955
0.2
2
4
6
8
1.2
1
0
2
4
6
8
660
1.4
1.2
1
0.4
0.2
2
R = 0.9631
0
2
4
6
8
2
R = 0.9365
2
4
6
8
0.2
1
0.1
0.8
0
880
R2 = 0.969
0
900
0.6
0.4
y = 0.0315x + 0.7718
0.2
R2 = 0.3231
2
4
6
8
10
2
4
6
8
1
ESRG2006
R = 0.4494
2
4
6
8
990
-0.1
3
0.8
0.6
y = -0.0334x + 0.946
0.2
0
2
4
6
8
10
y = -0.0113x + 0.9151
0.2
R2 = 0.1931
0
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2
4
500
6
8
0
1.4
4
1.2
1
2
4
6
8
1
1.2
1
0.8
2
4
6
8
Cycle #
10
4
6
8
10
800
1.2
1
y = -0.0362x + 1.2038
R2 = 0.9295
0
2
4
6
8
10
2
1
0.8
0.4
y = -0.0262x + 0.898
R2 = 0.5785
0.2
0
0
2
4
6
8
1.2
10
5
1
0.8
0.4
y = -0.0278x + 1.0432
0.2
R2 = 0.7672
0
0
2
0.6
2
R = 0.9612
10
R2 = 0.7563
1.4
10
0.6
y = -0.0275x + 1.1808
8
0
0
10
6
y = -0.0247x - 0.3811
0.4
0.2
R2 = 0.6407
R2 = 0.7826
8
4
0.6
y = -0.028x + 1.0441
6
2
550
0
10
y = -0.0349x + 0.6818
0.2
4
-0.2
-0.3
-0.4
-0.5
-0.6
-0.7
-0.8
-0.9
-1
0.8
0.4
2
0
10
770
R2 = 0.9543
0
0
8
0
0.4
0.2
R2 = 0.834
0.4
y = -0.0423x + 0.1396
0.8
0.6
0.4
6
0.6
-0.2
10
4
1
10
-0.4
0
2
0.8
10
700
2
-0.3
0
0
8
y = -0.0191x + 1.1435
0
10
1.2
y = -0.0248x + 1.2094
6
1.2
1
1.2
1
0.4
0.2
4
0
0
10
0
1.2
0
2
1.4
1.4
0.8
0.6
R2 = 0.914
0.4
0.2
y = -0.0638x + 1.1581
0
0
y = -0.0394x + 0.9657
0.8
0.6
0.6
y = -0.0519x + 1.2394
0.4
0.2
10
R2 = 0.8841
0
0.6
0
10
0.8
0.8
0.6
8
0
10
600
1.4
6
440
0.2
R2 = 0.8231
0
0
4
1
0.6
y = -0.0232x + 1.0039
2
0.8
0.4
0
R2 = 0.6806
1.2
y = -0.0349x + 1.153
0.4
0.2
y = -0.0198x + 1.4241
1.2
0
10
0.6
0.4
0.2
2
1.2
300
1.2
1
0.6
1.3
0
1.4
0.8
1.25
R2 = 0.9259
R = 0.1803
0
1.4
1.35
0.2
2
10
220
1.45
y = -0.0315x + 0.8658
y = -0.0273x + 1.0205
0
0
200
0.4
0.4
0.2
R2 = 0.9406
1
0.8
0.6
0.8
0.6
Log 10 (pmol)
1.4
0
2
4
6
8
10
0
2
4
6
8
ABRF
10
Initial Yield vs Linearity
Short Peptide (C* + C)
180
150
120
Initial Yield (pmol)
90
60
30
0
0
0.2
0.4
0.6
0.8
0.6
0.8
1
Long Peptide (B)
30
25
20
15
10
5
0
0
0.2
0.4
Linearity (R2)
1
Linearity (R2 > 0.9)
Short Peptide - 5 labs (22%)
Long Peptide - 10 labs (43%)
Accuracy ( 50% from expected)
Short Peptide (40-120 pmol) - 16 labs (70%)
Long Peptide (10-30 pmol) - 19 labs (83%)
Initial Yield vs Amount Analyzed
200
Short Peptide (C* + C)
150
Initial Yield (pmol)
100
50
0
0%
30
50%
100%
Long Peptide (B)
20
10
0
0%
50%
100%
Percent of Test Sample Sequenced
Initial Yield
Edman Sequencing vs
Mass Spectrometry
V
o
y
a
g
e
r
S
p
e
c
#
1
[
B
P
=
1
3
3
2
.
