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

1. Elution Profiles of 3meHis, Me2Lys & Me3Lys on cLC (21ml/L Premix vs 14.5ml/L Premix) ABI Procise HT Tri-Me Lys Di-Me Lys N-me Ala 3-Me His Homo Citr Cys 3meHis 3meHis vs Ala (Std & Cycle 4) lag Ala + 3meHis 21ml/L 14.5ml/L lag Ala D N S Q T G E H A R Y P M V W F I K L 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 minutes ABI Procise cLC Homo Citr 3-Me His Di-Me Lys Tri-Me Lys N-me Ala Cys Me2Lys vs Me3Lys (Cycle 2 & 11) Me2Lys - 2 Me2Lys - 2 lag Tyr Me3Lys - 11 14.5ml/L 21ml/L D N S Q T G E H A R Y P M V W F I K L % Loaded by Facility lag Tyr Me3Lys - 11 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 minutes 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