Skip to main content

Advertisement

Springer Nature Link
Log in

Can Edman degradation be used for quantification? Isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry and the long-term stability of 20 phenylthiohydantoin-amino acids

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Edman degradation is a well-known method for obtaining amino acid (AA) sequences from a peptide by means of sequential reactions that release the N-terminal AAs from the peptide as a phenylthiohydantoin (PTH) derivative. Because of unexpected loss during the reaction and handling, there are few reports of use of this reaction for quantification. This manuscript describes the development of isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry for 20 PTH-AA derivatives, and long-term stability testing of PTH-AAs to ensure quantitative quality in the reaction. The 20 corresponding [13C6]-PTH-AAs were prepared by use of a one-pot reaction involving a mixture of [13C6]-Edman reagent and 20 AAs. Good linearity was observed for standard curves for the PTH-AAs, using the corresponding [13C6]-PTH-AAs as internal standards (1–100 pmol per injection, r 2 = 0.989–1.000). Serum albumin (human), pepsin (porcine stomach mucosa), α-casein (bovine milk), ribonuclease A (bovine), lysozyme (chicken egg white), and insulin (bovine) subjected to Edman degradation were examined as model proteins and peptides for N-terminal AA analysis. The results of the impurity test were satisfactory. Yield from the entire reaction with human serum albumin was estimated to be at least 75 %, indicating great potential for absolute quantification of proteins without protein standards.

ᇵ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+
from $39.99 /Month
  • Starting from 10 chapters or articles per month
  • Access and download chapters and articles from more than 300k books and 2,500 journals
  • Cancel anytime
View plans

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Explore related subjects

Discover the latest articles and news from researchers in related subjects, suggested using machine learning.

References

  1. Edman P (1950) Method for determination of the amino acid sequence in peptides. Acta Chem Scand 4:283–293

    Article  CAS  Google Scholar 

  2. Edman P, Begg G (1967) A protein sequenator. Eur J Biochem 1:80–91

    Article  CAS  Google Scholar 

  3. Chen W, Yin X, Mu J, Yin Y (2007) Subfemtomole level protein sequencing by Edman degradation carried out in a microfluidic chip. Chem Commun (Camb) 2488–2490

  4. Chang JY, Creaser EH, Bentley KW (1976) 4-NN-dimethylaminoazobenzene 4′-isothiocyanate, a new chromophoric reagent for protein sequence analysis. Biochem J 153:607–611

    CAS  Google Scholar 

  5. Jin SW, Chen GX, Palacz Z, Wittmann-Liebold B (1986) A new sensitive Edman-type reagent: 4-(N-1-dimethylaminonaphthalene-5-sulfonylamino)phenyl isothiocyanate. Its synthesis and application for micro-sequencing of polypeptides. FEBS Lett 198:150–154

    Article  CAS  Google Scholar 

  6. Muramoto K, Nokihara K, Ueda A, Kamiya H (1994) Gas-phase microsequencing of peptides and proteins with a fluorescent Edman-type reagent, fluorescein isothiocyanate. Biosci Biotechnol Biochem 58:300–304

    Article  CAS  Google Scholar 

  7. http://www.abrf.org/index.cfm/group.show/EdmanSequencing.29.htm

  8. Buckel S, Cook R, Crawford J, Dupont D, Madden B, Neveu J, Steinke L, Fernandez J (2002) ABRF-2002ESRG, a difficult sequence: analysis of a PVDF-bound known protein with a heterogeneous amino-terminus. J Biomol Tech 13:246–257

    Google Scholar 

  9. Mische SM, Yüksel KÜ, Mende-Mueller LM, Matsudaira P, Crimmins DL, Andrews P (1993) Protein sequencing of post-translationally modified peptides and proteins: Design, characterization and results of ABRF-92SEQ. In: Hogue-Angeletti R (ed) Techniques in protein chemistry IV. Academic Press Inc., San Diego, pp 453–461

    Google Scholar 

  10. Chait BT, Wang R, Beavis RC, Kent SBH (1993) Protein ladder sequencing. Science 262:89–92

    Article  CAS  Google Scholar 

  11. Aebersold R, Bures EJ, Namchuk M, Goghari MH, Shushan B, Covey TC (1992) Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry. Protein Sci 1:494–503

