* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Identification of Modified Amino Acids by Edman Sequencing
Survey
Document related concepts
Metalloprotein wikipedia , lookup
Point mutation wikipedia , lookup
Fatty acid synthesis wikipedia , lookup
Fatty acid metabolism wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Citric acid cycle wikipedia , lookup
Protein structure prediction wikipedia , lookup
Proteolysis wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Genetic code wikipedia , lookup
Peptide synthesis wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Transcript
J.W. Leone – Pfizer Inc., St. Louis, MO, United States B.J. Madden – Mayo Clinic College of Medicine, Rochester, MN, United States D. Brune – Arizona State University, Tempe, AZ, United States J. Pohl – Emory University, Atlanta, GA, United States R. Kobayashi – UT MD Anderson Cancer Center, Houston, TX, United States W.S. Lane and J.M. Neveu – Harvard University, Cambridge, MA, United States N.D. Denslow – University of Florida, Gainesville, FL, United States. ABRF ESRG 2005 Study: Identification of Modified Amino Acids by Edman Sequencing Submitted Amino Acid Calls by Each Participating Facility Tyr Arg Ala 124 Tyr (Arg) 125 Tyr 126 Tyr Materials and Methods 127 Tyr The ESRG 2005 peptide was synthesized on a Milligen-Biosearch 9050+ peptide synthesizer using Fmoc chemistry. Synthesis was performed on Fmoc-Arg-PEGPS 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-3Me-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. 128* Tyr 129 Tyr 130 Tyr (biotinylLys) (Me2Lys) 131 Tyr 132* 133 134 Tyr Tyr Tyr 135 Tyr 136 Tyr 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. 137 Tyr 18 ABI 49X-HT (1-10 years old: avg age of 7 yrs) 7 ABI 49X-cLC (5-10 years old: avg age of 6.7 yrs) 1 ABI 477A (10 yrs old) 22 used all instrument manufacturer reagents 1 used some mfg. R1, R2c, R4, R5, & Premix 1 said almost mfg. reagents 1 said partially 1 said S4 home made 21 GFF, 4 PVDF, 1 said both GFF and PVDF (probably answered in general) Chemistry Cycle All users seemed to use the cycles that matched the support (ie. GFF or PVDF) except one PVDF with GFF cycle DTT in S2 6 yes and 20 no Other Additives 1TCEP to R4A, 1 DTT in R4A & 1 DTT in S3 Sample Solvent 13 used 0.1% TFA/30% acetonitrile 4 used 0.1% TFA/20% acetonitrile 2 used 0.1% TFA/ water 1 used 1% TFA/ 60% acetonitrile 1 used 0.01%TFA/ 50% acetonitrile 1 used 0.0005% TFA/ 0.05%HAc/ 30% acetonitrile 1 used 30% acetonitrile/ water 3 users were unclear Ranged from 2 to 100% Me3Lys Arg X Gly Gly Gly N-MeAla X Me3Lys Tyr Tyr Tyr Lys Lys Lys His His His X X (Cystine) Ala Ala Ala (Cystine) X X Tyr Tyr Tyr (N-MeAla) X Tyr Gly Gly Gly (Me3Lys) X X Tyr Tyr Tyr Ala Ala Ala (isoAsp) X Ala X Lys His Cys-SPam Ala pSer Tyr Gly Me2Arg Tyr Ala Ala Ala (3MeHis) Lys His X Ala (Cystine) Tyr Gly Me3Lys Tyr Ala X (X) Ala 3MeHis Lys His (X) Ala Gly (X) Me2Lys Ala X Arg Ala Me3Lys Me2Lys Cystine X Tyr X Gly Cys N-MeAla 3MeHis isoAsp Me3Lys (Me2Lys) His HomoCit Ala NHydroxyMet HydroxySuccinyl- Lan CAM-Met pSer Me2Lys Lys sulfone Lys Lys Arg X Tyr HydroxyPro