8
,
4
2
0
8
1
]
4.2E+4
100
1290.81
90
80
1332.83
60
50
40
30
20
V
0
1276.0
o
y
a
g
e
r
S
p
e
c
#
1
[
B
P
=
1
3
3
2
.
8
,
4
2
0
8
1
]
2336.1
100
10
1292.6
1309.2
1325.8
1342.4
90
0
1359.0
80
Mass (m/z)
70
% In ten sity
% In ten sity
% Intensity
70
MALDI-TOF
M+H
60
2949.73
50
40
30
20
10
0
2944.0
2948.2
2952.4
2956.6
Mass (m/z)
Mass (m/z)
2960.8
0
2965.0
Mass Spec Ratio
Short (C* + C) vs. Long (B) Peptide
120
Ratio
100
80
60
4/1
ratio
Ave. = 22.4/1
Std Dev. = 30.6
40
20
100
110
200
330
400
440
500
600
700
770
800
880
1
2
3
4
0
Lab ID
Instrumentation
MALDI
16
ESI or Ion Trap 5
Not reported
3
3 of 16 labs were between
3/1 and 5/1
(Not enough data to
separate MS instrumentation)
The Modified Amino Acid
Acetyl-Lysine
Cycle 4
DPTU
Ac-K
K
Y
D
N
Q
S TG E
PTH Standard
H A
R
Y
P
MV
DPTU
K
W F
I L
13 of 18 labs correctly identified the modified peptide
4
3.5
3
2.5
2
1.5
1
0.5
0
1/1
ratio
Ave. = 1.5/1
100
110
200
220
300
330
400
440
500
550
600
700
770
800
880
900
1
2
3
4
5
Ratio
Area Ratio
Short (C* vs C) Peptide
Lab ID
16 of 21 labs were between
0.5/1 and 1.5/1
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Ave. = 2/1
1/1
ratio
100
110
200
330
400
440
500
600
700
770
800
1
2
3
4
Ratio
Mass Spec Ratio
Short (C* vs C) Peptide
Lab ID
8 of 15 labs were between
0.5/1 and 1.5/1
Conclusions
- Edman sequencing tends to underestimate
initial yields by approximately 1/3. Most
lab data was within a factor of 2 from the
expected accuracy.
- Edman Sequencing produced very accurate
peptide to peptide ratios. Mass
Spectrometry produced far less accurate
ratio data.
- The majority of labs were able to identify
the modified amino acid – Acetylated lysine
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 Henriette Remmer from the
ABRF Peptide Standards committee for
supplying two of the synthetic peptides.
Thanks also to Renee Schrauben for removing
identifiers from the responding laboratories.
Congratulations to
“Joe Leone”
On his election as
ESRG Chair!
Suggested Future ESRG Studies
1. Deblocking a Protein
2. Sequencing a Very Large Protein on a PVDF
Membrane
3. Internal Cleavage Analysis
4. Repetitive Yield Study
5. Protein Mixture or an Antibody
6. Very Hydrophobic Peptides
7. More Tips on Unusual Amino Acids
8. Phosphorylated and/or Glycosylated Proteins
9. Alkylated Cysteine
10. Evaluation of PVDF Membranes and Transfer
Techniques
11. Low Protein Concentrations (< 2 pmol)
12. Quantitation of Post-Translational Modifications