1. Edman Sequencing as a Method For Polypeptide Quantitation
ABRF 2006 ESRG Study
Edman Sequencing as a Method
For Polypeptide Quantitation

2. 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

3. 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

4. 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 20 pmol KAQYARSVLLEKDAEPD
ILELATGYR
- both unmodified peptides were supplied by the
ABRF Peptide Standards Committee

5. 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

6. Sample Preparation (cont.)
3. Three peptides were mixed
a L C*, 59mL 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

7. 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

8. Sequencer Information
Manufacturer 8 ABI 49X-Ht
and Model (5-14 yrs, Ave = 8.6)
10 ABI 49X-cLC
(0-11 yrs, Ave. = 7.0)
Reagents 15 all Manufacturers reagents
2 used some (R1, R2C, R4, R5
and Premix)
1 S4 was homemade
TFA Cleavage 15 liquid, 3 gas-phase
Sample Support 15 GFF, 3 PVDF
DTT in S2 8 yes, 10 no
Other additives 1 TCEP to R5

9. Labs Participating in ESRG Studies
Participating Labs
Number of
Year

10. Accuracy of Identification
Short
Peptide
(C* + C) K
Ac-K R Q A V L L Y S G R
Correct Calls
Incorrect Calls
Accuracy
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
Long
Peptide
(B)
Cycle

11. 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
Long Peptide
y = x
R2 =
y = x
R2 = 0.834
3. I.Y. = (antilog of y-intercept)/(% of sample tested)
R.Y. = (antilog of slope)

12. Initial Yield Performance
Short Peptide (C* + C)
80 pmol
Ave.
= 53.6pmol
67.0%
Initial Yield (pmol)
Long Peptide (B)
20 pmol
Ave.
= 13.1pmol
65.6%
Lab ID

13. Short (C* + C) vs. Long (B) Peptide
Initial Yield Ratio
Short (C* + C) vs. Long (B) Peptide
4/1
ratio
Ave.
= 4.27/1
Ratio
Lab ID
20 of 23 labs were between 3/1 and 5/1 ratios

14. 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

15. Initial Yield - Short Peptide (C* + C) Slope Calculations
Sequence - RQAK*VLLYSGR
100
110
200
220
300
400
440
500
550
330
Log 10 (pmol)
600
660
770
800
700
880
900
990 1 2 3 4 5
ABRF
ESRG2006
Cycle #

16. Initial Yield - Long Peptide (B) Slope Calculations
Sequence - KAQYARSVLLEKDAEPDILELATGYR
100
110
200
220
300
330
400
440
550
500
Log 10 (pmol)
600
660
770
800
700
990 1 2
880
900 3 4 5
ESRG2006
ABRF
Cycle #

17. Initial Yield vs Linearity
Short Peptide (C* + C)
Initial Yield (pmol)
Long Peptide (B)
Linearity (R2)

18. Linearity (R2 > 0.9) Accuracy ( 50% from expected)
Short Peptide - 5 labs (22%)
Long Peptide - 10 labs (43%)
Accuracy ( 50% from expected)
Short Peptide ( pmol) - 16 labs (70%)
Long Peptide (10-30 pmol) - 19 labs (83%)

19. Initial Yield vs Amount Analyzed
Short Peptide (C* + C)
Initial Yield (pmol)
Long Peptide (B)
Percent of Test Sample Sequenced

20. Initial Yield Edman Sequencing vs Mass Spectrometry
MALDI-TOF
M+H
% Intensity
Mass (m/z)

21. Short (C* + C) vs. Long (B) Peptide
Mass Spec Ratio
Short (C* + C) vs. Long (B) Peptide
Ave. = 22.4/1
Std Dev. = 30.6
4/1
ratio
Ratio
Lab ID
Instrumentation
3 of 16 labs were between
3/1 and 5/1
(Not enough data to
separate MS instrumentation)
MALDI 16
ESI or Ion Trap 5
Not reported 3

22. The Modified Amino Acid
Acetyl-Lysine
Cycle 4
Ac-K
DPTU K Y
PTH Standard D N Q S T G E H A Y K R P M V W
DPTU F I L
13 of 18 labs correctly identified the modified peptide

23. Area Ratio Short (C* vs C) Peptide
Ave. = 1.5/1
1/1
ratio
Ratio
Lab ID
16 of 21 labs were between
0.5/1 and 1.5/1

24. Mass Spec Ratio Short (C* vs C) Peptide
Ave. = 2/1
1/1
ratio
Ratio
Lab ID
8 of 15 labs were between
0.5/1 and 1.5/1

25. 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

26. 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.

27. Congratulations to
“Joe Leone”
On his election as
ESRG Chair!

28. 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