Gly Abu Tyr Cys Tyr (NmethylAla) Gly (Me2Lys) Tyr Ala (MeLys) Ala 3MeHis Lys His (CAM-Cys) Ala (Cystine) Tyr (Me2Lys) Gly (Me3Lys) Tyr Ala (isoAsp) Ala (3MeHis) Lys His (HomoCit) Ala (Cys) Tyr (N-MeAla) Gly (Me3Lys) Tyr Ala (isoAsp) (Me2Lys) Ala 3MeHis Lys His HomoCit Ala Cystine Tyr Me3Lys Gly N-MeAla Tyr Ala (iso-Asp) Me2Lys 3MeHis (Arg) Ala Ala Ala 3MeHis N-MeAla (3MeHis) Lys Lys Lys His His His HomoCit isoAsp X Ala Ala Ala Cystine (Ser) (MeLys) Tyr Tyr Tyr Me3Lys HomoCit X Gly Gly Gly N-MeAla Cystine X Tyr Tyr Tyr Ala Ala Ala isoAsp Ala X (N-MeAla) Ala 3MeHis Lys (HomoCit)(isoAsp) Ala Cys Tyr X Gly (Tyr) Ala Glu Ala 3MeHis Lys His Ala Cystine Tyr Me3Lys Gly N-MeAla Tyr Ala (iso-Asp) (3MeHis) Lys His (HomoCit) Ala (Cystine) Tyr (Me2Lys) Gly (Me3Lys) Tyr Ala (iso-Asp) (N-MeAla) Ala -0.30 -0.27 -0.22 -0.20 -0.18 -0.17 -0.13 -0.04 0.00 0.12 0.16 0.33 0.38 0.40 0.55 0.63 0.66 0.68 0.70 494-HT 494-HT Av Std Dev of Full RT RTnA's 0.03 0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.00 0.02 0.01 0.00 0.01 0.00 0.01 0.02 0.02 0.03 0.03 4.19 4.61 5.24 5.52 5.74 6.00 6.48 7.69 8.22 9.78 10.36 12.63 13.34 13.65 15.61 16.64 17.08 17.36 17.61 494-cLC 494-cLC Average 494-cLC Av Std Dev of RTnA's Full RT RTnA's n=10 -0.30 -0.27 -0.23 -0.21 -0.19 -0.17 -0.14 -0.05 0.00 0.11 0.16 0.33 0.38 0.41 0.57 0.62 0.66 0.68 0.70 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.01 0.00 0.01 0.00 0.01 0.01 0.01 0.01 0.01 6.14 6.59 7.34 7.68 7.92 8.24 8.77 10.13 10.89 12.57 13.39 16.06 16.86 17.23 19.84 20.66 21.15 21.48 21.79 477 RTnA's n=1 -0.24 -0.21 -0.17 -0.16 -0.14 -0.12 -0.10 0.00 0.00 0.13 0.16 0.37 0.42 0.44 0.61 0.66 0.70 0.73 0.76 477 Full RT 7.91 8.50 9.35 9.75 10.10 10.50 11.00 13.00 13.10 15.80 16.64 21.10 22.20 22.60 26.15 27.30 28.23 28.73 29.38 Porton RTnA's n=1 -0.31 -0.28 -0.23 -0.21 -0.20 -0.16 -0.15 -0.05 0.00 0.12 0.15 0.36 0.39 0.40 0.57 0.62 0.71 0.67 0.69 % loaded % loaded 90 80 70 60 50 20 40 30 10 20 10 01 Mean 21.35% 3 1 3 5 5 7 7 9 9 11 11 Val* Arg* Porton Full RT 6.26 6.75 7.46 7.79 8.02 8.60 8.88 10.49 11.23 13.23 13.61 16.93 17.49 17.72 20.44 21.23 22.66 22.00 22.31 Time Lines for Elution of Standard and Modified Amino Acids on the Procise HT and Procise cLC 30 25 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. ABI Procise HT 20 X = "X" + "-" 15 3-Me His Homo Citr Tri-Me Di-Me N-me Lys Lys Cys Ala W = PW + TW C = PC + TC D N SQTG E H A R Y P MV W F I KL 10 5 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 minutes 0 ABI Procise cLC Homo Citr D N 3-Me His Di-Me Tri-Me Lys Lys Cys N-me Ala SQT G E H A R Y P M V W F I K L Average PTH Yields 4.00 200 Normalized Retention Times for Amino Acids in Peptide % of Sample Loaded 30 Me3Lys) Arg* Accuracy of Identification 100 40 (Me2 or Val* HydroxypThr Lys (3MeHis)(HomoCit) His HomoCit Ala Me3Lys) Ala Me2Lys (iso-Asp) (Me2 or 70 0 % Loaded Tyr Tyr Tyr 90 50 isoAsp X X Cys X (Arg) (2) 100 60 Ala Ala Ala His HomoCit Ala His (Cys-S-Pam) Ala His X Ala 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 minutes cLC HT % Loaded by Facility 80 Gla Tyr 13 15 13 17 15 17 19 21 participant # 19 23 21 23 150 Relative Peak Area Sample Support Ala Cystine di m D N S Q T G E H A R Y P M V W F I K L Sequencers Information 23 liquid, 2 gas phase and 1 both Tyr Ala Arg* Ty r 494-HT Average RTnA's n=23 Kosower, N.S. and E. M. Kosower (1987) Formation of disulfides with diamide. Meth. Enzymol. 