ABRF ESRG 2005 Study: Identification of Modified Amino Acids by Edman Sequencing

1
J.W. Leone Pfizer Inc., St. Louis, MO, United
StatesB.J. Madden Mayo Clinic College of
Medicine, Rochester, MN, United StatesD. Brune
Arizona State University, Tempe, AZ, United
StatesJ. Pohl Emory University, Atlanta, GA,
United StatesR. Kobayashi UT MD Anderson
Cancer Center, Houston, TX, United States W.S.
Lane and J.M. Neveu Harvard University,
Cambridge, MA, United StatesN.D. Denslow
University of Florida, Gainesville, FL, United
States.
ABRF ESRG 2005 Study Identification ofModified
Amino Acids by Edman Sequencing
Submitted Amino Acid Calls by Each Participating
Facility
Abstract The Edman Sequencing Research Group
(ESRG) of the Association of Biomolecular
Resource Facilities (ABRF) has directed numerous
studies focused on various aspects of Edman
degradation of proteins and peptides. 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 ABRF ESRG 2005
sample is a continuation of a similar study
conducted with the ESRG 2004 sample in which
laboratories were asked to identify the sequence
of a synthetic peptide containing both standard
amino acids and posttranslationally modified or
uncommon amino acids that are occasionally
encountered in submitted samples. Laboratories
requesting a sample were provided with 1 nanomole
of an 18 amino acid synthetic peptide and asked
to provide amino acid assignments at each cycle
along with the retention time and peak area.
Details about instruments and parameters used in
the analysis were also collected. Participants
were also provided with several modified amino
acid elution references posted on the ESRG
website, and had the option of viewing a list of
the modified amino acids present in this peptide.
Together with the ESRG 2004 results, this study
will provide a valuable reference for Edman
sequencing laboratories of the retention times of
uncommonly encountered amino acids which will be
accessible at the ABRF ESRG website. Materials
and Methods The ESRG 2005 peptide was synthesized
on a Milligen-Biosearch 9050 peptide synthesizer
using Fmoc chemistry. Synthesis was performed on
Fmoc-Arg-PEG-PS resin (Applied Biosystems) using
a 4-fold molar excess (0.8 mmole) of each Fmoc
amino acid and the HATU coupling reagent, except
in the cases of Fmoc-3-Me-His-OH and Fmoc
Lys(Me3)-OH, which were available in smaller
amounts. In these cases, PyAOP (0.7 mmole) was
used as the coupling reagent. Three amino acids,
Fmoc-Lys(Me2)-OH, Fmoc Lys(Me3)-OH, and
Fmoc-3-Me-His-OH, were injected manually during
the synthesis all others were dissolved and
injected automatically. Coupling times for the
methylated Lys residues were extended to one
hour, and coupling of Gly to N-Me-Ala was 45
minutes all others were for 30 minutes. The
synthetic peptide was cleaved from the resin with
92.5 TFA with 2.5 each of triisopropylsilane,
ethanedithiol, and water, precipitated by adding
diethyl ether, and dried under vacuum. The
monomeric peptide was purified by reversed phase
HPLC on a Phenomenex C12 proteo column (1 cm x 25
cm) using a gradient of 10-20 acetonitrile in
water containing 10 mM TFA over 20 minutes. The
peptide was then dissolved in 25 mM Na Phosphate,
pH 7.6, at a final concentration of 1.8 mM
(determined form its 280 nm absorbance) followed
immediately by adding diamide to a final
concentration of 0.9 mM to cause dimerization
(Kosower et al., 1987). After allowing the
reaction to proceed for several minutes, the
diamide concentration was increased to 1.3 mM, in
order to drive the reaction to completion. The
dimeric peptide was then purified by reversed
phase HPLC as described for the monomer, using a
gradient of 12 to 22 acetonitrile in water
containing 10 mM TFA over 20 minutes. The
dimeric nature of the product was indicated by
(1) elution at a different (higher) percentage of
acetonitrile during reversed phase HPLC, (2) the
presence of a peak with twice the molecular mass
of the monomer in MALDI-TOF mass spectrometry,
and (3) and earlier elution time than the monomer
in size exclusion chromatography on a Phenomenex
SEC 2000 column (0.78 x 60 cm). Reference Kosower
, N.S. and E. M. Kosower (1987) Formation of
disulfides with diamide. Meth. Enzymol. 143,
264-270.
Accuracy of Identification
Time Lines for Elution of Standard and Modified
Amino Acids on the Procise HT and Procise cLC
Normalized retention times of standards relative
to Ala by instrument type
To compensate for variations in the actual
elution times of amino acid standards, the
retention time (RT) for each standard was
normalized to Ala using the following procedure
The retention time of Ala was subtracted from
the retention time of each amino acid and this
was divided by the total time interval between
Asp and Leu (the first and last standards to
elute).  RTnA is the decimal fraction of this
interval between the time when each amino acid
eluted and the time when Ala eluted, with
negative values indicating amino acids eluting
before Ala.  Std Dev RTnA is the calculated
standard deviation for each RTnA value.  Av Full
RT were determined by multiplying the RT
difference between Asp and Leu by RTnA and adding
the product to the RT for Ala. In cases where
data were available from only one instrument of a
particular type, only RTnA values and Full RT
are reported.Results in this table include data
from instruments operated by members of the Edman
Sequencing Research Group. 
Average PTH Yields
Normalized Retention Times for Amino Acids in
Peptide
Effect of Premix Concentration on Elution Profiles
of Sample Loaded

• Conclusions
• Relative retention times of the modified amino
  acids between similar instruments were very
  consistent.
• Sequencing and elution properties of the modified
  amino acids on the ABI Procise HT and cLC were
  well characterized. In addition we have profiles
  for these amino acids on the single participating
  ABI 477 and Porton sequencer.
• Assignment of the positively charged modified
  amino acids proved to be challenging due to their
  poor behavior on silica based reverse phase
  supports

Acknowledgements Thanks to all the participating
laboratories who agreed to run this sample and
who were willing to share the data with our
committee. Without their contributions this
study would not have been possible. Thanks to
Bachem and AnaSpec for generously contributing
modified amino acids to this study. Thanks to
Melinda Miller for removing identifiers from the
contributing laboratories