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

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 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.

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

“…HPLC fractions were collected, dried and converted to graphite as previously described (Buchholz et al, [2000]; Getachew et al, [2006]; Miyashita et al, [2001]). 14 C contents in the graphite samples were quantified by AMS (Ognibene et al, [2002]).…”

“… a) Reported values were converted to concentrations (mM) as previously described (11), b) P < 0.05 compared to present study wild type, c) P > 0.1 compared to present study wild type, d) 0.05 < P< 0.10 compared to present study wild type, e) P < 0.05 compared to NAD + under normal growth, f) P < 0.05 compared to NADH under normal growth, g) 0.05 < P< 0.10 compared to qpt1Δ NADH levels under normal growth, h) P > 0.10 compared to npt1Δ NADH levels under normal growth, i) P < 0.05 compared to NAD + :NADH ratio under normal growth conditions, j) P > 0.1 compared to NAD + :NADH ratio under normal growth. …”

Applications of accelerator mass spectrometry for pharmacological and toxicological research

“…Those factors make AMS a powerful new tool in drug development in pre-Phase I clinical trials. Furthermore, areas where AMS can be exploited are continually expanding; recently reported new applications include N-terminal sequencing of unknown proteins, when coupled with Edman degradation (Miyashita et al, 2001) and radioimmunoassays, where AMS offers the necessary sensitivity of conventional assays without generating radioactive waste (Shan et al, 2000;Lu et al, 2002).…”

GAS-HYBRID SOURCE 14C-AMS:Application to terrestrial ecosystem, biological, and environmental research

“…The development of the MWI was driven by the need for basic analytical tools for biomedical studies with a focus on metabolites, enzyme dynamics, elucidation and quantification of biochemistry pathways, and developing data-sets that are appropriate to test flux balance models [e.g., Bucholz et al, 1999;Miyashita et al, 2001;Stewart et al, 2010;Vogel et al, 2001]. The MWI is based on the design of Sessions et al, [2005] The design and initial optimization of the MWI focused on tracer-based applications in the biological sciences where 1-2µl drops of HPLC separated analytes are, via a micro-capillary needle, placed on the wire, dried, and converted to CO 2 .…”