Attomole level protein sequencing by Edman degradation coupled with accelerator mass spectrometry.
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 (14)C. The transgenic transferase was labeled by growing transformed Escherichia coli on [(14)C]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.
Miyashita
M
M
Department of Entomology, Molecular Structure Facility, Cancer Center, and Department of Internal Medicine, University of California, Davis, CA 95616, USA.
Presley
J M
JM
Buchholz
B A
BA
Lam
K S
KS
Lee
Y M
YM
Vogel
J S
JS
Hammock
B D
BD
D002250:Carbon Radioisotopes; D005982:Glutathione Transferase / Q000737:chemistry*; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
D002250:Carbon Radioisotopes; D005982:Glutathione Transferase