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

1. 20 pmol 80 pmol A: Average = 53.6 pmol, 67% B: Average = 13.1 pmol, 65.6% 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 C+C*; 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 KAQYARSVLLEKDAEPDILELATGYR [ 1 ] both unmodified peptides were supplied by the ABRF Peptide Standards Committee R Q A X V L L Y S G R 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 C+C*, 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) √ - 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 1.49±0.56, 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.