Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry

Masahiro Miyashita, Jack M. Presley, Bruce A. Buchholz, Kit Lam, Young Moo Lee, John S. Vogel, Bruce D. Hammock

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Edman degradation remains the primary method for determining the sequence of proteins. In this study, accelerator mass spectrometry was used to determine the N-terminal sequence of glutathione S-transferase at the attomole level with zeptomole precision using a tracer of 14C. The transgenic transferase was labeled by growing transformed Escherichia coli on [14C]glucose and purified by microaffinity chromatography. An internal standard of peptides on a solid phase synthesized to release approximately equal amounts of all known amino acids with each cycle were found to increase yield of gas phase sequencing reactions and subsequent semimicrobore HPLC as did a lactoglobulin carrier. This method is applicable to the sequencing of proteins from cell culture and illustrates a path to more general methods for determining N-terminal sequences with high sensitivity.

Original languageEnglish (US)
Pages (from-to)4403-4408
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number8
DOIs
StatePublished - Apr 10 2001

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Protein Sequence Analysis
Mass Spectrometry
Lactoglobulins
Transferases
Glutathione Transferase
Chromatography
Cell Culture Techniques
Gases
High Pressure Liquid Chromatography
Escherichia coli
Amino Acids
Glucose
Peptides
Proteins

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry. / Miyashita, Masahiro; Presley, Jack M.; Buchholz, Bruce A.; Lam, Kit; Lee, Young Moo; Vogel, John S.; Hammock, Bruce D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 98, No. 8, 10.04.2001, p. 4403-4408.

Research output: Contribution to journalArticle

Miyashita, Masahiro ; Presley, Jack M. ; Buchholz, Bruce A. ; Lam, Kit ; Lee, Young Moo ; Vogel, John S. ; Hammock, Bruce D. / Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry. In: Proceedings of the National Academy of Sciences of the United States of America. 2001 ; Vol. 98, No. 8. pp. 4403-4408.
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