    Article  CAS  Google Scholar 

  12. Bures EJ, Nika H, Chow DT, Morrison HD, Hess D, Aebersold R (1995) Synthesis of the protein-sequencing reagent 4-(3-pyridinylmethylaminocarboxypropyl) phenyl isothiocyanate and characterization of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins. Anal Biochem 224:364–372

    Article  CAS  Google Scholar 

  13. Ducret A, Bures EJ, Aebersold R (1997) High-sensitivity detection of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by capillary liquid chromatography–microelectrospray ion trap mass spectrometry. J Protein Chem 16:323–328

    Article  CAS  Google Scholar 

  14. Marekov LN, Steinert PM (2003) Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Mass Spectrom 38:373–377

    Article  CAS  Google Scholar 

  15. Thakkar A, Wavreille AS, Pei D (2006) Traceless capping agent for peptide sequencing by partial Edman degradation and mass spectrometry. Anal Chem 78:5935–5939

    Article  CAS  Google Scholar 

  16. Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1:376–386

    Article  CAS  Google Scholar 

  17. Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999

    Article  CAS  Google Scholar 

  18. Li J, Steen H, Gygi SP (2003) Protein profiling with Cleavable Isotope-Coded Affinity Tag (cICAT) reagents. Mol Cell Proteomics 2:1198–1204

    Article  CAS  Google Scholar 

  19. Zieske LR (2006) A perspective on the use of iTRAQ reagent technology for protein complex and profiling studies. J Exp Bot 57:1501–1508

    Article  CAS  Google Scholar 

  20. Leng J, Wang H, Zhang L, Zhang J, Wang H, Guo Y (2013) A highly sensitive isotope-coded derivatization method and its application for the mass spectrometric analysis of analytes containing the carboxyl group. Anal Chim Acta 758:114–121

    Article  CAS  Google Scholar 

  21. Guidance for Industry, Q5E Comparability of biotechnological/biological products subject to changes in their manufacturing process, June 2005, ICH. (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm073476.pdf)

  22. Oe T, Ackermann BL, Inoue K, Berna MJ, Garner CO, Gelfanova V, Dean RA, Siemers ER, Holtzman DM, Farlow MR, Blair IA (2006) Quantitative analysis of amyloid beta peptides in cerebrospinal fluid of Alzheimer’s disease patients by immunoaffinity purification and stable isotope dilution liquid chromatography/negative electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 20:3723–3735

    Article  CAS  Google Scholar 

  23. Kamiie J, Ohtsuki S, Iwase R, Ohmine K, Katsukura Y, Yanai K, Sekine Y, Uchida Y, Ito S, Terasaki T (2008) Quantitative atlas of membrane transporter proteins: development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharm Res 25:1469–1483

    Article  CAS  Google Scholar 

  24. Ciccimaro E, Hanks SK, Yu KH, Blair IA (2009) Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry. Anal Chem 81:3304–3313

    Article  CAS  Google Scholar 

  25. Brune DC, Hampton B, Kobayashi R, Leone JW, Linse KD, Pohl J, Thoma RS, Denslow ND (2007) ABRF ESRG 2006 study: Edman sequencing as a method for polypeptide quantitation. J Biomol Tech 18:306–320

    CAS  Google Scholar 

  26. Oe T, Kambouris SJ, Walker VE, Meng Q, Recio L, Wherli S, Chaudhary AK, Blair IA (1999) Persistence of N7-(2,3,4-trihydroxybutyl)guanine adducts in the livers of mice and rats exposed to 1,3-butadiene. Chem Res Toxicol 12:247–257

    Article  CAS  Google Scholar 

  27. Oe T, Tian Y, O’Dwyer PJ, Roberts DW, Malone MD, Bailey CJ, Blair IA (2002) A validated liquid chromatography/tandem mass spectrometry assay for cis-amminedichloro(2-methylpyridine)platinum(II) in human plasma ultrafiltrate. Anal Chem 74:591–599

