ABRF ESRG 2006 Study: Edman Sequencing as a Method for Polypeptide Quantitation

1
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.
Table 1 Submitted Amino Acid Calls by
Participating Facilities
Figure 1 Accuracy of Identification. A.
Peptides CC B. Peptide B.

• 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

Abstract 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.
Nature of the Test Sample The test sample
contained a mixture of 3 Peptides (100 pmol/vial)
lyophilized Ratio C 40
pmol RQAKVLLYSGR
2
C 40 pmol RQA(Ac-Lys)VLLYSGR

2 B 20 pmol KAQYARSVLLEKDAEPDILE
LATGYR 1
both unmodified peptides were supplied by the
ABRF Peptide Standards Committee
G R
R
Q
A
X
V
L L
Y S
KAQYARSVLLEKDAEPDILELATGYR
X AcLys or K
Figure 2. Graphical Representation of Initial
Yields A Peptides C C
B Peptide B
Materials and Methods 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 CC, 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.
Sequencer Information

Figure 3. Graphical Representation of Yield
Ratios A (Ac-Lys/Lys) Ratio at cycle 4
B Initial Yield Ratio (C C/B)
Q
Figure 4. Graphical Representation Yield Ratios
from Mass Spectrometry A Mass Spec Ratio
(C/C) B Mass Spec
Ratio (C C/B)
v - 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-?-propion-
amide hyP - Hydroxyproline hyL -
5-Hydroxylysine dmR - Dimethylarginine nc - no
call fi - failed injection
Table 2 Initial and RepetitiveYields, Calculated
Peptide Ratios, and Mass Spectrometry Peak Areas
from Participating Facility

• Results and Conclusions
• 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 1.490.56,
  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).

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.