143, 264-270. TFA Cleavage Abu, Canavn., MeArg OMeLys, OMeGlu Val* Normalized retention times of standards relative to Ala by instrument type Reference: Reagents isoAsp (1) Comment: Coelutes with Ser (2) Comment: Cystine (3) Comment: Order of residues 3,4,8, and 15: 8<15<3<4. Would be very easy to call all Ala. * These facilities correstly assigned residues 17 and 18 as Val and Arg, respectively, by MS/MS. 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). Manufacturer and Model Ala As p 123 (3MeHis) X X Tyr a (N-MeAla) Ala X Ala X Ala N-MeAla iso Tyr Tyr Tyr Gly X Al 120 121 122 Me3Lys Ala Ty r Ala Ala Ala His CAMMet Gly Tyr X a Me2Lys Arg N-MeAla Cystine (Me3Lys) 18 Arg eA l Tyr Tyr Tyr Ala Gly 17 Val Nm 117 118 119 HomoCit 16 isoAsp X isoAsp Glu G ly Ala His 15 Ala Ala X (3) Ala eL ys Tyr 3MeHis Lys NAcetylLys Lys 3MeHis Lys (Ala) Lys(1) HomoCit Lys Cystine (N-MeAla) 14 Tyr Tyr Tyr Me3Lys N-MeAla m 116 OMeThr X 13 N-MeAla Arg X Ty r Ala Ala Me3Lys X X 3MeCys 12 Gly Gly Gly Gly tri Me2Lys X 11 a Cy st in e Tyr His 10 Tyr Tyr Tyr Tyr Al 115* 9 Cystine X Cystine Tyr H Ho is m oC it Ala 8 Ala Ala X Ala Ly s (Me-His) 7 HomoCit X X HomoCit is Tyr 6 His His X His eH 114 4 5 3MeHis Lys Ala Lys Ala Lys 3MeHis Me2Lys X Me2Lys a Me2Lys Arg Arg iso-Asp 3 Ala Ala X Ala 3M 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. 2 Al 1 expected Tyr 111 Tyr 112 Tyr 113 Tyr eL ys Abstract 25 25 participant # Effect of Premix Concentration on Elution Profiles Elution Profiles of 3meHis, Me2Lys & Me3Lys on cLC 100 (21ml/L Premix vs 14.5ml/L Premix) 3meHis 3meHis vs 21ml/L lag Ala lag Ala + 3meHis Ala (Std & Cycle 4) 14.5ml/L 50 Description of the Sample The sample was a synthetic, cysteine-9 disulfide-linked 18-mer peptide with the following sequence: H N H H3 C N CH3 H 3C N H2N O H3 C NH N N CH3 CH3 S S H H N H OH O R2 -N,N-Dimethyl Lysine N H H OH CH3 O H R4 3- Methyl Histidine N H OH O R7 -N-Carbamyl Lysine H OH N H O R9 Cystine H OH N O H H N OH OH N H O R13 N-Methyl Alanine R11 -N,N,N- Trimethyl Lysine Ala Tyr NmeAla Gly trimeLys Tyr Cystine 1 Ala HomoCit His Lys 3MeHis Ala Me2Lys Me2Lys - 2 vs Me3Lys - 11 Me3Lys (Cycle 2 & 11) Me2Lys - 2 lag Tyr 14.5ml/L 21ml/L lag Tyr Me3Lys - 11 1. Relative retention times of the modified amino acids between similar instruments were very consistent. CH3 OH Tyr Conclusions Structures of the modified amino acids are shown below: O dimeLys 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Tyr-[Me2-Lys]-Ala-[3-Me-His]-Lys-His-[homoCit]-Ala-Cys-Tyr-[Me3-Lys]-Gly-[N-Me-Ala]-Tyr-Ala-[isoAsp]-Val –Arg S-S Tyr-[Me2-Lys]-Ala-[3-Me-His]-Lys-His-[homoCit]-Ala-Cys-Tyr-[Me3-Lys]-Gly-[N-Me-Ala]-Tyr-Ala-[isoAsp]-Val –Arg 0 O HO O R16 iso-Aspartic Acid 2. 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. 3. 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