    Article  CAS  Google Scholar 

  28. Oe T, Maekawa M, Satoh R, Lee SH, Goto T (2010) Combining [13C6]-phenylisothiocyanate and the Edman degradation reaction: a possible breakthrough for absolute quantitative proteomics together with protein identification. Rapid Commun Mass Spectrom 24:173–179

    Article  CAS  Google Scholar 

  29. Ares JJ, Fowble JW, Urchek TG, Miller DD (1987) Synthesis and application of 13C enriched phenylisothiocyanate as a 13C NMR protein probe. Biochem Biophys Res Commun 142:1064–1071

    Article  CAS  Google Scholar 

  30. Guillaume E, Panchaud A, Affolter M, Desvergnes V, Kussmann M (2006) Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification. Proteomics 6:2338–2349

    Article  CAS  Google Scholar 

  31. Leng JP, Wang HY, Zhang L, Zhang J, Wang H, Cai TT, Yao JT, Guo YL (2011) Integration of high accuracy N-terminus identification in peptide sequencing and comparative protein analysis via isothiocyanate-based isotope labeling reagent with ESI ion-trap TOF MS. J Am Soc Mass Spectrom 22:1204–1213

    Article  CAS  Google Scholar 

  32. Leng J, Zhu D, Wu D, Zhu T, Zhao N, Guo Y (2012) Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 26:2555–2562

    Article  CAS  Google Scholar 

  33. Anonymous (2010) Novel approach to absolute protein quantitation. Expert Rev Proteomics 7:13–13

    Google Scholar 

  34. Smith GF, Murray M (1968) Direct spectrophotometric quantitation of phenylthiohydantoin derivatives of amino acids from thin layers of silica gel. Anal Biochem 23:183–195

    Article  CAS  Google Scholar 

  35. Glajch JL, Gluckman JC, Charikofsky JG, Minor JM, Kirkland JJ (1985) Simultaneous selectivity optimization of mobile and stationary phases in reversed-phased liquid chromatography for isocratic separations of phenylthiohydantoin amino acid derivatives. J Chromatogr A 318:23–39

    Article  CAS  Google Scholar 

  36. Tsunasawa S, Kondo J, Sakiyama F (1985) Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate. J Biochem 97:701–704

    CAS  Google Scholar 

  37. Zhou J, Hefta S, Lee TD (1997) High sensitivity analysis of phenylthiohydantoin amino acid derivatives by electrospray mass spectrometry. J Am Soc Mass Spectrom 8:1165–1174

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by the Asahi Glass Foundation through a grant to T.O. (2010–2011, “Novel analytical platform to enable both absolute quantification and identification of unknown proteins in biological samples”) and through their continuing support for the restoration of our laboratories damaged by the Great East Japan Earthquake. The authors also thank Shionogi & Co., Ltd. (Osaka, Japan), Astellas Pharma Inc. (Tokyo, Japan), and Professor Ian A. Blair (University of Pennsylvania, Philadelphia, PA) for donating two used TSQ 7000 s and an LCQ Deca, respectively.

Author information

Authors and Affiliations

  1. Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan

    Ryo Satoh, Takaaki Goto, Seon Hwa Lee & Tomoyuki Oe

Authors
  1. Ryo Satoh
    View author publications

    Search author on:PubMed Google Scholar

  2. Takaaki Goto
    View author publications

    Search author on:PubMed Google Scholar

  3. Seon Hwa Lee
    View author publications

    Search author on:PubMed Google Scholar

  4. Tomoyuki Oe
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Tomoyuki Oe.

Additional information

Published in the topical collection Amino Acid Analysis with guest editor Toshimasa Toyo’oka.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 1.63 MB)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Satoh, R., Goto, T., Lee, S.H. et al. Can Edman degradation be used for quantification? Isotope-dilution liquid chromatography–electrospray ionization tandem mass spectrometry and the long-term stability of 20 phenylthiohydantoin-amino acids. Anal Bioanal Chem 405, 8001–8010 (2013). https://doi.org/10.1007/s00216-013-6901-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-013-6901-6

Keywords

Profiles

  1. Tomoyuki Oe View author profile