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Synthesis, Evaluation and Application of a Panel of Novel Reagents for Stepwise Degradation of Polypeptides

  • Edward J. Bures
  • Heinz Nika
  • David T. Chow
  • Daniel Hess
  • Hamish D. Morrison
  • Michael Bartlet-Jones
  • Darryl J. C. Pappin
  • Ruedi Aebersold

Abstract

The Edman degradation (Edman, 1949) has been the most successful, general and widely used technique for the determination of the amino acid sequence of proteins and peptides. As a benefit of this distinction, over the last four decades the method has been refined to a high degree of perfection. Nevertheless, sequencing with phenyl isothiocyanate (PITC) suffers from a few practical limitations. First, the extinction coefficient of the phenylthiohydantoins (PTH’s) limits sequencing sensitivity. Currently, routine sequencing in most laboratories requires low picomole amounts of sample applied to the sequencer. Second, UV-absorbing products which may co-elute with PTH’s during high performance liquid chromatography (HPLC) separation have a tendency to obscure the specific PTH signals during high sensitivity sequencing. Third, with the exception of select cases (Wettenhall et al, 1991; Meyer et al, 1990, 1991; Aebersold et al, 1991; Gooley et al, 1991; Pisano et al, 1993), modified and unnatural amino acids of known structure are difficult to identify and de-novo characterization of such residues by UV absorbance detection alone is extremely difficult.

Keywords

High Performance Liquid Chromatography High Performance Liquid Chromatography Quaternary Amine Amino Acid Derivative Unnatural Amino Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

PITC

phenyl isothiocyanate

PTH

phenylthiohydantoin

HPLC

high-performance liquid chromatography

ESI-MS

electrospray ionization mass spectrometer/metry

MS/MS

tandem mass spectrometer/metry

PETMA-PITC

3-[4’(ethylene-N,N,N-trimethylamino)-phenyl]-2-isothiocyanate

PITC-311

4-(3 pyridylmethylaminocarboxypropyl)-phenyl isothiocyanate

RP-HPLC

reverse-phase high-performance liquid chromatography

TFA

trifluoroacetic acid

MeCN

acetonitrile.

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References

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Copyright information

© Springer Science+Business Media New York 1995

Authors and Affiliations

  • Edward J. Bures
    • 1
  • Heinz Nika
    • 1
  • David T. Chow
    • 1
  • Daniel Hess
    • 1
  • Hamish D. Morrison
    • 1
  • Michael Bartlet-Jones
    • 2
  • Darryl J. C. Pappin
    • 2
  • Ruedi Aebersold
    • 1
  1. 1.Biomedical Research CentreUniversity of BritishColumbiaCanada
  2. 2.Imperial Cancer Research FundLondonUK

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