Absolute quantification of Corynebacterium glutamicum glycolytic and anaplerotic enzymes by QconCAT

Abstract

Determination of absolute protein amounts using quantitative concatemers (QconCAT's) has already been successfully demonstrated for various species including human, animal and yeast. Interestingly, application of the QconCAT methodology for the determination of cytoplasmic enzyme concentrations in a prokaryote has not been described so far. This study is concerned with a novel targeted approach for the absolute quantification of 10 key enzymes from the central carbon metabolism of the industrially important organism Corynebacterium glutamicum. We demonstrate a method that enables complete cell lysis of this robust soil bacterium, thus allowing for accurate quantification of cytoplasmic enzymes. By linking measured enzyme amounts with respective biovolume data, intracellular enzyme concentrations were estimated, which are of special importance for any systems biology approach studying C. glutamicum's metabolism at the mechanistic level. To our knowledge this is the first report of applying the QconCAT methodology for determining intracellular enzyme concentrations in a prokaryote.