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

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

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

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

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

Sample Preparation (cont.) 3. Three peptides were mixed a. 15.25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

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

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

Labs Participating in ESRG Studies Participating Labs Number of Year

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

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 = - 0.0338x + 1.57 R2 = 0.9284 y = - 0.0334x + 0.946 R2 = 0.834 3. I.Y. = (antilog of y-intercept)/(% of sample tested) R.Y. = (antilog of slope)

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

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

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

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 #

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 #

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

Linearity (R2 > 0.9) Accuracy ( 50% from expected) Short Peptide - 5 labs (22%) Long Peptide - 10 labs (43%) Accuracy ( 50% from expected) Short Peptide (40-120 pmol) - 16 labs (70%) Long Peptide (10-30 pmol) - 19 labs (83%)

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

Initial Yield Edman Sequencing vs Mass Spectrometry 1290.81 1332.83 2949.73 MALDI-TOF M+H % Intensity Mass (m/z)

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

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

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

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

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

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.

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

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